W. E. Moerner
Harry S. Mosher Professor and Professor, by courtesy, of Applied Physics
Chemistry
Web page: http://web.stanford.edu/group/moerner
Bio
W. E. (William Esco) Moerner, the Harry S. Mosher Professor of Chemistry and Professor by courtesy of Applied Physics, has conducted research in physical chemistry, biophysics, and the optical properties of single molecules, and is actively involved in the development of 2D and 3D super-resolution imaging for cell biology. Imaging studies include viral RNA and proteins in infected cells, protein superstructures in bacteria and mammalian cells, and studies of chromatin organization. Using powerful microscopes optimized for tracking of single objects in cells, the motions of proteins, DNA, and RNA are being measured in three dimensions in real time to understand organization, processing and binding interactions. A previous research area concerns precise analysis of photodynamics of single trapped biomolecules in solution, with applications to photosynthesis, protein-protein interactions, and transport measurements.
Born on June 24, 1953 at Parks Air Force Base in Pleasanton, California, Professor Moerner was raised in San Antonio, Texas. He attended Washington University as a Langsdorf Engineering Fellow, graduating in 1975 with degrees in Physics and Electrical Engineering (both B.S. with top honors), and Mathematics (A.B. summa cum laude). His doctoral research in physics at Cornell University (M.S. 1978, Ph.D. 1982) employed tunable infrared lasers to explore infrared vibrational modes of impurities in crystals. In 1982, he moved from New York to San Jose, California to join the IBM Research Division developing spectral holeburning for frequency domain optical storage and photorefractivity for dynamic hologram formation. After 13 years at IBM, Dr. Moerner accepted a position as Distinguished Professor of Physical Chemistry at UC San Diego, where he broadened his research to include biological systems and biophysics. Recruited to the Stanford Chemistry Department faculty in 1997, he served as Chair of the department from 2011 to 2014.
Professor Moerner’s scientific contributions were recognized with the 2014 Nobel Prize in Chemistry "for the development of super-resolved fluorescence microscopy." One method to surpass the optical diffraction limit (PALM/STORM) uses single-molecule imaging combined with an active control mechanism to keep the concentration of emitting molecules at a very low level, followed by sequential localization to reconstruct the underlying structure. The fundamentals of this idea came from early work in the Moerner lab: optical detection and imaging of single molecules (1989) combined with blinking and switching at low temperature, as well as the discovery of optical control and blinking of single copies of green fluorescent protein at room temperature (1997). Among many other honors and awards, Professor Moerner was elected fellow of the American Physical Society, Optical Society of America, American Association for the Advancement of Science, American Academy of Arts and Sciences, SPIE; and member of the National Academy of Sciences.
Today, the Moerner Laboratory uses laser spectroscopy and microscopy of single molecules to probe biological processes, one molecule at a time. Primary thrusts include development and application of fluorescence microscopy far beyond the optical diffraction limit by PALM/STORM, 3D single-molecule tracking in complex cellular environments, invention and validation of methods for precise and accurate 3D optical microscopy in cells, and imaging of viral RNA and proteins during cellular infection by SARS-CoV-2 viruses. Through a variety of collaborations, these approaches are applied to explore protein and oligonucleotide localization patterns in mammalian cells and bacteria, define the organization of cell invasion proteins in parasites such as Toxoplasma gondii, and develop correlative super-resolution optical imaging with cryo-EM enhanced by suitable switchable fluorescent proteins for 77K.
Please visit the Moerner Lab home page for more information.
Academic Appointments
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Professor, Chemistry
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Professor (By courtesy), Applied Physics
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Member, Bio-X
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Faculty Fellow, Sarafan ChEM-H
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Member, Wu Tsai Neurosciences Institute
Administrative Appointments
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Faculty Fellow, ChEM-H at Stanford (2014 - Present)
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Chairman, Department of Chemistry, Stanford University (2011 - 2014)
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Member, Advisory Board, Center for Biological Imaging at Stanford (2010 - 2015)
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Member, Board of Scientific Counselors, NIBIB (2010 - 2014)
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Chair, University Health and Safety Committee (2008 - 2010)
Honors & Awards
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Nobel Prize in Chemistry, Nobel Foundation (2014)
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Wu Zheng Kai Chemistry Prize, Fudan University (2018)
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Distinguished Eagle Scout Award, Boy Scouts of America (2017)
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INSPIRE Award for Excellence, San Antonio Independent School District (2016)
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Julio Palmaz Award for Innovation in Healthcare and Biosciences, BioMed SA (2015)
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Peter Debye Award in Physical Chemistry, American Chemical Society (2013)
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Irving Langmuir Prize in Chemical Physics, American Physical Society (2009)
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Wolf Prize in Chemistry, Wolf Foundation of Israel (2008)
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Member, National Academy of Sciences (2007)
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Earle K. Plyler Prize in Molecular Spectroscopy, American Physical Society (2001)
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Fellow, Optical Society of America (1992)
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Fellow, American Physical Society (1992)
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National Winner, Roger I. Wilkinson Outstanding Young Electrical Engineer Award, Eta Kappa Nu (1985)
Boards, Advisory Committees, Professional Organizations
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Trustee, Society for Science and the Public (2018 - Present)
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Member, Advisory Board, Institute of Atomic and Molecular Sciences, Academica Sinica, Taiwan (2003 - Present)
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Member, Scientific Advisory Board, Welch Foundation (2017 - 2021)
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Member, International Advisory Board, Angewandte Chemie (2017 - 2019)
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Member, Corporation Visiting Committee, Department of Chemistry, Massachusetts Institute of Technology (2013 - 2017)
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Editorial Advisory Board Member, Journal of Physical Chemistry (2013 - 2015)
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Member, Board of Scientific Counselors, National Institute of Biomedical Imaging and Bioengineering (2010 - 2014)
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Member, DOE Workshop on Single-Molecule Research in the New Millenium (2005 - 2005)
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Advisory Editor, Single Molecules (2000 - 2002)
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Member, NIH-NIGMS Workshop on Single Molecule Detection and Manipulation (2000 - 2000)
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Member, FAMOS Update Panel, National Research Council (1999 - 2002)
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Member, NIH Bioengineering Symposium Panel on Imaging at the Molecular and Cellular Levels (1998 - 1998)
Professional Education
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Ph.D., Cornell University, Physics (1982)
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M.S., Cornell University, Physics (1978)
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B. S., Washington University, Physics (1975)
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B. S., Washington University, Electrical Engineering (1975)
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A. B., Washington University, Mathematics (1975)
Community and International Work
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Amateur Radio Emergency Service
Topic
Emergency Communications
Populations Served
Santa Clara County
Ongoing Project
Yes
Opportunities for Student Involvement
No
Current Research and Scholarly Interests
Most biophysical or chemical experiments in condensed matter measure the average behavior of a huge number, N, of molecules, where N may range from millions to billions to Avogadro's Number. At the same time, most theoretical models are intended to describe the behavior of a single molecule interacting with its surroundings, and averaging over the number of molecules N is normally required to compute an observable. Using precision laser spectroscopic techniques, we have been detecting and probing the detailed properties of individual impurity molecules hidden deep inside a cell, in a protein, or even in a liquid, i.e., the ultimate limit of N=1. This was first done in the Moerner Lab in 1989, and has since expanded dramatically to include many groups around the world. A key reason for doing this is to explore heterogeneity that is normally obscured by ensemble averaging.
Studying one individual molecule in a solid means we are working with an extremely small number of moles of material. You might be aware that the international standards organization, IUPAC, has defined several new prefixes: zepto- for 1E-21, and yocto- for 1E-24. Thus 1 molecule is equivalent to 1.66 yoctomoles. But we think this is unwieldy. Thus we define a new prefix guaca- so that (with apologies to Prof. Avogadro)
1 guacamole = 1 / ( Avocado's Number) of moles.
More seriously, it is worth recalling that each molecule we are probing is only 1 or 2 nanometers in size. This means that when we use a laser to select one probe molecule, we can sense details of the immediate local environment of a truly nanoscopic probe.
To achieve this extreme reduction of the concentration and reach the single-molecule level, we use either (a) extremely low concentrations and diffraction-limited confocal, TIRF, or far-field microscopy, or (b) near field optical excitation to pump sample volumes much smaller than the diffraction limit, or (c) superresolution imaging by single-molecule active control. By studying a large number of individual molecules one at a time, we are able not only to observe how the usual ensemble average behavior is formed, but also to see unexpected, surprising behavior normally hidden by the usual ensemble averaging.
The phenomena under study include protein localization patternd in bacteria, chaperonin proteins, and new fluorophores for active-control superresolution imaging. By dispersing the emitted light, even the vibrational mode spectrum of a single molecule may be measured! By measuring correlations in the emitted photon stream, fast dynamics including environmental fluctuations, or the purely quantum-mechanical behavior termed photon antibunching may be probed. In biomolecules, we observe fascinating differences in behavior due to conformational states, local environments, or enzymatic cycle, all of which are obscured in large N experiments.
Importantly, a single molecule can be viewed as a probe of its immediate local nanoenvironment on the scale on the order of the molecular size (~1 nm). Because single molecules are nanoscale emitters, when active control is used to turn molecules on and off, it is possible to build up a super-resolution image of the sample, far beyond the optical diffraction limit, typically on the 40 nm scale. Several advanced optical techniques for obtaining thee-dimensional information from single-molecule photoswitching are underdevelopment, and we apply these methods to imaging a variety of cellular structures in bacteria and in mammalian cells and to tracking of RNA in living yeast.
2024-25 Courses
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Independent Studies (9)
- Advanced Undergraduate Research
CHEM 190 (Aut, Win, Spr, Sum) - Curricular Practical Training
APPPHYS 291 (Aut, Win, Spr, Sum) - Directed Instruction/Reading
CHEM 90 (Aut, Win, Spr, Sum) - Directed Reading in Biophysics
BIOPHYS 399 (Aut, Win, Spr, Sum) - Directed Studies in Applied Physics
APPPHYS 290 (Aut, Win, Spr, Sum) - Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum) - Research
PHYSICS 490 (Aut, Win, Spr, Sum) - Research and Special Advanced Work
CHEM 200 (Aut, Win, Spr, Sum) - Research in Chemistry
CHEM 301 (Aut, Win, Spr, Sum)
- Advanced Undergraduate Research
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Prior Year Courses
2022-23 Courses
- Advanced Physical Chemistry - Single Molecules and Light
CHEM 275 (Spr)
2021-22 Courses
- Advanced Physical Chemistry - Single Molecules and Light
CHEM 275 (Spr)
- Advanced Physical Chemistry - Single Molecules and Light
Stanford Advisees
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Doctoral Dissertation Reader (AC)
Sarah E Holmes, Erica Liu -
Postdoctoral Faculty Sponsor
Leonid Andronov, Andrew Barentine, Pierre Jouchet, Michelle Kueppers -
Doctoral Dissertation Advisor (AC)
Ashwin Balaji
All Publications
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Exploring Transient States of PAmKate to Enable Improved Cryogenic Single-Molecule Imaging.
Journal of the American Chemical Society
2024
Abstract
Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nanoscale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) that photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remain a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find that a transient state forms with a lifetime of 2 ms under cryogenic conditions, which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for the study of other FPs at cryogenic temperatures.
View details for DOI 10.1021/jacs.4c05632
View details for PubMedID 39388715
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Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung.
The Journal of experimental medicine
2024; 221 (6)
Abstract
Early stages of deadly respiratory diseases including COVID-19 are challenging to elucidate in humans. Here, we define cellular tropism and transcriptomic effects of SARS-CoV-2 virus by productively infecting healthy human lung tissue and using scRNA-seq to reconstruct the transcriptional program in "infection pseudotime" for individual lung cell types. SARS-CoV-2 predominantly infected activated interstitial macrophages (IMs), which can accumulate thousands of viral RNA molecules, taking over 60% of the cell transcriptome and forming dense viral RNA bodies while inducing host profibrotic (TGFB1, SPP1) and inflammatory (early interferon response, CCL2/7/8/13, CXCL10, and IL6/10) programs and destroying host cell architecture. Infected alveolar macrophages (AMs) showed none of these extreme responses. Spike-dependent viral entry into AMs used ACE2 and Sialoadhesin/CD169, whereas IM entry used DC-SIGN/CD209. These results identify activated IMs as a prominent site of viral takeover, the focus of inflammation and fibrosis, and suggest targeting CD209 to prevent early pathology in COVID-19 pneumonia. This approach can be generalized to any human lung infection and to evaluate therapeutics.
View details for DOI 10.1084/jem.20232192
View details for PubMedID 38597954
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Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles.
Nature communications
2024; 15 (1): 4644
Abstract
The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.
View details for DOI 10.1038/s41467-024-48991-x
View details for PubMedID 38821943
View details for PubMedCentralID 7951565
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Exploring transient states of PAmKate to enable improved cryogenic single-molecule imaging.
bioRxiv : the preprint server for biology
2024
Abstract
Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nano-scale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) which photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remains a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find a transient state forms with a lifetime of ~2 ms which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for study of other FPs at cryogenic temperatures.
View details for DOI 10.1101/2024.04.24.590965
View details for PubMedID 38712218
View details for PubMedCentralID PMC11071506
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Stimulated emission does not radiate in a pure dipole pattern
OPTICA
2024; 11 (4): 464-470
View details for DOI 10.1364/OPTICA.515226
View details for Web of Science ID 001234696100006
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Solution-phase sample-averaged single-particle spectroscopy of quantum emitters with femtosecond resolution.
Nature materials
2024
Abstract
The development of many quantum optical technologies depends on the availability of single quantum emitters with near-perfect coherence. Systematic improvement is limited by a lack of understanding of the microscopic energy flow at the single-emitter level and ultrafast timescales. Here we utilize a combination of fluorescence correlation spectroscopy and ultrafast spectroscopy to capture the sample-averaged dynamics of defects with single-particle sensitivity. We employ this approach to study heterogeneous emitters in two-dimensional hexagonal boron nitride. From milliseconds to nanoseconds, the translational, shelving, rotational and antibunching features are disentangled in time, which quantifies the normalized two-photon emission quantum yield. Leveraging the femtosecond resolution of this technique, we visualize electron-phonon coupling and discover the acceleration of polaronic formation on multi-electron excitation. Corroborated with theory, this translates to the photon fidelity characterization of cascaded emission efficiency and decoherence time. Our work provides a framework for ultrafast spectroscopy in heterogeneous emitters, opening new avenues of extreme-scale characterization for quantum applications.
View details for DOI 10.1038/s41563-024-01855-7
View details for PubMedID 38589542
View details for PubMedCentralID 5615041
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Revealing the 3D nanoscale organization of MyosinH in the apical complex of toxoplasma gondii through single-molecule localization microscopy with the double-helix point spread function
CELL PRESS. 2024: 30A-31A
View details for Web of Science ID 001194120700146
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Localization of viral RNA and proteins in the SARS-CoV-2 replication organelles revealed by super-resolution microscopy
CELL PRESS. 2024: 464A
View details for Web of Science ID 001194120702650
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Single-molecule orientation and position measurement assisted by deep learning and point spread function engineering
CELL PRESS. 2024: 154A
View details for Web of Science ID 001194120701033
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Proving stimulated emission radiates directionally.
ArXiv
2023
Abstract
Stimulated Emission (StE) remains relatively unused as an image-forming signal despite having potential advantages over fluorescence in speed, coherence, and ultimately resolution. Several ideas for the radiation pattern and directionality of StE remain prevalent, namely whether a single molecule would radiate like a simple dipole, or whether its emission direction depends on the driving field. Previous StE imaging has been carried out in transmission, which would collect signal either way. Here, we introduce the StE driving field (the probe) at an angle, using total internal reflection to avoid incident probe light and its specular reflections in our detection path. In this non-collinear detection configuration which also collects some fluorescence from the sample, we observe fluorescence depletion even in the spectral window where an increase in detected signal from StE would be expected if StE radiated like a simple dipole. We further demonstrate that this directionality is a property of StE and not due to ensemble effects from multiple emitters. Our study clarifies a critical characteristic of StE for optimal microscope design, optical cooling, and more.
View details for PubMedID 38196744
View details for PubMedCentralID PMC10775344
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Combining deep learning approaches and point spread function engineering for simultaneous 3D position and 3D orientation measurements of fluorescent single molecules
OPTICS COMMUNICATIONS
2023; 542
View details for DOI 10.1016/j.optcom.2023.129589
View details for Web of Science ID 001010300700001
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Combining deep learning approaches and point spread function engineering for simultaneous 3D position and 3D orientation measurements of fluorescent single molecules.
Optics communications
2023; 542
Abstract
Point Spread Function (PSF) engineering is an effective method to increase the sensitivity of single-molecule fluorescence images to specific parameters. Classical phase mask optimization approaches have enabled the creation of new PSFs that can achieve, for example, localization precision of a few nanometers axially over a capture range of several microns with bright emitters. However, for complex high-dimensional optimization problems, classical approaches are difficult to implement and can be very time-consuming for computation. The advent of deep learning methods and their application to single-molecule imaging has provided a way to solve these problems. Here, we propose to combine PSF engineering and deep learning approaches to obtain both an optimized phase mask and a neural network structure to obtain the 3D position and 3D orientation of fixed fluorescent molecules. Our approach allows us to obtain an axial localization precision around 30 nanometers, as well as an orientation precision around 5 degrees for orientations and positions over a one micron depth range for a signal-to-noise ratio consistent with what is typical in single-molecule cellular imaging experiments.
View details for DOI 10.1016/j.optcom.2023.129589
View details for PubMedID 37396964
View details for PubMedCentralID PMC10310311
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Advanced Cryogenic Light Microscopy Stage to Enable 3D Super-resolved Cryogenic Correlative Light and Electron Microscopy.
Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
2023; 29 (Supplement_1): 1941
View details for DOI 10.1093/micmic/ozad067.1005
View details for PubMedID 37612900
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Structural and photophysical characterization of the small ultra-red fluorescent protein.
Nature communications
2023; 14 (1): 4155
Abstract
The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.
View details for DOI 10.1038/s41467-023-39776-9
View details for PubMedID 37438348
View details for PubMedCentralID 2763207
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Characterization of mApple as a Red Fluorescent Protein for Cryogenic Single-Molecule Imaging with Turn-Off and Turn-On Active Control Mechanisms.
The journal of physical chemistry. B
2023
Abstract
Single-molecule superresolution microscopy is a powerful tool for the study of biological structures on size scales smaller than the optical diffraction limit. Imaging samples at cryogenic temperatures (77 K) reduces the quantum yield of photobleaching for many fluorescent labels, yielding localization precisions below 10 nm. Cryogenic imaging further enables correlation with cryogenic electron tomography. A key limitation in applying methods such as PALM and STORM to samples maintained at 77 K is the limited number of fluorophores known to undergo efficient turn-on and turn-off mechanisms necessary to control the sparsity of active emitters. We find that mApple, a red-emitting fluorescent protein, undergoes a novel turn-off mechanism in response to simultaneous illumination with two colors of light. This turn-off mechanism enables localization of many individual molecules in initially bright samples, but the final density of localizable emitters is limited by relatively inefficient turn-on (photoactivation). Bulk excitation and emission spectroscopy shows that mApple has access to two distinct emissive states as well as dark states accessible optically or through changes in pH. The bright and stable emission of mApple enables widefield collection of single-molecule emission spectra, which highlight the complex nature and environmental sensitivity of states observed in red fluorescent proteins.
View details for DOI 10.1021/acs.jpcb.2c08995
View details for PubMedID 36943356
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Ratiometric sensing of redox environments inside individual carboxysomes trapped in solution
CELL PRESS. 2023: 304A
View details for Web of Science ID 000989629701600
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Ratiometric sensing of redox environments inside individual carboxysomes trapped in solution.
Biophysical journal
2023; 122 (3S1): 304a
View details for DOI 10.1016/j.bpj.2022.11.1712
View details for PubMedID 36783520
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Multicolor super-resolution imaging to study human coronavirus RNA during cellular infection.
Biophysical journal
2023; 122 (3S1): 16a
View details for DOI 10.1016/j.bpj.2022.11.313
View details for PubMedID 36782799
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Multicolor super-resolution imaging to study human coronavirus RNA during cellular infection
CELL PRESS. 2023: 16A
View details for Web of Science ID 000989629700076
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Exploring Masses and Internal Mass Distributions of Single Carboxysomes in Free Solution Using Fluorescence and Interferometric Scattering in an Anti-Brownian Trap.
The journal of physical chemistry. B
2022
Abstract
Carboxysomes are self-assembled bacterial microcompartments that facilitate carbon assimilation by colocalizing the enzymes of CO2 fixation within a protein shell. These microcompartments can be highly heterogeneous in their composition and filling, so measuring the mass and loading of an individual carboxysome would allow for better characterization of its assembly and function. To enable detailed and extended characterizations of single nanoparticles in solution, we recently demonstrated an improved interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which tracks the position of a single nanoparticle via its scattering of a near-infrared beam and applies feedback to counteract its Brownian motion. Importantly, the scattering signal can be related to the mass of nanoscale proteinaceous objects, whose refractive indices are well-characterized. We calibrate single-particle scattering cross-section measurements in the ISABEL trap and determine individual carboxysome masses in the 50-400 MDa range by analyzing their scattering cross sections with a core-shell model. We further investigate carboxysome loading by combining mass measurements with simultaneous fluorescence reporting from labeled internal components. This method may be extended to other biological objects, such as viruses or extracellular vesicles, and can be combined with orthogonal fluorescence reporters to achieve precise physical and chemical characterization of individual nanoscale biological objects.
View details for DOI 10.1021/acs.jpcb.2c05939
View details for PubMedID 36282790
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Metallic Support Films Reduce Optical Heating in Cryogenic Correlative Light and Electron Tomography.
Journal of structural biology
2022: 107901
Abstract
Super-resolved cryogenic correlative light and electron tomography is an emerging method that provides both the single-molecule sensitivity and specificity of fluorescence imaging, and the molecular scale resolution and detailed cellular context of tomography, all in vitrified cells preserved in their native hydrated state. Technical hurdles that limit these correlative experiments need to be overcome for the full potential of this approach to be realized. Chief among these is sample heating due to optical excitation which leads to devitrification, a phase transition from amorphous to crystalline ice. Here we show that much of this heating is due to the material properties of the support film of the electron microscopy grid, specifically the absorptivity and thermal conductivity. We demonstrate through experiment and simulation that the properties of the standard holey carbon electron microscopy grid lead to substantial heating under optical excitation. In order to avoid devitrification, optical excitation intensities must be kept orders of magnitude lower than the intensities commonly employed in room temperature super-resolution experiments. We further show that the use of metallic films, either holey gold grids, or custom made holey silver grids, alleviate much of this heating. For example, the holey silver grids permit 20* the optical intensities used on the standard holey carbon grids. Super-resolution correlative experiments conducted on holey silver grids under these increased optical excitation intensities have a corresponding increase in the rate of single-molecule fluorescence localizations. This results in an increased density of localizations and improved correlative imaging without deleterious effects from sample heating.
View details for DOI 10.1016/j.jsb.2022.107901
View details for PubMedID 36191745
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Identification and Demonstration of roGFP2 as an Environmental Sensor for Cryogenic Correlative Light and Electron Microscopy.
Journal of structural biology
2022: 107881
Abstract
Cryogenic correlative light and electron microscopy (cryo-CLEM) seeks to leverage orthogonal information present in two powerful imaging modalities. While recent advances in cryogenic electron microscopy (cryo-EM) allow for the visualization and identification of structures within cells at the nanometer scale, information regarding the cellular environment, such as pH, membrane potential, ionic strength etc. that influence the observed structures remains absent. Fluorescence microscopy can potentially be used to reveal this information when specific labels, known as fluorescent biosensors, are used, but there has been minimal use of such biosensors in cryo-CLEM to date. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. At room temperature, the ratio of roGFP2 emission brightness when excited at 425 nm or 488 nm is known to report on the local redox potential. When samples containing roGFP2 are rapidly cooled to 77K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. By expressing an inducible roGFP2-PopZ fusion we can visualize individual microdomains in the context of their redox environment.
View details for DOI 10.1016/j.jsb.2022.107881
View details for PubMedID 35811036
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Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution.
The journal of physical chemistry letters
2022: 4455-4462
Abstract
Diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individual particles and measure their physical and chemical properties. To overcome this, we previously developed the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which uses scattering to localize a particle and applies electrokinetic forces that counteract Brownian motion, thus enabling extended observation. Here we present an improved ISABEL trap that incorporates a near-infrared scatter illumination beam and rapidly interleaves 405 and 488 nm fluorescence excitation reporter beams. With the ISABEL trap, we monitored the internal redox environment of individual carboxysomes labeled with the ratiometric redox reporter roGFP2. Carboxysomes widely vary in scattering contrast (reporting on size) and redox-dependent ratiometric fluorescence. Furthermore, we used redox sensing to explore the chemical kinetics within intact carboxysomes, where bulk measurements may contain unwanted contributions from aggregates or interfering fluorescent proteins. Overall, we demonstrate the ISABEL trap's ability to sensitively monitor nanoscale biological objects, enabling new experiments on these systems.
View details for DOI 10.1021/acs.jpclett.2c00782
View details for PubMedID 35549289
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Fast and parallel nanoscale 3D tracking of heterogeneous mammalian chromatin dynamics.
Molecular biology of the cell
2022: mbcE21100514
Abstract
Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel 3D tracking of multiple chromosomal loci of choice over many thousands of frames on various time scales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking of both fast dynamics (50 Hz) and over time scales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent alpha, and a transition to more isotropic dynamics in 3D after such treatment. We think that in the future our methodology will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.
View details for DOI 10.1091/mbc.E21-10-0514
View details for PubMedID 35352962
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A bottom-up perspective on photodynamics and photoprotection in light-harvesting complexes using anti-Brownian trapping.
The Journal of chemical physics
2022; 156 (7): 070901
Abstract
Single-molecule fluorescence spectroscopy allows direct, real-time observation of dynamic photophysical changes in light harvesting complexes. The Anti-Brownian ELectrokinetic (ABEL) trap is one such single-molecule method with useful advantages. This approach is particularly well-suited to make detailed spectroscopic measurements of pigment-protein complexes in a solution phase because it enables extended-duration single-molecule observation by counteracting Brownian motion. This Perspective summarizes recent contributions by the authors and others that have utilized the unique capabilities of the ABEL trap to advance our understanding of phycobiliproteins and the phycobilisome complex, the primary light-harvesting apparatus of cyanobacteria. Monitoring the rich spectroscopic data from these measurements, which include brightness, fluorescence lifetime, polarization, and emission spectra, among other measurable parameters, has provided direct characterization of pigments and energy transfer pathways in the phycobilisome, spanning scales from single pigments and monomeric phycobiliproteins to higher order oligomers and protein-protein interactions of the phycobilisome complex. Importantly, new photophysical states and photodynamics were observed to modulate the flow of energy through the phycobilisome and suggest a previously unknown complexity in phycobilisome light harvesting and energy transport with a possible link to photoadaptive or photoprotective functions in cyanobacteria. Beyond deepening our collective understanding of natural light-harvesting systems, these and future discoveries may serve as inspiration for engineering improved artificial light-harvesting technologies.
View details for DOI 10.1063/5.0079042
View details for PubMedID 35183095
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Autobiography of W. E. (William Esco) Moerner.
The journal of physical chemistry. B
2022; 126 (6): 1159
View details for DOI 10.1021/acs.jpcb.2c00137
View details for PubMedID 35172583
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Characterizing the distribution of myosin H in the apical complex of conoid protruded and conoid retracted Toxoplasma gondii
CELL PRESS. 2022: 409A
View details for Web of Science ID 000759523002535
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Custom metallic electron microscopy grids reduce sample heating in super-resolved cryogenic correlative light and electron microscopy experiments
CELL PRESS. 2022: 128
View details for Web of Science ID 000759523000617
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roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy
CELL PRESS. 2022: 128
View details for Web of Science ID 000759523000618
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Simultaneous position and orientation measurements of single molecules using deep learning and PSF engineering approaches
CELL PRESS. 2022: 412A-413A
View details for Web of Science ID 000759523002552
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Redox sensing inside individual carboxysomes in the ISABEL trap
CELL PRESS. 2022: 104
View details for Web of Science ID 000759523000503
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Characterizing physical properties of single carboxysomes in the Interferometric Scattering Anti-Brownian ELectrokinetic trap
CELL PRESS. 2022: 431A
View details for Web of Science ID 000759523002641
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Exploring cell-surface nanopillar interactions with 3D superresolution microscopy
CELL PRESS. 2022: 278A
View details for Web of Science ID 000759523001617
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Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection.
bioRxiv : the preprint server for biology
2022
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Employing the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multicolor RNA-immunoFISH and visualized their localization patterns within the cell. The exquisite resolution of our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive framework that supports investigations of coronavirus fundamental biology and therapeutic effects.
View details for DOI 10.1101/2021.06.09.447760
View details for PubMedID 34127974
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ATP-responsive biomolecular condensates tune bacterial kinase signaling.
Science advances
2022; 8 (7): eabm6570
Abstract
Biomolecular condensates formed via liquid-liquid phase separation enable spatial and temporal organization of enzyme activity. Phase separation in many eukaryotic condensates has been shown to be responsive to intracellular adenosine triphosphate (ATP) levels, although the consequences of these mechanisms for enzymes sequestered within the condensates are unknown. Here, we show that ATP depletion promotes phase separation in bacterial condensates composed of intrinsically disordered proteins. Enhanced phase separation promotes the sequestration and activity of a client kinase enabling robust signaling and maintenance of viability under the stress posed by nutrient scarcity. We propose that a diverse repertoire of condensates can serve as control knobs to tune enzyme sequestration and reactivity in response to the metabolic state of bacterial cells.
View details for DOI 10.1126/sciadv.abm6570
View details for PubMedID 35171683
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Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection.
Cell reports methods
2022: 100170
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Employing the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multicolor RNA-immunoFISH and visualized their localization patterns within the cell. The 20nm resolution achieved by of our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive imaging framework that will enable future investigations of coronavirus fundamental biology and therapeutic effects.
View details for DOI 10.1016/j.crmeth.2022.100170
View details for PubMedID 35128513
View details for PubMedCentralID PMC8806145
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Genome-Wide CRISPR screens reveal specific ligands for glycan-binding immune checkpoint receptors
OXFORD UNIV PRESS INC. 2021: 1682-1683
View details for Web of Science ID 000754737200022
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A localized adaptor protein performs distinct functions at the Caulobacter cell poles.
Proceedings of the National Academy of Sciences of the United States of America
2021; 118 (13)
Abstract
Asymmetric cell division generates two daughter cells with distinct characteristics and fates. Positioning different regulatory and signaling proteins at the opposing ends of the predivisional cell produces molecularly distinct daughter cells. Here, we report a strategy deployed by the asymmetrically dividing bacterium Caulobacter crescentus where a regulatory protein is programmed to perform distinct functions at the opposing cell poles. We find that the CtrA proteolysis adaptor protein PopA assumes distinct oligomeric states at the two cell poles through asymmetrically distributed c-di-GMP: dimeric at the stalked pole and monomeric at the swarmer pole. Different polar organizing proteins at each cell pole recruit PopA where it interacts with and mediates the function of two molecular machines: the ClpXP degradation machinery at the stalked pole and the flagellar basal body at the swarmer pole. We discovered a binding partner of PopA at the swarmer cell pole that together with PopA regulates the length of the flagella filament. Our work demonstrates how a second messenger provides spatiotemporal cues to change the physical behavior of an effector protein, thereby facilitating asymmetry.
View details for DOI 10.1073/pnas.2024705118
View details for PubMedID 33753507
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A localized adaptor protein performs distinct functions at the Caulobacter cell poles
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2021; 118 (13)
View details for Web of Science ID 000637394200069
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Genome-wide CRISPR screens reveal a specific ligand for the glycan-binding immune checkpoint receptor Siglec-7.
Proceedings of the National Academy of Sciences of the United States of America
2021; 118 (5)
Abstract
Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan-receptor interactions in living cells.
View details for DOI 10.1073/pnas.2015024118
View details for PubMedID 33495350
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Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale.
Annual review of physical chemistry
2021
Abstract
We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 72 is April 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
View details for DOI 10.1146/annurev-physchem-090319-051546
View details for PubMedID 33441030
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Exploring Cell Surface-Nanopillar Interactions with 3D Super-Resolution Microscopy.
ACS nano
2021
Abstract
Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used three-dimensional (3D) nanostructures such as nanopillars to imprint well-defined membrane curvatures (the "nano-bio interface"). In these studies, proteins and their interactions were probed by two-dimensional fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone-oil immersion objective and 3D double-helix point spread function microscopy. We carefully adjust the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R (the reciprocal of the radius) membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.
View details for DOI 10.1021/acsnano.1c05313
View details for PubMedID 34582687
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Viewpoint: Single Molecules at 31: What's Next?
Nano letters
2020
View details for DOI 10.1021/acs.nanolett.0c04042
View details for PubMedID 33170016
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Super-resolution Microscopy with Single Molecules in Biology and Beyond-Essentials, Current Trends, and Future Challenges.
Journal of the American Chemical Society
2020
Abstract
Single-molecule super-resolution microscopy has developed from a specialized technique into one of the most versatile and powerful imaging methods of the nanoscale over the past two decades. In this perspective, we provide a brief overview of the historical development of the field, the fundamental concepts, the methodology required to obtain maximum quantitative information, and the current state of the art. Then, we will discuss emerging perspectives and areas where innovation and further improvement are needed. Despite the tremendous progress, the full potential of single-molecule super-resolution microscopy is yet to be realized, which will be enabled by the research ahead of us.
View details for DOI 10.1021/jacs.0c08178
View details for PubMedID 33034452
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Addressing systematic errors in axial distance measurements in single-emitter localization microscopy
OPTICS EXPRESS
2020; 28 (13): 18616–32
View details for DOI 10.1364/OE.391496
View details for Web of Science ID 000545130800006
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Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter.
Proceedings of the National Academy of Sciences of the United States of America
2020
Abstract
Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of 30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.
View details for DOI 10.1073/pnas.2001849117
View details for PubMedID 32513734
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Deep learning in single-molecule microscopy: fundamentals, caveats, and recent developments [Invited].
Biomedical optics express
2020; 11 (3): 1633–61
Abstract
Deep learning-based data analysis methods have gained considerable attention in all fields of science over the last decade. In recent years, this trend has reached the single-molecule community. In this review, we will survey significant contributions of the application of deep learning in single-molecule imaging experiments. Additionally, we will describe the historical events that led to the development of modern deep learning methods, summarize the fundamental concepts of deep learning, and highlight the importance of proper data composition for accurate, unbiased results.
View details for DOI 10.1364/BOE.386361
View details for PubMedID 32206433
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Cryogenic Superresolution Fluorescence Correlated with Cryogenic Electron Tomography: Combining Specific Labeling and High Resolution
CELL PRESS. 2020: 20A–21A
View details for Web of Science ID 000513023200098
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Continuous, Topologically Guided Protein Crystallization Drives Self-Assembly of a Bacterial Surface Layer
CELL PRESS. 2020: 201A–202A
View details for Web of Science ID 000513023201258
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Robust Modulation of a Bacterial Kinase by Protein Phase Separation
CELL PRESS. 2020: 203A
View details for Web of Science ID 000513023201267
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Selective sequestration of signalling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus.
Nature microbiology
2020
Abstract
Selective recruitment and concentration of signalling proteins within membraneless compartments is a ubiquitous mechanism for subcellular organization1-3. The dynamic flow of molecules into and out of these compartments occurs on faster timescales than for membrane-enclosed organelles, presenting a possible mechanism to control spatial patterning within cells. Here, we combine single-molecule tracking and super-resolution microscopy, light-induced subcellular localization, reaction-diffusion modelling and a spatially resolved promoter activation assay to study signal exchange in and out of the 200nm cytoplasmic pole-organizing protein popZ (PopZ) microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus4-8. Two phospho-signalling proteins, the transmembrane histidine kinase CckA and the cytoplasmic phosphotransferase ChpT, provide the only phosphate source for the cell fate-determining transcription factor CtrA9-18. We find that all three proteins exhibit restricted rates of entry into and escape from the microdomain as well as enhanced phospho-signalling within, leading to a submicron gradient of activated CtrA-P19 that is stable and sublinear. Entry into the microdomain is selective for cytosolic proteins and requires a binding pathway to PopZ. Our work demonstrates how nanoscale protein assemblies can modulate signal propagation with fine spatial resolution, and that in Caulobacter, this modulation serves to reinforce asymmetry and differential cell fate of the two daughter cells.
View details for DOI 10.1038/s41564-019-0647-7
View details for PubMedID 31959967
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Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule super-resolution microscopy.
Molecular biology of the cell
2020: mbcE19090499
Abstract
Primary cilia in many cell types contain a peri-axonemal sub-compartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development including left-right asymmetry and renal tissue homeostasis. Here we combine super-resolution imaging of human RPE1 cells, a classic model for studying primary cilia in vitro, with a genetic dissection of the protein-protein binding relationships that organize compartment assembly to develop a new structural model. We observe that INVS is the core structural determinant of a compartment composed of novel fibril-like substructures, which we identify here by 3D single-molecule super-resolution imaging. We find that NEK8 and ANKS6 depend on INVS for localization to these fibrillar assemblies and that ANKS6-NEK8 density within the compartment is regulated by NEK8. Together, NEK8 and ANKS6 are required downstream of INVS to localize and concentrate NPHP3 within the compartment. In the absence of these upstream components, NPHP3 is redistributed within cilia. These results provide a more detailed structure for the inversin compartment and introduce a new example of a membraneless compartment organized by protein-protein interactions. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
View details for DOI 10.1091/mbc.E19-09-0499
View details for PubMedID 31895004
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T-Plastin reinforces membrane protrusions to bridge matrix gaps during cell migration.
Nature communications
2020; 11 (1): 4818
Abstract
Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.
View details for DOI 10.1038/s41467-020-18586-3
View details for PubMedID 32968060
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Interferometric scattering for fluorescence-free electrokinetic trapping of single nanoparticles in free solution
SPIE-INT SOC OPTICAL ENGINEERING. 2020
View details for DOI 10.1117/12.2546638
View details for Web of Science ID 000546225400012
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Cryogenic Correlative Single-Particle Photoluminescence Spectroscopy and Electron Tomography for Investigation of Nanomaterials.
Angewandte Chemie (International ed. in English)
2020
Abstract
Cryogenic single-particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complimentary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single-particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in-plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases the onset of the splitting corresponded to changes in the in-plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.
View details for DOI 10.1002/anie.202002856
View details for PubMedID 32330371
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Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation.
Proceedings of the National Academy of Sciences of the United States of America
2020
Abstract
Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
View details for DOI 10.1073/pnas.2007297117
View details for PubMedID 32989128
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Cryogenic single-molecule active control microscopy with a photoactivatable fluorescent protein
SPIE-INT SOC OPTICAL ENGINEERING. 2020
View details for DOI 10.1117/12.2546333
View details for Web of Science ID 000546225400006
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Opposing Effects of Cohesin and Transcription on CTCF Organization Revealed by Super-resolution Imaging.
Molecular cell
2020
Abstract
CCCTC-binding factor (CTCF) and cohesin play critical roles in organizing mammalian genomes into topologically associating domains (TADs). Here, by combining genetic engineering with quantitative super-resolution stimulated emission depletion (STED) microscopy, we demonstrate that in living cells, CTCF forms clusters typically containing 2-8 molecules. A fraction of CTCF clusters, enriched for those with ≥3 molecules, are coupled with cohesin complexes with a characteristic physical distance suggestive of a defined molecular interaction. Acute degradation of the cohesin unloader WAPL or transcriptional inhibition (TI) result in increased CTCF clustering. Furthermore, the effect of TI on CTCF clusters is alleviated by the acute loss of the cohesin subunit SMC3. Our study provides quantitative characterization of CTCF clusters in living cells, uncovers the opposing effects of cohesin and transcription on CTCF clustering, and highlights the power of quantitative super-resolution microscopy as a tool to bridge the gap between biochemical and genomic methodologies in chromatin research.
View details for DOI 10.1016/j.molcel.2020.10.001
View details for PubMedID 33091336
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Accurate phase retrieval of complex 3D point spread functions with deep residual neural networks.
Applied physics letters
2019; 115 (25): 251106
Abstract
Phase retrieval, i.e., the reconstruction of phase information from intensity information, is a central problem in many optical systems. Imaging the emission from a point source such as a single molecule is one example. Here, we demonstrate that a deep residual neural net is able to quickly and accurately extract the hidden phase for general point spread functions (PSFs) formed by Zernike-type phase modulations. Five slices of the 3D PSF at different focal positions within a two micrometer range around the focus are sufficient to retrieve the first six orders of Zernike coefficients.
View details for DOI 10.1063/1.5125252
View details for PubMedID 32127719
View details for PubMedCentralID PMC7043838
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Accurate phase retrieval of complex 3D point spread functions with deep residual neural networks
APPLIED PHYSICS LETTERS
2019; 115 (25)
View details for DOI 10.1063/1.5125252
View details for Web of Science ID 000505535900036
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Quantitative super-resolution microscopy of the mammalian glycocalyx
AMER CHEMICAL SOC. 2019
View details for Web of Science ID 000525055501242
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Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation.
Proceedings of the National Academy of Sciences of the United States of America
2019
Abstract
The cell cycle-regulated methylation state of Caulobacter DNA mediates the temporal control of transcriptional activation of several key regulatory proteins. Temporally controlled synthesis of the CcrM DNA methyltransferase and Lon-mediated proteolysis restrict CcrM to a specific time in the cell cycle, thereby allowing the maintenance of the hemimethylated state of the chromosome during the progression of DNA replication. We determined that a chromosomal DNA-based platform stimulates CcrM degradation by Lon and that the CcrM C terminus both binds to its DNA substrate and is recognized by the Lon protease. Upon asymmetric cell division, swarmer and stalked progeny cells employ distinct mechanisms to control active CcrM. In progeny swarmer cells, CcrM is completely degraded by Lon before its differentiation into a replication-competent stalked cell later in the cell cycle. In progeny stalked cells, however, accumulated CcrM that has not been degraded before the immediate initiation of DNA replication is sequestered to the cell pole. Single-molecule imaging demonstrated physical anticorrelation between sequestered CcrM and chromosomal DNA, thus preventing DNA remethylation. The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression.
View details for DOI 10.1073/pnas.1906119116
View details for PubMedID 31315982
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Physical Principles of Membrane Shape Regulation by the Glycocalyx
CELL
2019; 177 (7): 1757-+
View details for DOI 10.1016/j.cell.2019.04.017
View details for Web of Science ID 000471256800016
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Interferometric Scattering Enables Fluorescence-Free Electrokinetic Trapping of Single Nanoparticles in Free Solution.
Nano letters
2019
Abstract
Anti-Brownian traps confine single particles in free solution by closed-loop feedback forces that directly counteract Brownian motion. Extended-duration measurements on trapped objects allow detailed characterization of photophysical and transport properties as well as observation of infrequent or rare dynamics. However, this approach has been generally limited to particles that can be tracked by fluorescence emission. Here we present the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which uses interferometric scattering rather than fluorescence to monitor particle position. By decoupling the ability to track (and therefore trap) a particle from collection of its spectroscopic data, the ISABEL trap enables confinement and extended study of single particles that do not fluoresce, only weakly fluoresce, or exhibit intermittent fluorescence or photobleaching. This new technique significantly expands the range of nanoscale objects that may be investigated at the single-particle level in free solution.
View details for DOI 10.1021/acs.nanolett.9b01514
View details for PubMedID 31117762
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Physical Principles of Membrane Shape Regulation by the Glycocalyx.
Cell
2019
Abstract
Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.
View details for PubMedID 31056282
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Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2019; 116 (12): 5550-5557
View details for DOI 10.1073/pnas.1816747116
View details for Web of Science ID 000461679000056
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Single-molecule trapping and spectroscopy reveals photophysical heterogeneity of phycobilisomes quenched by Orange Carotenoid Protein.
Nature communications
2019; 10 (1): 1172
Abstract
The Orange Carotenoid Protein (OCP) is a cytosolic photosensor that is responsible for non-photochemical quenching (NPQ) of the light-harvesting process in most cyanobacteria. Upon photoactivation by blue-green light, OCP binds to the phycobilisome antenna complex, providing an excitonic trap to thermally dissipate excess energy. At present, both the binding site and NPQ mechanism of OCP are unknown. Using an Anti-Brownian ELectrokinetic (ABEL) trap, we isolate single phycobilisomes in free solution, both in the presence and absence of activated OCP, to directly determine the photophysics and heterogeneity of OCP-quenched phycobilisomes. Surprisingly, we observe two distinct OCP-quenched states, with lifetimes 0.09ns (6% of unquenched brightness) and 0.21ns (11% brightness). Photon-by-photon Monte Carlo simulations of exciton transfer through the phycobilisome suggest that the observed quenched states are kinetically consistent with either two or one bound OCPs, respectively, underscoring an additional mechanism for excitation control in this key photosynthetic unit.
View details for PubMedID 30862823
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Single-molecule trapping and spectroscopy reveals photophysical heterogeneity of phycobilisomes quenched by Orange Carotenoid Protein
NATURE COMMUNICATIONS
2019; 10
View details for DOI 10.1038/s41467-019-09084-2
View details for Web of Science ID 000460930600004
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Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol.
Proceedings of the National Academy of Sciences of the United States of America
2019
Abstract
The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.
View details for PubMedID 30819883
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A Bacterial Biomolecular Condensate Sequesters a Signaling Pathway that Drives Spatial Regulation of Gene Expression and Asymmetric Cell Division
CELL PRESS. 2019: 453A
View details for DOI 10.1016/j.bpj.2018.11.2446
View details for Web of Science ID 000460779802279
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Providing 3D for Super-Resolution Microscopy and Single-Particle Tracking in Cells with Single Molecules
CELL PRESS. 2019: 331A
View details for DOI 10.1016/j.bpj.2018.11.1803
View details for Web of Science ID 000460779801652
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Multi-Step 2D Protein Crystallization via Structural Changes within an Ordered Lattice
CELL PRESS. 2019: 194A
View details for DOI 10.1016/j.bpj.2018.11.1077
View details for Web of Science ID 000460779800965
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Protein Self-Assembly Drives Surface Layer Biogenesis and Maintenance in C. crescentus
CELL PRESS. 2019: 159A
View details for DOI 10.1016/j.bpj.2018.11.881
View details for Web of Science ID 000460779800789
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Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy
BIOPHYSICAL JOURNAL
2019; 116 (2): 319-329
View details for DOI 10.1016/j.bpj.2018.11.3136
View details for Web of Science ID 000456327100014
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Viewpoint: Localization Microscopy of Single Molecules Enhanced by 3D Imaging and Light Sheet Illumination.
Journal of physics D: Applied physics
2019; 52 (1)
Abstract
Single-molecule super-resolution fluorescence microscopy has produced a revolution in the optical resolution available for study of the nanoscale details of cells and materials. The early work on two-dimensional imaging yielded images of samples that spanned only a small axial range on the order of ~500 nm. Much more useful information can be obtained in complex extended samples using three-dimensional (3D) single-molecule localization. Without 3D capability, visualization of structures extending in the axial direction can easily be missed or confused and the understanding of the science compromised. A variety of methods for obtaining 3D super-resolution images have been devised, each with their own strengths and weaknesses, ranging from point-spread-function engineering to steep axial illumination gradients. Imaging thicker samples, such as mammalian cells and tissue, in all three dimensions presents additional challenges due to increased background and large volumes to image. Light sheet illumination is a method that allows for selective irradiation of the image plane, and its inherent optical sectioning capability allows for imaging of biological samples with reduced background, photobleaching, and photodamage. Beyond these developments, there is a continuing need for better controllable fluorophores, additional novel optical system designs, and enhanced mathematical and statistical signal processing ideas to accurately extract the maximum information available from single emitters in the shortest possible time.
View details for DOI 10.1088/1361-6463/aae632
View details for PubMedID 31160828
View details for PubMedCentralID PMC6544387
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NANOSCALE ELUCIDATION OF THE INVASION APPARATUS OF APICOMPLEXAN PARASITES
AMER SOC TROP MED & HYGIENE. 2019: 620
View details for Web of Science ID 000507364505127
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Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx
Developmental Cell
2019; 50 (1): 57-72
View details for DOI 10.1016/j.devcel.2019.04.035
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Accurate and rapid background estimation in single-molecule localization microscopy using the deep neural network BGnet.
Proceedings of the National Academy of Sciences of the United States of America
2019
Abstract
Background fluorescence, especially when it exhibits undesired spatial features, is a primary factor for reduced image quality in optical microscopy. Structured background is particularly detrimental when analyzing single-molecule images for 3-dimensional localization microscopy or single-molecule tracking. Here, we introduce BGnet, a deep neural network with a U-net-type architecture, as a general method to rapidly estimate the background underlying the image of a point source with excellent accuracy, even when point-spread function (PSF) engineering is in use to create complex PSF shapes. We trained BGnet to extract the background from images of various PSFs and show that the identification is accurate for a wide range of different interfering background structures constructed from many spatial frequencies. Furthermore, we demonstrate that the obtained background-corrected PSF images, for both simulated and experimental data, lead to a substantial improvement in localization precision. Finally, we verify that structured background estimation with BGnet results in higher quality of superresolution reconstructions of biological structures.
View details for DOI 10.1073/pnas.1916219117
View details for PubMedID 31871202
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Topologically-guided continuous protein crystallization controls bacterial surface layer self-assembly.
Nature communications
2019; 10 (1): 2731
Abstract
Many bacteria and most archaea possess a crystalline protein surface layer (S-layer), which surrounds their growing and topologically complicated outer surface. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. By labeling, superresolution imaging, and tracking the S-layer protein (SLP) from C. crescentus, we show that 2D protein self-assembly is sufficient to build and maintain the S-layer in living cells by efficient protein crystal nucleation and growth. We propose a model supported by single-molecule tracking whereby randomly secreted SLP monomers diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated at the edges of growing 2D S-layer crystals. Surface topology creates crystal defects and boundaries, thereby guiding S-layer assembly. Unsupervised assembly poses challenges for therapeutics targeting S-layers. However, protein crystallization as an evolutionary driver rationalizes S-layer diversity and raises the potential for biologically inspired self-assembling macromolecular nanomaterials.
View details for DOI 10.1038/s41467-019-10650-x
View details for PubMedID 31227690
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Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx.
Developmental cell
2019
Abstract
The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.
View details for DOI 10.1016/j.devcel.2019.04.035
View details for PubMedID 31105009
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Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy.
Biophysical journal
2018
Abstract
Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of 250nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (2-6 mum long and 200-300nm in diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened, with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, two-color, two-dimensional stimulated emission depletion microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the Smoothened-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses stimulated emission depletion to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.
View details for PubMedID 30598282
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Identification of PAmKate as a Red Photoactivatable Fluorescent Protein for Cryogenic Super-Resolution Imaging.
Journal of the American Chemical Society
2018; 140 (39): 12310–13
Abstract
Single-molecule super-resolution fluorescence microscopy conducted in vitrified samples at cryogenic temperatures offers enhanced localization precision due to reduced photobleaching rates, a chemical-free and rapid fixation method, and the potential of correlation with cryogenic electron microscopy. Achieving cryogenic super-resolution microscopy requires the ability to control the sparsity of emissive labels at cryogenic temperatures. Obtaining this control presents a key challenge for the development of this technique. In this work, we identify a red photoactivatable protein, PAmKate, which remains activatable at cryogenic temperatures. We characterize its activation as a function of temperature and find that activation is efficient at cryogenic and room temperatures. We perform cryogenic super-resolution experiments in situ, labeling PopZ, a protein known to assemble into a microdomain at the poles of the model bacterium Caulobacter crescentus. We find improved localization precision at cryogenic temperatures compared to room temperature by a factor of 4, attributable to reduced photobleaching.
View details for PubMedID 30222332
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Identification of PAmKate as a Red Photoactivatable Fluorescent Protein for Cryogenic Super-Resolution Imaging
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2018; 140 (39): 12310-12313
View details for DOI 10.1021/jacs.8b05960
View details for Web of Science ID 000446920100001
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Quantitative super-resolution microscopy reveals the architecture of the mammalian glycocalyx and its changes during cancer progression
AMER CHEMICAL SOC. 2018
View details for Web of Science ID 000447600001117
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Resolving Mixtures in Solution by Single-Molecule Rotational Diffusivity
NANO LETTERS
2018; 18 (8): 5279-5287
View details for DOI 10.1021/acs.nanolett.8b02280
View details for Web of Science ID 000441478300091
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Resolving Mixtures in Solution by Single-Molecule Rotational Diffusivity.
Nano letters
2018
Abstract
Sensing the size of individual molecules in an ensemble has proven to be a powerful tool to investigate biomolecular interactions and association-dissociation processes. In biologically relevant solution environments, molecular size is often sensed by translational or rotational diffusivity. The rotational diffusivity is more sensitive to the size and conformation of the molecules as it is inversely proportional to the cube of the hydrodynamic radius, as opposed to the inverse linear dependence of the translational diffusion coefficient. Single-molecule rotational diffusivity has been measured with time-resolved fluorescence anisotropy decay, but the ability to sense different sizes has been restricted by the limited number of photons available or has required surface attachment to observe each molecule longer, and the attachment may be perturbative. To address these limitations, we show how to measure and monitor single-molecule rotational diffusivity by combining the solution-phase Anti-Brownian ELectrokinetic (ABEL) trap and maximum likelihood analysis of time-resolved fluorescence anisotropy based on the information inherent in each detected photon. We demonstrate this approach by resolving a mixture of single- and double-stranded fluorescently labeled DNA molecules at equilibrium, freely rotating in a native solution environment. The rotational diffusivity, fluorescence brightness and lifetime, and initial and steady-state anisotropy are simultaneously determined for each trapped single DNA molecule. The time resolution and precision of this method are analyzed using statistical signal analysis and simulations. We present key parameters that define the usefulness of a particular fluorescent label for extracting molecular size information from single-molecule rotational diffusivity measurements.
View details for PubMedID 30001492
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Light sheet approaches for improved precision in 3D localization-based super-resolution imaging in mammalian cells [Invited]
OPTICS EXPRESS
2018; 26 (10): 13122–47
Abstract
The development of imaging techniques beyond the diffraction limit has paved the way for detailed studies of nanostructures and molecular mechanisms in biological systems. Imaging thicker samples, such as mammalian cells and tissue, in all three dimensions, is challenging due to increased background and volumes to image. Light sheet illumination is a method that allows for selective irradiation of the image plane, and its inherent optical sectioning capability allows for imaging of biological samples with reduced background, photobleaching, and photodamage. In this review, we discuss the advantage of combining single-molecule imaging with light sheet illumination. We begin by describing the principles of single-molecule localization microscopy and of light sheet illumination. Finally, we present examples of designs that successfully have married single-molecule super-resolution imaging with light sheet illumination for improved precision in mammalian cells.
View details for DOI 10.1364/OE.26.013122
View details for Web of Science ID 000432457600088
View details for PubMedID 29801343
View details for PubMedCentralID PMC6005674
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Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2018; 115 (16): E3712–E3721
Abstract
We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in Escherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E's function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.
View details for PubMedID 29610352
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Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase
JOURNAL OF CHEMICAL PHYSICS
2018; 148 (12): 123319
Abstract
Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 μM range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ∼0.1 s-1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.
View details for PubMedID 29604852
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In Situ Imaging of Spatial Organization of Accessible Chromatin at the Nanoscale with ATAC-see and Single-Molecule Super-Resolution Fluorescence Microscopy
CELL PRESS. 2018: 539A
View details for Web of Science ID 000430563200448
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Precise Measurement of Single -Molecule Rotational Diffusivity in Solution
CELL PRESS. 2018: 170A
View details for Web of Science ID 000430439600102
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Cryogenic Dissection of the Phycobilisome's Electronic Structure
CELL PRESS. 2018: 169A
View details for Web of Science ID 000430439600094
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Single-Molecule Measurements of Quenching and Photophysical Heterogeneity in Phycobiliproteins
CELL PRESS. 2018: 522A–523A
View details for Web of Science ID 000430563200363
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Probing Asymmetric Behavior of a Cell Cycle Regupatory Protein in Live Caulobacter using Single-Molecule Imaging
CELL PRESS. 2018: 350A
View details for Web of Science ID 000430450000249
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Quantitative Super-Resolution Imaging Reveals Mammalian Glycocalyx Dynamics
CELL PRESS. 2018: 537A–538A
View details for Web of Science ID 000430563200441
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Quantifying Nanoscale Morphological Features of the Primary Cilium Membrane using Super-Resolution Fluorescence Microscopy
CELL PRESS. 2018: 268A
View details for Web of Science ID 000430439600595
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A Red Fluorescent Protein for Cryogenic Single-Molecule Superresolution Imaging
CELL PRESS. 2018: 529A–530A
View details for Web of Science ID 000430563200402
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Two-Color Sted Microscopy to Visualize S-Layer Biogenesis in Caulobacter Crescentus
CELL PRESS. 2018: 613A
View details for Web of Science ID 000430563300065
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Spatial Organization and Dynamics of RNA Processing in Caulobacter Crescentus
CELL PRESS. 2018: 251A
View details for Web of Science ID 000430439600509
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A Polar Matrix Microdomain Constrains Diffusion and Regulates Intracellular Signaling
CELL PRESS. 2018: 548A
View details for Web of Science ID 000430563200493
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Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.
Proceedings of SPIE--the International Society for Optical Engineering
2018; 10500
Abstract
To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.
View details for PubMedID 29681676
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3D single-molecule super-resolution microscopy with a tilted light sheet
NATURE COMMUNICATIONS
2018; 9: 123
Abstract
Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.
View details for PubMedID 29317629
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The story of single molecules, from hole-burning and early FM spectroscopy in solids, to super-resolution nanoscopy in cells and beyond
E D P SCIENCES. 2018
View details for DOI 10.1051/epjconf/201819001001
View details for Web of Science ID 000463775100001
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Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells
SPIE-INT SOC OPTICAL ENGINEERING. 2018
View details for DOI 10.1117/12.2288443
View details for Web of Science ID 000456502700008
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Single-Molecule Imaging of Wnt3A Protein Diffusion on Living Cell Membranes
BIOPHYSICAL JOURNAL
2017; 113 (12): 2762–67
Abstract
Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labeled Wnt3A transiently and dynamically associates with the membranes of Drosophila Schneider 2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2 receptor-plus (S2R+) cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes, and represent, to our knowledge, a new system for investigating the dynamics of Wnt transport.
View details for PubMedID 29262368
View details for PubMedCentralID PMC5925569
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Observation of live chromatin dynamics in cells via 3D localization microscopy using Tetrapod point spread functions
BIOMEDICAL OPTICS EXPRESS
2017; 8 (12): 5735–48
Abstract
We report the observation of chromatin dynamics in living budding yeast (Saccharomyces cerevisiae) cells, in three-dimensions (3D). Using dual color localization microscopy and employing a Tetrapod point spread function, we analyze the spatio-temporal dynamics of two fluorescently labeled DNA loci surrounding the GAL locus. From the measured trajectories, we obtain different dynamical characteristics in terms of inter-loci distance and temporal variance; when the GAL locus is activated, the 3D inter-loci distance and temporal variance increase compared to the inactive state. These changes are visible in spite of the large thermally- and biologically-driven heterogeneity in the relative motion of the two loci. Our observations are consistent with current euchromatin vs. heterochromatin models.
View details for PubMedID 29296501
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Measurement-based estimation of global pupil functions in 3D localization microscopy
OPTICS EXPRESS
2017; 25 (7): 7945-7959
Abstract
We report the use of a phase retrieval procedure based on maximum likelihood estimation (MLE) to produce an improved, experimentally calibrated model of a point spread function (PSF) for use in three-dimensional (3D) localization microscopy experiments. The method estimates a global pupil phase function (which includes both the PSF and system aberrations) over the full axial range from a simple calibration scan. The pupil function is used to refine the PSF model and hence enable superior localizations from experimental data. To demonstrate the utility of the procedure, we apply it to experimental data acquired with a microscope employing a tetrapod PSF with a 6 µm axial range. The phase-retrieved model demonstrates significant improvements in both accuracy and precision of 3D localizations relative to the model based on scalar diffraction theory. The localization precision of the phase-retrieved model is shown to be near the limits imposed by estimation theory, and the reproducibility of the procedure is characterized and discussed. Code which performs the phase retrieval algorithm is provided.
View details for DOI 10.1364/OE.25.007945
View details for Web of Science ID 000398536000071
View details for PubMedID 28380911
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Ultra-photostable, genetically directed fluoromodule enables STED nanoscopy and long time scale single protein tracks in live bacteria
AMER CHEMICAL SOC. 2017
View details for Web of Science ID 000430568500763
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Story of single-molecule detection and spectroscopy and the surprises leading to super-resolution microscopy and beyond
AMER CHEMICAL SOC. 2017
View details for Web of Science ID 000430569107694
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Direct Single-Molecule Measurements of Phycocyanobilin Photophysics in Monomeric C-Phycocyanin
CELL PRESS. 2017: 471A
View details for DOI 10.1016/j.bpj.2016.11.2527
View details for Web of Science ID 000402375700326
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Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking.
Chemical reviews
2017
Abstract
Single-molecule super-resolution fluorescence microscopy and single-particle tracking are two imaging modalities that illuminate the properties of cells and materials on spatial scales down to tens of nanometers or with dynamical information about nanoscale particle motion in the millisecond range, respectively. These methods generally use wide-field microscopes and two-dimensional camera detectors to localize molecules to much higher precision than the diffraction limit. Given the limited total photons available from each single-molecule label, both modalities require careful mathematical analysis and image processing. Much more information can be obtained about the system under study by extending to three-dimensional (3D) single-molecule localization: without this capability, visualization of structures or motions extending in the axial direction can easily be missed or confused, compromising scientific understanding. A variety of methods for obtaining both 3D super-resolution images and 3D tracking information have been devised, each with their own strengths and weaknesses. These include imaging of multiple focal planes, point-spread-function engineering, and interferometric detection. These methods may be compared based on their ability to provide accurate and precise position information on single-molecule emitters with limited photons. To successfully apply and further develop these methods, it is essential to consider many practical concerns, including the effects of optical aberrations, field dependence in the imaging system, fluorophore labeling density, and registration between different color channels. Selected examples of 3D super-resolution imaging and tracking are described for illustration from a variety of biological contexts and with a variety of methods, demonstrating the power of 3D localization for understanding complex systems.
View details for DOI 10.1021/acs.chemrev.6b00629
View details for PubMedID 28151646
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Experimental Demonstration of Sparsity-Based Single-Shot Fluorescence Imaging at Sub-wavelength Resolution
IEEE. 2017
View details for Web of Science ID 000427296200163
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Direct single-molecule measurements of phycocyanobilin photophysics in monomeric C-phycocyanin.
Proceedings of the National Academy of Sciences of the United States of America
2017; 114 (37): 9779–84
Abstract
Phycobilisomes are highly organized pigment-protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment-protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from Spirulina platensis in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higher-order complexes.
View details for PubMedID 28847963
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Super-Resolution Microscopy and Single-Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule.
Current protocols in cell biology
2017; 75: 4.32.1–4.32.22
Abstract
Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically fuses the protein of interest to a photostable fluorescent tag. Several labeling schemes are available to accomplish this. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types. However, their use in bacteria has been limited due to challenges imposed by a complex bacterial cell wall. Here, we describe the use of a genetically encoded photostable fluoromodule that can be targeted to cytosolic and membrane proteins in the Gram negative bacterium Caulobacter crescentus. Additionally, we summarize methods to use this fluoromodule for single protein imaging and super-resolution microscopy using stimulated emission depletion. © 2017 by John Wiley & Sons, Inc.
View details for PubMedID 28627757
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Multicolour localization microscopy by point-spread-function engineering
NATURE PHOTONICS
2016; 10 (9): 590-594
Abstract
Super-resolution microscopy has revolutionized cellular imaging in recent years1-4. Methods relying on sequential localization of single point emitters enable spatial tracking at ~10-40 nm resolution. Moreover, tracking and imaging in three dimensions is made possible by various techniques, including point-spread-function (PSF) engineering5-9 -namely, encoding the axial (z) position of a point source in the shape that it creates in the image plane. However, a remaining challenge for localization-microscopy is efficient multicolour imaging - a task of the utmost importance for contextualizing biological data. Normally, multicolour imaging requires sequential imaging10, 11, multiple cameras12, or segmented dedicated fields of view13, 14. Here, we demonstrate an alternate strategy, the encoding of spectral information (colour), in addition to 3D position, directly in the image. By exploiting chromatic dispersion, we design a new class of optical phase masks that simultaneously yield controllably different PSFs for different wavelengths, enabling simultaneous multicolour tracking or super-resolution imaging in a single optical path.
View details for Web of Science ID 000382800500012
View details for PubMedCentralID PMC5391844
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Super-resolution Imaging of Live Bacteria Cells Using a Genetically Directed, Highly Photostable Fluoromodule.
Journal of the American Chemical Society
2016; 138 (33): 10398-10401
Abstract
The rapid development in fluorescence microscopy and imaging techniques has greatly benefited our understanding of the mechanisms governing cellular processes at the molecular level. In particular, super-resolution microscopy methods overcome the diffraction limit to observe nanoscale cellular structures with unprecedented detail, and single-molecule tracking provides precise dynamic information about the motions of labeled proteins and oligonucleotides. Enhanced photostability of fluorescent labels (i.e., maximum emitted photons before photobleaching) is a critical requirement for achieving the ultimate spatio-temporal resolution with either method. While super-resolution imaging has greatly benefited from highly photostable fluorophores, a shortage of photostable fluorescent labels for bacteria has limited its use in these small but relevant organisms. In this study, we report the use of a highly photostable fluoromodule, dL5, to genetically label proteins in the Gram-negative bacterium Caulobacter crescentus, enabling long-time-scale protein tracking and super-resolution microscopy. dL5 imaging relies on the activation of the fluorogen Malachite Green (MG) and can be used to label proteins sparsely, enabling single-protein detection in live bacteria without initial bleaching steps. dL5-MG complexes emit 2-fold more photons before photobleaching compared to organic dyes such as Cy5 and Alexa 647 in vitro, and 5-fold more photons compared to eYFP in vivo. We imaged fusions of dL5 to three different proteins in live Caulobacter cells using stimulated emission depletion microscopy, yielding a 4-fold resolution enhancement compared to diffraction-limited imaging. Importantly, dL5 fusions to an intermediate filament protein CreS are significantly less perturbative compared to traditional fluorescent protein fusions. To the best of our knowledge, this is the first demonstration of the use of fluorogen activating proteins for super-resolution imaging in live bacterial cells.
View details for DOI 10.1021/jacs.6b05943
View details for PubMedID 27479076
View details for PubMedCentralID PMC4996739
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Story of single molecules and the surprises leading to super-resolution microscopy and beyond
AMER CHEMICAL SOC. 2016
View details for Web of Science ID 000431460201022
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Removal of single-molecule localization bias using a metasurface polarization filter
AMER CHEMICAL SOC. 2016
View details for Web of Science ID 000431460403029
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Multicolour localization microscopy by point-spread-function engineering.
Nature photonics
2016; 10: 590-594
Abstract
Super-resolution microscopy has revolutionized cellular imaging in recent years1-4. Methods relying on sequential localization of single point emitters enable spatial tracking at ~10-40 nm resolution. Moreover, tracking and imaging in three dimensions is made possible by various techniques, including point-spread-function (PSF) engineering5-9 -namely, encoding the axial (z) position of a point source in the shape that it creates in the image plane. However, a remaining challenge for localization-microscopy is efficient multicolour imaging - a task of the utmost importance for contextualizing biological data. Normally, multicolour imaging requires sequential imaging10, 11, multiple cameras12, or segmented dedicated fields of view13, 14. Here, we demonstrate an alternate strategy, the encoding of spectral information (colour), in addition to 3D position, directly in the image. By exploiting chromatic dispersion, we design a new class of optical phase masks that simultaneously yield controllably different PSFs for different wavelengths, enabling simultaneous multicolour tracking or super-resolution imaging in a single optical path.
View details for DOI 10.1038/nphoton.2016.137
View details for PubMedID 28413434
View details for PubMedCentralID PMC5391844
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Removing orientation-induced localization biases in single-molecule microscopy using a broadband metasurface mask
NATURE PHOTONICS
2016; 10 (7): 459-?
Abstract
Nanoscale localization of single molecules is a crucial function in several advanced microscopy techniques, including single-molecule tracking and wide-field super-resolution imaging 1. To date, a central consideration of such techniques is how to optimize the precision of molecular localization. However, as these methods continue to push toward the nanometre size scale, an increasingly important concern is the localization accuracy. In particular, single fluorescent molecules emit with an anisotropic radiation pattern of an oscillating electric dipole, which can cause significant localization biases using common estimators 2-5. Here we present the theory and experimental demonstration of a solution to this problem based on azimuthal filtering in the Fourier plane of the microscope. We do so using a high efficiency dielectric metasurface polarization/phase device composed of nanoposts with sub-wavelength spacing 6. The method is demonstrated both on fluorophores embedded in a polymer matrix, and in dL5 protein complexes that bind Malachite green 7, 8.
View details for DOI 10.1038/NPHOTON.2016.93
View details for Web of Science ID 000378839600011
View details for PubMedCentralID PMC5001689
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Enhanced DNA imaging using super-resolution microscopy and simultaneous single-molecule orientation measurements
OPTICA
2016; 3 (6): 659-666
Abstract
Single-molecule orientation measurements provide unparalleled insight into a multitude of biological and polymeric systems. We report a simple, high-throughput technique for measuring the azimuthal orientation and rotational dynamics of single fluorescent molecules, which is compatible with localization microscopy. Our method involves modulating the polarization of an excitation laser, and analyzing the corresponding intensities emitted by single dye molecules and their modulation amplitudes. To demonstrate our approach, we use intercalating and groove-binding dyes to obtain super-resolved images of stretched DNA strands through binding-induced turn-on of fluorescence. By combining our image data with thousands of dye molecule orientation measurements, we develop a means of probing the structure of individual DNA strands, while also characterizing dye-DNA interactions. This approach may hold promise as a method for monitoring DNA conformation changes resulting from DNA-binding proteins.
View details for DOI 10.1364/OPTICA.3.000659
View details for Web of Science ID 000378847400017
View details for PubMedCentralID PMC5050005
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Removing Orientation-Induced Localization Biases in Single-Molecule Microscopy Using a Broadband Metasurface Mask.
Nature photonics
2016; 10: 459-462
Abstract
Nanoscale localization of single molecules is a crucial function in several advanced microscopy techniques, including single-molecule tracking and wide-field super-resolution imaging 1. To date, a central consideration of such techniques is how to optimize the precision of molecular localization. However, as these methods continue to push toward the nanometre size scale, an increasingly important concern is the localization accuracy. In particular, single fluorescent molecules emit with an anisotropic radiation pattern of an oscillating electric dipole, which can cause significant localization biases using common estimators 2-5. Here we present the theory and experimental demonstration of a solution to this problem based on azimuthal filtering in the Fourier plane of the microscope. We do so using a high efficiency dielectric metasurface polarization/phase device composed of nanoposts with sub-wavelength spacing 6. The method is demonstrated both on fluorophores embedded in a polymer matrix, and in dL5 protein complexes that bind Malachite green 7, 8.
View details for DOI 10.1038/nphoton.2016.93
View details for PubMedID 27574529
View details for PubMedCentralID PMC5001689
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Three-Dimensional Super-Resolution Imaging of the RNA Degradation Machinery in Caulobacter Crescentus
CELL PRESS. 2016: 163A–164A
View details for DOI 10.1016/j.bpj.2015.11.913
View details for Web of Science ID 000375093800305
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Seeing Single Molecules, from Early Spectroscopy in Solids, to Super-Resolution Microscopy, to 3D Dynamics of Biomolecules in Cells
CELL PRESS. 2016: 4A
View details for DOI 10.1016/j.bpj.2015.11.069
View details for Web of Science ID 000375093500024
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3D Single-Molecule Super-Resolution Fluorescence Microscopy with the Corkscrew Point Spread Function
CELL PRESS. 2016: 176A
View details for DOI 10.1016/j.bpj.2015.11.980
View details for Web of Science ID 000375093800367
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Delayed emergence of subdiffraction-sized mutant huntingtin fibrils following inclusion body formation.
Quarterly reviews of biophysics
2016; 49
Abstract
Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington's Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several μm) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 µm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular.
View details for PubMedID 26350150
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Enhanced DNA Imaging Using Super-Resolution Microscopy and Simultaneous Single-Molecule Orientation Measurements
IEEE. 2016
View details for Web of Science ID 000391286401350
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A Diffusion Trap at the Caulobacter Cell Poles Leads to Spatially Resolved Transcription.
AMER SOC CELL BIOLOGY. 2016
View details for Web of Science ID 000394259500008
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Single-molecule microscopy reveals constrained diffusion by a polar matrix microdomain.
AMER SOC CELL BIOLOGY. 2016
View details for Web of Science ID 000394259500434
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Super-resolution microscopy reveals protuberance at the ciliary tip when retrograde transport is impaired.
AMER SOC CELL BIOLOGY. 2016
View details for Web of Science ID 000396047200449
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A Diffusion Trap at the Caulobacter Cell Poles Leads to Spatially Resolved Transcription.
AMER SOC CELL BIOLOGY. 2016
View details for Web of Science ID 000396046900594
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A genetically-directed, photostable fluoromodule for sub-diffraction imaging and single protein tracking in live bacteria.
AMER SOC CELL BIOLOGY. 2016
View details for Web of Science ID 000396047100674
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Two-color fast scanning STED microscopy of live bacteria cells.
AMER SOC CELL BIOLOGY. 2016
View details for Web of Science ID 000396047100665
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Enhanced DNA imaging using super-resolution microscopy and simultaneous single-molecule orientation measurements.
Optica
2016; 3 (6): 3-6
Abstract
Single-molecule orientation measurements provide unparalleled insight into a multitude of biological and polymeric systems. We report a simple, high-throughput technique for measuring the azimuthal orientation and rotational dynamics of single fluorescent molecules, which is compatible with localization microscopy. Our method involves modulating the polarization of an excitation laser, and analyzing the corresponding intensities emitted by single dye molecules and their modulation amplitudes. To demonstrate our approach, we use intercalating and groove-binding dyes to obtain super-resolved images of stretched DNA strands through binding-induced turn-on of fluorescence. By combining our image data with thousands of dye molecule orientation measurements, we develop a means of probing the structure of individual DNA strands, while also characterizing dye-DNA interactions. This approach may hold promise as a method for monitoring DNA conformation changes resulting from DNA-binding proteins.
View details for PubMedID 27722186
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Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy.
Optica
2015; 2 (11): 985-993
Abstract
The localization of single fluorescent molecules enables the imaging of molecular structure and dynamics with subdiffraction precision and can be extended to three dimensions using point spread function (PSF) engineering. However, the nanoscale accuracy of localization throughout a 3D single-molecule microscope's field of view has not yet been rigorously examined. By using regularly spaced subdiffraction apertures filled with fluorescent dyes, we reveal field-dependent aberrations as large as 50-100 nm and show that they can be corrected to less than 25 nm over an extended 3D focal volume. We demonstrate the applicability of this technique for two engineered PSFs, the double-helix PSF and the astigmatic PSF. We expect these results to be broadly applicable to 3D single-molecule tracking and superresolution methods demanding high accuracy.
View details for DOI 10.1364/OPTICA.2.000985
View details for PubMedID 26973863
View details for PubMedCentralID PMC4782984
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Dissecting pigment architecture of individual photosynthetic antenna complexes in solution
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2015; 112 (45): 13880-13885
Abstract
Oligomerization plays a critical role in shaping the light-harvesting properties of many photosynthetic pigment-protein complexes, but a detailed understanding of this process at the level of individual pigments is still lacking. To study the effects of oligomerization, we designed a single-molecule approach to probe the photophysical properties of individual pigment sites as a function of protein assembly state. Our method, based on the principles of anti-Brownian electrokinetic trapping of single fluorescent proteins, step-wise photobleaching, and multiparameter spectroscopy, allows pigment-specific spectroscopic information on single multipigment antennae to be recorded in a nonperturbative aqueous environment with unprecedented detail. We focus on the monomer-to-trimer transformation of allophycocyanin (APC), an important antenna protein in cyanobacteria. Our data reveal that the two chemically identical pigments in APC have different roles. One (α) is the functional pigment that red-shifts its spectral properties upon trimer formation, whereas the other (β) is a "protective" pigment that persistently quenches the excited state of α in the prefunctional, monomer state of the protein. These results show how subtleties in pigment organization give rise to functionally important aspects of energy transfer and photoprotection in antenna complexes. The method developed here should find immediate application in understanding the emergent properties of other natural and artificial light-harvesting systems.
View details for DOI 10.1073/pnas.1514027112
View details for Web of Science ID 000364470300055
View details for PubMedID 26438850
View details for PubMedCentralID PMC4653145
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Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy
REVIEWS OF MODERN PHYSICS
2015; 87 (4)
View details for DOI 10.1103/RevModPhys.87.1183
View details for Web of Science ID 000363246100003
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Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy
OPTICA
2015; 2 (11): 985-993
Abstract
The localization of single fluorescent molecules enables the imaging of molecular structure and dynamics with subdiffraction precision and can be extended to three dimensions using point spread function (PSF) engineering. However, the nanoscale accuracy of localization throughout a 3D single-molecule microscope's field of view has not yet been rigorously examined. By using regularly spaced subdiffraction apertures filled with fluorescent dyes, we reveal field-dependent aberrations as large as 50-100 nm and show that they can be corrected to less than 25 nm over an extended 3D focal volume. We demonstrate the applicability of this technique for two engineered PSFs, the double-helix PSF and the astigmatic PSF. We expect these results to be broadly applicable to 3D single-molecule tracking and superresolution methods demanding high accuracy.
View details for DOI 10.1364/OPTICA.2.000985
View details for Web of Science ID 000365738100013
View details for PubMedCentralID PMC4782984
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Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1.
Proceedings of the National Academy of Sciences of the United States of America
2015; 112 (27): 8320-8325
Abstract
Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation.
View details for DOI 10.1073/pnas.1510094112
View details for PubMedID 26100903
View details for PubMedCentralID PMC4500289
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Chromosomal locus tracking with proper accounting of static and dynamic errors
PHYSICAL REVIEW E
2015; 91 (6)
Abstract
The mean-squared displacement (MSD) and velocity autocorrelation (VAC) of tracked single particles or molecules are ubiquitous metrics for extracting parameters that describe the object's motion, but they are both corrupted by experimental errors that hinder the quantitative extraction of underlying parameters. For the simple case of pure Brownian motion, the effects of localization error due to photon statistics ("static error") and motion blur due to finite exposure time ("dynamic error") on the MSD and VAC are already routinely treated. However, particles moving through complex environments such as cells, nuclei, or polymers often exhibit anomalous diffusion, for which the effects of these errors are less often sufficiently treated. We present data from tracked chromosomal loci in yeast that demonstrate the necessity of properly accounting for both static and dynamic error in the context of an anomalous diffusion that is consistent with a fractional Brownian motion (FBM). We compare these data to analytical forms of the expected values of the MSD and VAC for a general FBM in the presence of these errors.
View details for DOI 10.1103/PhysRevE.91.062716
View details for Web of Science ID 000357028500009
View details for PubMedCentralID PMC4533921
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Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions.
Nano letters
2015; 15 (6): 4194-4199
Abstract
We employ a novel framework for information-optimal microscopy to design a family of point spread functions (PSFs), the Tetrapod PSFs, which enable high-precision localization of nanoscale emitters in three dimensions over customizable axial (z) ranges of up to 20 μm with a high numerical aperture objective lens. To illustrate, we perform flow profiling in a microfluidic channel and show scan-free tracking of single quantum-dot-labeled phospholipid molecules on the surface of living, thick mammalian cells.
View details for DOI 10.1021/acs.nanolett.5b01396
View details for PubMedID 25939423
View details for PubMedCentralID PMC4462996
-
Chromosomal locus tracking with proper accounting of static and dynamic errors.
Physical review. E, Statistical, nonlinear, and soft matter physics
2015; 91 (6): 062716-?
Abstract
The mean-squared displacement (MSD) and velocity autocorrelation (VAC) of tracked single particles or molecules are ubiquitous metrics for extracting parameters that describe the object's motion, but they are both corrupted by experimental errors that hinder the quantitative extraction of underlying parameters. For the simple case of pure Brownian motion, the effects of localization error due to photon statistics ("static error") and motion blur due to finite exposure time ("dynamic error") on the MSD and VAC are already routinely treated. However, particles moving through complex environments such as cells, nuclei, or polymers often exhibit anomalous diffusion, for which the effects of these errors are less often sufficiently treated. We present data from tracked chromosomal loci in yeast that demonstrate the necessity of properly accounting for both static and dynamic error in the context of an anomalous diffusion that is consistent with a fractional Brownian motion (FBM). We compare these data to analytical forms of the expected values of the MSD and VAC for a general FBM in the presence of these errors.
View details for PubMedID 26172745
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Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions
NANO LETTERS
2015; 15 (6): 4194-4199
Abstract
We employ a novel framework for information-optimal microscopy to design a family of point spread functions (PSFs), the Tetrapod PSFs, which enable high-precision localization of nanoscale emitters in three dimensions over customizable axial (z) ranges of up to 20 μm with a high numerical aperture objective lens. To illustrate, we perform flow profiling in a microfluidic channel and show scan-free tracking of single quantum-dot-labeled phospholipid molecules on the surface of living, thick mammalian cells.
View details for DOI 10.1021/acs.nanolett.5b01396
View details for Web of Science ID 000356316900081
View details for PubMedID 25939423
View details for PubMedCentralID PMC4462996
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Detecting biomolecular interactions and photodynamics in solution by suppression of Brownian motion
AMER CHEMICAL SOC. 2015
View details for Web of Science ID 000411183303562
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Single-Molecule Identification of Quenched and Unquenched States of LHCII
JOURNAL OF PHYSICAL CHEMISTRY LETTERS
2015; 6 (5): 860-867
Abstract
In photosynthetic light harvesting, absorbed sunlight is converted to electron flow with near-unity quantum efficiency under low light conditions. Under high light conditions, plants avoid damage to their molecular machinery by activating a set of photoprotective mechanisms to harmlessly dissipate excess energy as heat. To investigate these mechanisms, we study the primary antenna complex in green plants, light-harvesting complex II (LHCII), at the single-complex level. We use a single-molecule technique, the Anti-Brownian Electrokinetic trap, which enables simultaneous measurements of fluorescence intensity, lifetime, and spectra in solution. With this approach, including the first measurements of fluorescence lifetime on single LHCII complexes, we access the intrinsic conformational dynamics. In addition to an unquenched state, we identify two partially quenched states of LHCII. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in LHCII. Furthermore, one of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light.
View details for DOI 10.1021/acs.jpclett.5b00034
View details for Web of Science ID 000350843400017
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Single-Molecule Identification of Quenched and Unquenched States of LHCII.
journal of physical chemistry letters
2015; 6 (5): 860-867
Abstract
In photosynthetic light harvesting, absorbed sunlight is converted to electron flow with near-unity quantum efficiency under low light conditions. Under high light conditions, plants avoid damage to their molecular machinery by activating a set of photoprotective mechanisms to harmlessly dissipate excess energy as heat. To investigate these mechanisms, we study the primary antenna complex in green plants, light-harvesting complex II (LHCII), at the single-complex level. We use a single-molecule technique, the Anti-Brownian Electrokinetic trap, which enables simultaneous measurements of fluorescence intensity, lifetime, and spectra in solution. With this approach, including the first measurements of fluorescence lifetime on single LHCII complexes, we access the intrinsic conformational dynamics. In addition to an unquenched state, we identify two partially quenched states of LHCII. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in LHCII. Furthermore, one of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light.
View details for DOI 10.1021/acs.jpclett.5b00034
View details for PubMedID 26262664
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Determining the rotational mobility of a single molecule from a single image: a numerical study
OPTICS EXPRESS
2015; 23 (4): 4255-4276
Abstract
Measurements of the orientational freedom with which a single molecule may rotate or 'wobble' about a fixed axis have provided researchers invaluable clues about the underlying behavior of a variety of biological systems. In this paper, we propose a measurement and data analysis procedure based on a widefield fluorescence microscope image for quantitatively distinguishing individual molecules that exhibit varying degrees of rotational mobility. Our proposed technique is especially applicable to cases in which the molecule undergoes rotational motions on a timescale much faster than the framerate of the camera used to record fluorescence images. Unlike currently available methods, sophisticated hardware for modulating the polarization of light illuminating the sample is not required. Additional polarization optics may be inserted in the microscope's imaging pathway to achieve superior measurement precision, but are not essential. We present a theoretical analysis, and benchmark our technique with numerical simulations using typical experimental parameters for single-molecule imaging.
View details for DOI 10.1364/OE.23.004255
View details for Web of Science ID 000350872700041
View details for PubMedID 25836463
View details for PubMedCentralID PMC4394761
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Pigment-Specific Fluorescence Spectroscopy of Single Antenna Complexes in Solution
CELL PRESS. 2015: 368A
View details for DOI 10.1016/j.bpj.2014.11.2017
View details for Web of Science ID 000362849400261
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Single-Molecule Exploration of Photoprotective Mechanisms in Light-Harvesting Complexes
Conference on Single Molecule Spectroscopy and Superresolution Imaging VIII
SPIE-INT SOC OPTICAL ENGINEERING. 2015
View details for DOI 10.1117/12.2083628
View details for Web of Science ID 000353557600004
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Light Paves the Way to Single-Molecule Detection and Photocontrol, Foundations of Super-Resolution Microscopy
IEEE. 2015
View details for Web of Science ID 000370627101042
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Maximally Informative Point Spread Functions for 3D Super-Resolution Imaging
IEEE. 2015
View details for Web of Science ID 000370627102241
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An Azimuthal Polarizer Assures Localization Accuracy in Single-Molecule Super-Resolution Fluorescence Microscopy
IEEE. 2015
View details for Web of Science ID 000370627102240
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Single-molecule tracking and super-resolution imaging reveal a diffusion trap at the poles of Caulobacter crescentus
AMER SOC CELL BIOLOGY. 2015
View details for Web of Science ID 000209928500044
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The story of single molecules, from early spectroscopy in solids, to super-resolution microscopy, to 3D dynamics of biomolecules in cells.
AMER SOC CELL BIOLOGY. 2015
View details for Web of Science ID 000209928400004
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Single-Molecule Photocontrol and Nanoscopy
FAR-FIELD OPTICAL NANOSCOPY
2015; 14: 87-110
View details for DOI 10.1007/4243_2011_37
View details for Web of Science ID 000482949500005
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Single-molecule tracking of smoothened in the primary cilium
AMER SOC CELL BIOLOGY. 2015
View details for Web of Science ID 000209928500166
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Single-Molecule Spectroscopy, Imaging, and Photocontrol: Foundations for Super-Resolution Microscopy (Nobel Lecture).
Angewandte Chemie (International ed. in English)
2015; 54 (28): 8067–93
Abstract
The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super-resolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super-resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.
View details for PubMedID 26088273
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Single-molecule spectroscopy and imaging over the decades
FARADAY DISCUSSIONS
2015; 184: 9-36
Abstract
As of 2015, it has been 26 years since the first optical detection and spectroscopy of single molecules in condensed matter. This area of science has expanded far beyond the early low temperature studies in crystals to include single molecules in cells, polymers, and in solution. The early steps relied upon high-resolution spectroscopy of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral fine structure arising directly from the position-dependent fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 1990s, a variety of fascinating physical effects were observed for individual molecules, including imaging of the light from single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency. In the room temperature regime, researchers showed that bursts of light from single molecules could be detected in solution, leading to imaging and microscopy by a variety of methods. Studies of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. All of these early steps provided important fundamentals underpinning the development of super-resolution microscopy based on single-molecule localization and active control of emitting concentration. Current thrust areas include extensions to three-dimensional imaging with high precision, orientational analysis of single molecules, and direct measurements of photodynamics and transport properties for single molecules trapped in solution by suppression of Brownian motion. Without question, a huge variety of studies of single molecules performed by many talented scientists all over the world have extended our knowledge of the nanoscale and many microscopic mechanisms previously hidden by ensemble averaging.
View details for DOI 10.1039/c5fd00149h
View details for Web of Science ID 000366548800001
View details for PubMedID 26616210
View details for PubMedCentralID PMC4782608
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Delayed emergence of subdiffraction-sized mutant huntingtin fibrils following inclusion body formation
Quarterly Reviews of Biophysics
2015: 178-243
Abstract
The majority of studies of the living cell rely on capturing images using fluorescence microscopy. Unfortunately, for centuries, diffraction of light was limiting the spatial resolution in the optical microscope: structural and molecular details much finer than about half the wavelength of visible light (~200 nm) could not be visualized, imposing significant limitations on this otherwise so promising method. The surpassing of this resolution limit in far-field microscopy is currently one of the most momentous developments for studying the living cell, as the move from microscopy to super-resolution microscopy or 'nanoscopy' offers opportunities to study problems in biophysical and biomedical research at a new level of detail. This review describes the principles and modalities of present fluorescence nanoscopes, as well as their potential for biophysical and cellular experiments. All the existing nanoscopy variants separate neighboring features by transiently preparing their fluorescent molecules in states of different emission characteristics in order to make the features discernible. Usually these are fluorescent 'on' and 'off' states causing the adjacent molecules to emit sequentially in time. Each of the variants can in principle reach molecular spatial resolution and has its own advantages and disadvantages. Some require specific transitions and states that can be found only in certain fluorophore subfamilies, such as photoswitchable fluorophores, while other variants can be realized with standard fluorescent labels. Similar to conventional far-field microscopy, nanoscopy can be utilized for dynamical, multi-color and three-dimensional imaging of fixed and live cells, tissues or organisms. Lens-based fluorescence nanoscopy is poised for a high impact on future developments in the life sciences, with the potential to help solve long-standing quests in different areas of scientific research.
View details for DOI 10.1017/S0033583515000219
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Motion of chromosomal loci and the mean-squared displacement of a fractional Brownian motion in the presence of static and dynamic errors
Conference on Single Molecule Spectroscopy and Superresolution Imaging VIII
SPIE-INT SOC OPTICAL ENGINEERING. 2015
View details for DOI 10.1117/12.2079703
View details for Web of Science ID 000353557600002
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Super-resolution fluorescence of huntingtin reveals growth of globular species into short fibers and coexistence of distinct aggregates.
ACS chemical biology
2014; 9 (12): 2767-2778
Abstract
Polyglutamine-expanded huntingtin, the protein encoded by HTT mutations associated with Huntington's disease, forms aggregate species in vitro and in vivo. Elucidation of the mechanism of growth of fibrillar aggregates from soluble monomeric protein is critical to understanding the progression of Huntington's disease and to designing therapeutics for the disease, as well as for aggregates implicated in Alzheimer's and Parkinson's diseases. We used the technique of multicolor single-molecule, super-resolution fluorescence imaging to characterize the growth of huntingtin exon 1 aggregates. The huntingtin exon 1 aggregation followed a pathway from exclusively spherical or globular species of ∼80 nm to fibers ∼1 μm in length that increased in width, but not length, over time with the addition of more huntingtin monomers. The fibers further aggregated with one another into aggregate assemblies of increasing size. Seeds created by sonication, which were comparable in shape and size to the globular species in the pathway, were observed to grow through multidirectional elongation into fibers, suggesting a mechanism for growth of globular species into fibers. The single-molecule sensitivity of our approach made it possible to characterize the aggregation pathway across a large range of size scales, from monomers to fiber assemblies, and revealed the coexistence of different aggregate species (globular species, fibers, fiber assemblies) even at late time points.
View details for DOI 10.1021/cb500335w
View details for PubMedID 25330023
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Stimulated emission depletion (STED) microscopy reveals a sub-diffraction, 9-fold symmetric domain containing Cby1, Ahi1, and Ofd1 at the centriole-cilium interface
AMER SOC CELL BIOLOGY. 2014
View details for Web of Science ID 000352094104058
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Single-molecule tracking of Smoothened reveals binding in the primary cilium that is altered by pathway agonists.
AMER SOC CELL BIOLOGY. 2014
View details for Web of Science ID 000352094102257
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Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope.
Molecular biology of the cell
2014; 25 (22): 3619-3629
Abstract
Single-particle tracking has been applied to study chromatin motion in live cells, revealing a wealth of dynamical behavior of the genomic material once believed to be relatively static throughout most of the cell cycle. Here we used the dual-color three-dimensional (3D) double-helix point spread function microscope to study the correlations of movement between two fluorescently labeled gene loci on either the same or different budding yeast chromosomes. We performed fast (10 Hz) 3D tracking of the two copies of the GAL locus in diploid cells in both activating and repressive conditions. As controls, we tracked pairs of loci along the same chromosome at various separations, as well as transcriptionally orthogonal genes on different chromosomes. We found that under repressive conditions, the GAL loci exhibited significantly higher velocity cross-correlations than they did under activating conditions. This relative increase has potentially important biological implications, as it might suggest coupling via shared silencing factors or association with decoupled machinery upon activation. We also found that on the time scale studied (∼0.1-30 s), the loci moved with significantly higher subdiffusive mean square displacement exponents than previously reported, which has implications for the application of polymer theory to chromatin motion in eukaryotes.
View details for DOI 10.1091/mbc.E14-06-1127
View details for PubMedID 25318676
View details for PubMedCentralID PMC4230621
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Azimuthal Polarization Filtering for Accurate, Precise, and Robust Single-Molecule Localization Microscopy
NANO LETTERS
2014; 14 (11): 6407-6413
Abstract
Many single nanoemitters such as fluorescent molecules produce dipole radiation that leads to systematic position errors in both particle tracking and super-resolution microscopy. Via vectorial diffraction equations and simulations, we show that imaging only azimuthally polarized light in the microscope naturally avoids emission from the z-component of the transition dipole moment, resulting in negligible localization errors for all emitter orientations and degrees of objective lens misfocus. Furthermore, localization accuracy is maintained even in the presence of aberrations resulting from imaging in mismatched media.
View details for DOI 10.1021/nl502914k
View details for Web of Science ID 000345723800059
View details for PubMedID 25272093
View details for PubMedCentralID PMC4245985
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Small-molecule labeling of live cell surfaces for three-dimensional super-resolution microscopy.
Journal of the American Chemical Society
2014; 136 (40): 14003-14006
Abstract
Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10-20 nm.
View details for DOI 10.1021/ja508028h
View details for PubMedID 25222297
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Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function
MOLECULAR BIOLOGY OF THE CELL
2014; 25 (19): 2919-2933
Abstract
Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1(-/-) cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1(-/-) cells.
View details for DOI 10.1091/mbc.E14-02-0735
View details for Web of Science ID 000343124100004
View details for PubMedCentralID PMC4230582
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Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function.
Molecular biology of the cell
2014; 25 (19): 2919-2933
Abstract
Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1(-/-) cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1(-/-) cells.
View details for DOI 10.1091/mbc.E14-02-0735
View details for PubMedID 25103236
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Optimal point spread function design for 3D imaging.
Physical review letters
2014; 113 (13): 133902-?
Abstract
To extract from an image of a single nanoscale object maximum physical information about its position, we propose and demonstrate a framework for pupil-plane modulation for 3D imaging applications requiring precise localization, including single-particle tracking and superresolution microscopy. The method is based on maximizing the information content of the system, by formulating and solving the appropriate optimization problem--finding the pupil-plane phase pattern that would yield a point spread function (PSF) with optimal Fisher information properties. We use our method to generate and experimentally demonstrate two example PSFs: one optimized for 3D localization precision over a 3 μm depth of field, and another with an unprecedented 5 μm depth of field, both designed to perform under physically common conditions of high background signals.
View details for PubMedID 25302889
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Imaging-based approaches to the aggregation-prone mutant huntingtin protein in cellular contexts
WILEY-BLACKWELL. 2014: 32
View details for Web of Science ID 000359666800102
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Small-molecule labeling of live cell surfaces for 3D super-resolution microscopy
AMER CHEMICAL SOC. 2014
View details for Web of Science ID 000349165101765
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Multivariate single-molecule dynamics in solution by suppression of Brownian motion
AMER CHEMICAL SOC. 2014
View details for Web of Science ID 000349165102142
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Extending single-molecule microscopy using optical fourier processing.
journal of physical chemistry. B
2014; 118 (28): 8313-8329
Abstract
This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules.
View details for DOI 10.1021/jp501778z
View details for PubMedID 24745862
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Bacterial scaffold directs pole-specific centromere segregation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (19): E2046-E2055
Abstract
Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.
View details for DOI 10.1073/pnas.1405188111
View details for Web of Science ID 000335798000018
View details for PubMedCentralID PMC4024888
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A bisected pupil for studying single-molecule orientational dynamics and its application to three-dimensional super-resolution microscopy
APPLIED PHYSICS LETTERS
2014; 104 (19)
Abstract
A phase mask design that we term a "bisected pupil" (BSP) provides several advantages for single-molecule optical imaging. When using the BSP with a dual-polarization optical Fourier processing system, both the position and dipole orientation of individual fluorescent molecules may be measured from a single camera image. In the context of single-molecule super-resolution microscopy, this technique permits one to diagnose, and subsequently to remove imaging artifacts resulting from orientation-induced localization errors. If the molecules labeling a structure are rotationally mobile, thus mitigating dipole orientation errors, this technique enables super-resolution imaging in three dimensions. We present simulations and experimental verification.
View details for DOI 10.1063/1.4876440
View details for Web of Science ID 000336918600075
View details for PubMedCentralID PMC4032398
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A bisected pupil for studying single-molecule orientational dynamics and its application to three-dimensional super-resolution microscopy.
Applied physics letters
2014; 104 (19): 193701
Abstract
A phase mask design that we term a "bisected pupil" (BSP) provides several advantages for single-molecule optical imaging. When using the BSP with a dual-polarization optical Fourier processing system, both the position and dipole orientation of individual fluorescent molecules may be measured from a single camera image. In the context of single-molecule super-resolution microscopy, this technique permits one to diagnose, and subsequently to remove imaging artifacts resulting from orientation-induced localization errors. If the molecules labeling a structure are rotationally mobile, thus mitigating dipole orientation errors, this technique enables super-resolution imaging in three dimensions. We present simulations and experimental verification.
View details for DOI 10.1063/1.4876440
View details for PubMedID 24926098
View details for PubMedCentralID PMC4032398
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Robust hypothesis tests for detecting statistical evidence of two-dimensional and three-dimensional interactions in single-molecule measurements
PHYSICAL REVIEW E
2014; 89 (5)
Abstract
Experimental advances have improved the two- (2D) and three-dimensional (3D) spatial resolution that can be extracted from in vivo single-molecule measurements. This enables researchers to quantitatively infer the magnitude and directionality of forces experienced by biomolecules in their native environment. Situations where such force information is relevant range from mitosis to directed transport of protein cargo along cytoskeletal structures. Models commonly applied to quantify single-molecule dynamics assume that effective forces and velocity in the x,y (or x,y,z) directions are statistically independent, but this assumption is physically unrealistic in many situations. We present a hypothesis testing approach capable of determining if there is evidence of statistical dependence between positional coordinates in experimentally measured trajectories; if the hypothesis of independence between spatial coordinates is rejected, then a new model accounting for 2D (3D) interactions can and should be considered. Our hypothesis testing technique is robust, meaning it can detect interactions, even if the noise statistics are not well captured by the model. The approach is demonstrated on control simulations and on experimental data (directed transport of intraflagellar transport protein 88 homolog in the primary cilium).
View details for DOI 10.1103/PhysRevE.89.052705
View details for Web of Science ID 000335917500009
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Single-molecule motions enable direct visualization of biomolecular interactions in solution
NATURE METHODS
2014; 11 (5): 555-558
Abstract
Biomolecular interactions are generally accompanied by modifications in size and charge of biomolecules at the nanometer scale. Here we describe a single-molecule method to sense these changes in real time based on statistical learning of diffusive and electric field-induced motion parameters of a trapped molecule in solution. We demonstrate the approach by resolving a monomer-trimer mixture along a protein dissociation pathway and visualizing the binding-unbinding kinetics of a single DNA molecule.
View details for DOI 10.1038/NMETH.2882
View details for Web of Science ID 000335873400023
View details for PubMedID 24608179
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Robust hypothesis tests for detecting statistical evidence of two-dimensional and three-dimensional interactions in single-molecule measurements.
Physical review. E, Statistical, nonlinear, and soft matter physics
2014; 89 (5): 052705
Abstract
Experimental advances have improved the two- (2D) and three-dimensional (3D) spatial resolution that can be extracted from in vivo single-molecule measurements. This enables researchers to quantitatively infer the magnitude and directionality of forces experienced by biomolecules in their native environment. Situations where such force information is relevant range from mitosis to directed transport of protein cargo along cytoskeletal structures. Models commonly applied to quantify single-molecule dynamics assume that effective forces and velocity in the x,y (or x,y,z) directions are statistically independent, but this assumption is physically unrealistic in many situations. We present a hypothesis testing approach capable of determining if there is evidence of statistical dependence between positional coordinates in experimentally measured trajectories; if the hypothesis of independence between spatial coordinates is rejected, then a new model accounting for 2D (3D) interactions can and should be considered. Our hypothesis testing technique is robust, meaning it can detect interactions, even if the noise statistics are not well captured by the model. The approach is demonstrated on control simulations and on experimental data (directed transport of intraflagellar transport protein 88 homolog in the primary cilium).
View details for DOI 10.1103/PhysRevE.89.052705
View details for PubMedID 25353827
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The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL Trap.
Proceedings of SPIE--the International Society for Optical Engineering
2014; 8950: 89500H
Abstract
F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.
View details for DOI 10.1117/12.2042688
View details for PubMedID 25309100
View details for PubMedCentralID PMC4189113
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The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging.
Chemphyschem
2014; 15 (4): 587-599
Abstract
Numerous methods for determining the orientation of single-molecule transition dipole moments from microscopic images of the molecular fluorescence have been developed in recent years. At the same time, techniques that rely on nanometer-level accuracy in the determination of molecular position, such as single-molecule super-resolution imaging, have proven immensely successful in their ability to access unprecedented levels of detail and resolution previously hidden by the optical diffraction limit. However, the level of accuracy in the determination of position is threatened by insufficient treatment of molecular orientation. Here we review a number of methods for measuring molecular orientation using fluorescence microscopy, focusing on approaches that are most compatible with position estimation and single-molecule super-resolution imaging. We highlight recent methods based on quadrated pupil imaging and on double-helix point spread function microscopy and apply them to the study of fluorophore mobility on immunolabeled microtubules.
View details for DOI 10.1002/cphc.201300880
View details for PubMedID 24382708
View details for PubMedCentralID PMC3992256
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From "There's Plenty of Room at the Bottom" to Seeing What is Actually There
CHEMPHYSCHEM
2014; 15 (4): 547-549
View details for DOI 10.1002/cphc.201400097
View details for Web of Science ID 000332747500001
View details for PubMedID 24590957
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Quantifying the Spatial Organization of Bacterial Ribosomes using Three-Dimensional Super-Resolution Microscopy
CELL PRESS. 2014: 492A
View details for DOI 10.1016/j.bpj.2013.11.2753
View details for Web of Science ID 000337000402726
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Elucidation of the Photodynamics of Single Photosynthetic LH2 Complexes in Solution
CELL PRESS. 2014: 27A
View details for DOI 10.1016/j.bpj.2013.11.205
View details for Web of Science ID 000337000400137
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Single-Molecule Exploration of the Photodynamics of LHCII Complexes in Solution
CELL PRESS. 2014: 182A
View details for DOI 10.1016/j.bpj.2013.11.1030
View details for Web of Science ID 000337000401037
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Correlations in Chromatin Movement in Diploid Yeast Revealed by Two-Color Three-Dimensional Single-Particle Tracking using the Double-Helix Point Spread Function (DH-PSF) Microscope
CELL PRESS. 2014: 199A
View details for DOI 10.1016/j.bpj.2013.11.1174
View details for Web of Science ID 000337000401120
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Sensing the Association States of Single Biomolecules by Motion Analysis in an Electrokinetic Trap
CELL PRESS. 2014: 394A
View details for DOI 10.1016/j.bpj.2013.11.2225
View details for Web of Science ID 000337000402225
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Precise Measurement of the Relative Position of RNA Dimers within Virus-Like Particles using 2-Color 3D Super-Resolution Fluorescence Microscopy
CELL PRESS. 2014: 399A
View details for DOI 10.1016/j.bpj.2013.11.2252
View details for Web of Science ID 000337000402251
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The Aggregation-Prone Mutant Huntingtin Protein in a Cellular Context - Approaches by Super-Resolution Imaging
CELL PRESS. 2014: 683A
View details for DOI 10.1016/j.bpj.2013.11.3780
View details for Web of Science ID 000337000403803
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DNA Segregation and Partitioning in Caulobacter Crescentus: Super-Resolving Protein Colocalization at the Cell Pole
CELL PRESS. 2014: 59A–60A
View details for DOI 10.1016/j.bpj.2013.11.408
View details for Web of Science ID 000337000400306
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Quantitative Registration and Distribution Analysis of Multicolor 3D Super-Resolution Images of Proteins Reveals Nanoscale Spatial Organization
CELL PRESS. 2014: 203A
View details for DOI 10.1016/j.bpj.2013.11.1192
View details for Web of Science ID 000337000401138
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Single-Molecule Tracking of Smoothened in the Primary Cilium
CELL PRESS. 2014: 20A
View details for DOI 10.1016/j.bpj.2013.11.166
View details for Web of Science ID 000337000400103
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Single-molecule spectroscopy of photosynthetic proteins in solution: exploration of structure-function relationships
CHEMICAL SCIENCE
2014; 5 (8): 2933-2939
View details for DOI 10.1039/c4sc00582a
View details for Web of Science ID 000338652900002
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Single-molecule orientation measurements with a quadrated pupil
Conference on Single Molecule Spectroscopy and Superresolution Imaging VII
SPIE-INT SOC OPTICAL ENGINEERING. 2014
View details for DOI 10.1117/12.2042097
View details for Web of Science ID 000336479700009
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The regulatory switch of F-1-ATPase studied by single-molecule FRET in the ABEL Trap
Conference on Single Molecule Spectroscopy and Superresolution Imaging VII
SPIE-INT SOC OPTICAL ENGINEERING. 2014
Abstract
F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.
View details for DOI 10.1117/12.2042688
View details for Web of Science ID 000336479700007
View details for PubMedCentralID PMC4189113
-
Spectroscopic and transport measurements of single molecules in solution using an electrokinetic trap
Conference on Single Molecule Spectroscopy and Superresolution Imaging VII
SPIE-INT SOC OPTICAL ENGINEERING. 2014
View details for DOI 10.1117/12.2038320
View details for Web of Science ID 000336479700002
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Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging
NATURE REVIEWS MICROBIOLOGY
2014; 12 (1): 9-22
Abstract
The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field.
View details for DOI 10.1038/nrmicro3154
View details for Web of Science ID 000348329900001
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Quantifying Transient 3D Dynamical Phenomena of Single mRNA Particles in Live Yeast Cell Measurements
JOURNAL OF PHYSICAL CHEMISTRY B
2013; 117 (49): 15701-15713
Abstract
Single-particle tracking (SPT) has been extensively used to obtain information about diffusion and directed motion in a wide range of biological applications. Recently, new methods have appeared for obtaining precise (10s of nm) spatial information in three dimensions (3D) with high temporal resolution (measurements obtained every 4 ms), which promise to more accurately sense the true dynamical behavior in the natural 3D cellular environment. Despite the quantitative 3D tracking information, the range of mathematical methods for extracting information about the underlying system has been limited mostly to mean-squared displacement analysis and other techniques not accounting for complex 3D kinetic interactions. There is a great need for new analysis tools aiming to more fully extract the biological information content from in vivo SPT measurements. High-resolution SPT experimental data has enormous potential to objectively scrutinize various proposed mechanistic schemes arising from theoretical biophysics and cell biology. At the same time, methods for rigorously checking the statistical consistency of both model assumptions and estimated parameters against observed experimental data (i.e., goodness-of-fit tests) have not received great attention. We demonstrate methods enabling (1) estimation of the parameters of 3D stochastic differential equation (SDE) models of the underlying dynamics given only one trajectory; and (2) construction of hypothesis tests checking the consistency of the fitted model with the observed trajectory so that extracted parameters are not overinterpreted (the tools are applicable to linear or nonlinear SDEs calibrated from nonstationary time series data). The approach is demonstrated on high-resolution 3D trajectories of single ARG3 mRNA particles in yeast cells in order to show the power of the methods in detecting signatures of transient directed transport. The methods presented are generally relevant to a wide variety of 2D and 3D SPT tracking applications.
View details for DOI 10.1021/jp4064214
View details for Web of Science ID 000328529000047
View details for PubMedID 24015725
View details for PubMedCentralID PMC3865222
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Super-resolution fluorescence imaging with single molecules.
Current opinion in structural biology
2013; 23 (5): 778-787
Abstract
The ability to detect, image and localize single molecules optically with high spatial precision by their fluorescence enables an emergent class of super-resolution microscopy methods which have overcome the longstanding diffraction barrier for far-field light-focusing optics. Achieving spatial resolutions of 20-40nm or better in both fixed and living cells, these methods are currently being established as powerful tools for minimally-invasive spatiotemporal analysis of structural details in cellular processes which benefit from enhanced resolution. Briefly covering the basic principles, this short review then summarizes key recent developments and application examples of two-dimensional and three-dimensional (3D) multi-color techniques and faster time-lapse schemes. The prospects for quantitative imaging - in terms of improved ability to correct for dipole-emission-induced systematic localization errors and to provide accurate counts of molecular copy numbers within nanoscale cellular domains - are discussed.
View details for DOI 10.1016/j.sbi.2013.07.010
View details for PubMedID 23932284
View details for PubMedCentralID PMC3805708
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Rotational mobility of single molecules affects localization accuracy in super-resolution fluorescence microscopy.
Nano letters
2013; 13 (9): 3967-3972
Abstract
The asymmetric nature of single-molecule (SM) dipole emission patterns limits the accuracy of position determination in localization-based super-resolution fluorescence microscopy. The degree of mislocalization depends highly on the rotational mobility of SMs; only for SMs rotating within a cone half angle α > 60° can mislocalization errors be bounded to ≤10 nm. Simulations demonstrate how low or high rotational mobility can cause resolution degradation or distortion in super-resolution reconstructions.
View details for DOI 10.1021/nl304359p
View details for PubMedID 23360306
View details for PubMedCentralID PMC3696529
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Elucidation of the photodynamics of single LH2 proteins in solution
AMER CHEMICAL SOC. 2013
View details for Web of Science ID 000329618406344
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Photo-induced conformational flexibility in single solution-phase peridinin-chlorophyll-proteins.
journal of physical chemistry. A
2013; 117 (35): 8399-8406
Abstract
The peridinin-chlorophyll-protein (PCP) is an accessory light-harvesting complex found in red-tide dinoflagellates. PCP absorbs photons primarily in the blue-green spectral region via peridinin (Per) carotenoid pigments which then transfer excitations to chlorophyll (Chl) and ultimately downstream to photosystem II (PSII). Whereas the ultrafast dynamics of PCP are well-studied, much less is known about slower protein dynamics on time scales of milliseconds and seconds. Previous single-molecule studies of spectral emission and intensity have attached PCP to surfaces, but the native environment of PCP is in the lumen, meaning that a surface-attached environment could perturb its native conformations. To address this concern, we use the anti-Brownian electrokinetic (ABEL) trap to study single PCP monomers in solution for several seconds each. We measure, for the first time, simultaneous single-molecule intensity, lifetime, and spectral emission shifts for each trapped PCP monomer. The rate of reversible spectral redshifts depends linearly on irradiance over a factor of 30, indicating a light-induced mechanism which we attribute to a protein conformational change. Independent of these spectral shifts, our measurements of intensity and lifetime show reversible Chl quenching. In contrast to previous work, we show that this quenching cannot result from isolated photobleaching of Chl. These independent mechanisms arise from distinct conformational changes which maintain relatively stable fluorescence emission.
View details for DOI 10.1021/jp405790a
View details for PubMedID 23919352
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Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (27): 10899-10903
Abstract
Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.
View details for DOI 10.1073/pnas.1310222110
View details for Web of Science ID 000321978000022
View details for PubMedID 23776245
View details for PubMedCentralID PMC3704035
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Single-molecule orientation measurements with a quadrated pupil
OPTICS LETTERS
2013; 38 (9): 1521-1523
Abstract
This Letter presents a means of measuring the dipole orientation of a fluorescent, orientationally fixed single molecule, which uses a specially designed phase mask, termed a "quadrated pupil," conjugate to the back focal plane of a conventional wide-field microscope. The method leverages the spatial anisotropy of the far-field emission pattern of a dipole emitter and makes this anisotropy amenable to quantitative analysis at the image plane. In comparison to older image-fitting techniques that infer orientation by matching simulations to defocused or excessively magnified images, the quadrated pupil approach is more robust to minor modeling discrepancies and optical aberrations. Precision of 1°-5° is achieved in proof-of-concept experiments for both azimuthal (φ) and polar (θ) angles without defocusing. Since the phase mask is implemented on a liquid-crystal spatial light modulator that may be deactivated without any mechanical perturbation of the sample or imaging system, the technique may be readily integrated into clear aperture imaging studies.
View details for DOI 10.1364/OL.38.001521
View details for Web of Science ID 000318425600054
View details for PubMedID 23632538
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Lifetime and Spectrally Resolved Characterization of the Photodynamics of Single Fluorophores in Solution Using the Anti-Brownian Electrokinetic Trap
JOURNAL OF PHYSICAL CHEMISTRY B
2013; 117 (16): 4641-4648
Abstract
We report simultaneous determination of fluorescence intensity, lifetime, and emission spectrum over time scales on the order of seconds for single molecules in solution, using the anti-Brownian electrokinetic trap. We demonstrate the technique with trapped single fluorophores of Atto647N and Alexa647. Three emission states with distinct intensities, lifetimes, and emission peaks are found in the case of Atto647N. Transitions between states happen occasionally. We characterize the three states and quantify the transition probabilities between states using concurrent intensity, lifetime, and spectrum data. Alexa647, on the other hand, showed little dynamics. These results represent a significant advance in the ability to identify and characterize different dynamical states of single molecules in aqueous solution with high precision and millisecond time resolution.
View details for DOI 10.1021/jp308949d
View details for Web of Science ID 000318211600058
View details for PubMedID 23198678
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Award Address (Peter Debye Award in Physical Chemistry sponsored by EI du Pont de Nemours & Company). Single-molecule spectroscopy and imaging: 3D nanoscopy and biomolecular dynamics
AMER CHEMICAL SOC. 2013
View details for Web of Science ID 000324303603759
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Quantitative Multicolor Subdiffraction Imaging of Bacterial Protein Ultrastructures in Three Dimensions
NANO LETTERS
2013; 13 (3): 987-993
Abstract
We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.
View details for DOI 10.1021/nl304071h
View details for Web of Science ID 000316243800020
View details for PubMedID 23414562
View details for PubMedCentralID PMC3599789
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Easy-DHPSF open-source software for three-dimensional localization of single molecules with precision beyond the optical diffraction limit.
Protocol exchange
2013; 2013
Abstract
Automated processing of double-helix (DH) microscope images of single molecules (SMs) streamlines the protocol required to obtain super-resolved three-dimensional (3D) reconstructions of ultrastructures in biological samples by single-molecule active control microscopy. Here, we present a suite of MATLAB subroutines, bundled with an easy-to-use graphical user interface (GUI), that facilitates 3D localization of single emitters (e.g. SMs, fluorescent beads, or quantum dots) with precisions of tens of nanometers in multi-frame movies acquired using a wide-field DH epifluorescence microscope. The algorithmic approach is based upon template matching for SM recognition and least-squares fitting for 3D position measurement, both of which are computationally expedient and precise. Overlapping images of SMs are ignored, and the precision of least-squares fitting is not as high as maximum likelihood-based methods. However, once calibrated, the algorithm can fit 15-30 molecules per second on a 3 GHz Intel Core 2 Duo workstation, thereby producing a 3D super-resolution reconstruction of 100,000 molecules over a 20×20×2 μm field of view (processing 128×128 pixels × 20000 frames) in 75 min.
View details for PubMedID 25279136
View details for PubMedCentralID PMC4180084
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The double-helix point spread function enables precise and accurate measurement of 3D single-molecule localization and orientation.
Proceedings of SPIE--the International Society for Optical Engineering
2013; 8590: 85900
Abstract
Single-molecule-based super-resolution fluorescence microscopy has recently been developed to surpass the diffraction limit by roughly an order of magnitude. These methods depend on the ability to precisely and accurately measure the position of a single-molecule emitter, typically by fitting its emission pattern to a symmetric estimator (e.g. centroid or 2D Gaussian). However, single-molecule emission patterns are not isotropic, and depend highly on the orientation of the molecule's transition dipole moment, as well as its z-position. Failure to account for this fact can result in localization errors on the order of tens of nm for in-focus images, and ~50-200 nm for molecules at modest defocus. The latter range becomes especially important for three-dimensional (3D) single-molecule super-resolution techniques, which typically employ depths-of-field of up to ~2 μm. To address this issue we report the simultaneous measurement of precise and accurate 3D single-molecule position and 3D dipole orientation using the Double-Helix Point Spread Function (DH-PSF) microscope. We are thus able to significantly improve dipole-induced position errors, reducing standard deviations in lateral localization from ~2x worse than photon-limited precision (48 nm vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation we are able to improve from a lateral standard deviation of 116 nm (~4x worse than the precision, 28 nm) to 34 nm (within 6 nm).
View details for DOI 10.1117/12.2001671
View details for PubMedID 24817798
View details for PubMedCentralID PMC4013112
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Enzymatic activation of nitro-aryl fluorogens in live bacterial cells for enzymatic turnover-activated localization microscopy
CHEMICAL SCIENCE
2013; 4 (1): 220-225
Abstract
Many modern super-resolution imaging methods based on single-molecule fluorescence require the conversion of a dark fluorogen into a bright emitter to control emitter concentration. We have synthesized and characterized a nitro-aryl fluorogen which can be converted by a nitroreductase enzyme into a bright push-pull red-emitting fluorophore. Synthesis of model compounds and optical spectroscopy identify a hydroxyl-amino derivative as the product fluorophore, which is bright and detectable on the single-molecule level for fluorogens attached to a surface. Solution kinetic analysis shows Michaelis-Menten rate dependence upon both NADH and the fluorogen concentrations as expected. The generation of low concentrations of single-molecule emitters by enzymatic turnovers is used to extract subdiffraction information about localizations of both fluorophores and nitroreductase enzymes in cells. Enzymatic Turnover Activated Localization Microscopy (ETALM) is a complementary mechanism to photoactivation and blinking for controlling the emission of single molecules to image beyond the diffraction limit.
View details for DOI 10.1039/c2sc21074f
View details for Web of Science ID 000311971500023
View details for PubMedCentralID PMC3722058
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Measuring the 3D Position and Orientation of Single Molecules Simultaneously and Accurately with the Double Helix Microscope
IEEE. 2013
View details for Web of Science ID 000355262503100
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Enzymatic activation of nitro-aryl fluorogens in live bacterial cells for enzymatic turnover-activated localization microscopy†
Chemical science
2013; 42: 220-225
Abstract
Many modern super-resolution imaging methods based on single-molecule fluorescence require the conversion of a dark fluorogen into a bright emitter to control emitter concentration. We have synthesized and characterized a nitro-aryl fluorogen which can be converted by a nitroreductase enzyme into a bright push-pull red-emitting fluorophore. Synthesis of model compounds and optical spectroscopy identify a hydroxyl-amino derivative as the product fluorophore, which is bright and detectable on the single-molecule level for fluorogens attached to a surface. Solution kinetic analysis shows Michaelis-Menten rate dependence upon both NADH and the fluorogen concentrations as expected. The generation of low concentrations of single-molecule emitters by enzymatic turnovers is used to extract subdiffraction information about localizations of both fluorophores and nitroreductase enzymes in cells. Enzymatic Turnover Activated Localization Microscopy (ETALM) is a complementary mechanism to photoactivation and blinking for controlling the emission of single molecules to image beyond the diffraction limit.
View details for PubMedID 23894694
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The double-helix point spread function enables precise and accurate measurement of 3D single-molecule localization and orientation
Conference on Single Molecule Spectroscopy and Superresolution Imaging VI
SPIE-INT SOC OPTICAL ENGINEERING. 2013
Abstract
Single-molecule-based super-resolution fluorescence microscopy has recently been developed to surpass the diffraction limit by roughly an order of magnitude. These methods depend on the ability to precisely and accurately measure the position of a single-molecule emitter, typically by fitting its emission pattern to a symmetric estimator (e.g. centroid or 2D Gaussian). However, single-molecule emission patterns are not isotropic, and depend highly on the orientation of the molecule's transition dipole moment, as well as its z-position. Failure to account for this fact can result in localization errors on the order of tens of nm for in-focus images, and ~50-200 nm for molecules at modest defocus. The latter range becomes especially important for three-dimensional (3D) single-molecule super-resolution techniques, which typically employ depths-of-field of up to ~2 μm. To address this issue we report the simultaneous measurement of precise and accurate 3D single-molecule position and 3D dipole orientation using the Double-Helix Point Spread Function (DH-PSF) microscope. We are thus able to significantly improve dipole-induced position errors, reducing standard deviations in lateral localization from ~2x worse than photon-limited precision (48 nm vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation we are able to improve from a lateral standard deviation of 116 nm (~4x worse than the precision, 28 nm) to 34 nm (within 6 nm).
View details for DOI 10.1117/12.2001671
View details for Web of Science ID 000321741600013
View details for PubMedCentralID PMC4013112
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Probing Single Biomolecules in Solution Using the Anti-Brownian Electrokinetic (ABEL) Trap
ACCOUNTS OF CHEMICAL RESEARCH
2012; 45 (11): 1955-1964
Abstract
Single-molecule fluorescence measurements allow researchers to study asynchronous dynamics and expose molecule-to-molecule structural and behavioral diversity, which contributes to the understanding of biological macromolecules. To provide measurements that are most consistent with the native environment of biomolecules, researchers would like to conduct these measurements in the solution phase if possible. However, diffusion typically limits the observation time to approximately 1 ms in many solution-phase single-molecule assays. Although surface immobilization is widely used to address this problem, this process can perturb the system being studied and contribute to the observed heterogeneity. Combining the technical capabilities of high-sensitivity single-molecule fluorescence microscopy, real-time feedback control and electrokinetic flow in a microfluidic chamber, we have developed a device called the anti-Brownian electrokinetic (ABEL) trap to significantly prolong the observation time of single biomolecules in solution. We have applied the ABEL trap method to explore the photodynamics and enzymatic properties of a variety of biomolecules in aqueous solution and present four examples: the photosynthetic antenna allophycocyanin, the chaperonin enzyme TRiC, a G protein-coupled receptor protein, and the blue nitrite reductase redox enzyme. These examples illustrate the breadth and depth of information which we can extract in studies of single biomolecules with the ABEL trap. When confined in the ABEL trap, the photosynthetic antenna protein allophycocyanin exhibits rich dynamics both in its emission brightness and its excited state lifetime. As each molecule discontinuously converts from one emission/lifetime level to another in a primarily correlated way, it undergoes a series of state changes. We studied the ATP binding stoichiometry of the multi-subunit chaperonin enzyme TRiC in the ABEL trap by counting the number of hydrolyzed Cy3-ATP using stepwise photobleaching. Unlike ensemble measurements, the observed ATP number distributions depart from the standard cooperativity models. Single copies of detergent-stabilized G protein-coupled receptor proteins labeled with a reporter fluorophore also show discontinuous changes in emission brightness and lifetime, but the various states visited by the single molecules are broadly distributed. As an agonist binds, the distributions shift slightly toward a more rigid conformation of the protein. By recording the emission of a reporter fluorophore which is quenched by reduction of a nearby type I Cu center, we probed the enzymatic cycle of the redox enzyme nitrate reductase. We determined the rate constants of a model of the underlying kinetics through an analysis of the dwell times of the high/low intensity levels of the fluorophore versus nitrite concentration.
View details for DOI 10.1021/ar2003041
View details for PubMedID 22616716
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Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species
SCIENTIFIC REPORTS
2012; 2
Abstract
The identities of toxic aggregate species in Huntington's disease pathogenesis remain ambiguous. While polyQ-expanded huntingtin (Htt) is known to accumulate in compact inclusion bodies inside neurons, this is widely thought to be a protective coping response that sequesters misfolded conformations or aggregated states of the mutated protein. To define the spatial distributions of fluorescently-labeled Htt-exon1 species in the cell model PC12m, we employed highly sensitive single-molecule super-resolution fluorescence imaging. In addition to inclusion bodies and the diffuse pool of monomers and oligomers, fibrillar aggregates -100 nm in diameter and up to -1-2 µm in length were observed for pathogenic polyQ tracts (46 and 97 repeats) after targeted photo-bleaching of the inclusion bodies. These short structures bear a striking resemblance to fibers described in vitro. Definition of the diverse Htt structures in cells will provide an avenue to link the impact of therapeutic agents to aggregate populations and morphologies.
View details for DOI 10.1038/srep00895
View details for Web of Science ID 000311891000001
View details for PubMedID 23193437
View details for PubMedCentralID PMC3508451
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Simultaneous, accurate measurement of the 3D position and orientation of single molecules
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (47): 19087-19092
Abstract
Recently, single molecule-based superresolution fluorescence microscopy has surpassed the diffraction limit to improve resolution to the order of 20 nm or better. These methods typically use image fitting that assumes an isotropic emission pattern from the single emitters as well as control of the emitter concentration. However, anisotropic single-molecule emission patterns arise from the transition dipole when it is rotationally immobile, depending highly on the molecule's 3D orientation and z position. Failure to account for this fact can lead to significant lateral (x, y) mislocalizations (up to ∼50-200 nm). This systematic error can cause distortions in the reconstructed images, which can translate into degraded resolution. Using parameters uniquely inherent in the double-lobed nature of the Double-Helix Point Spread Function, we account for such mislocalizations and simultaneously measure 3D molecular orientation and 3D position. Mislocalizations during an axial scan of a single molecule manifest themselves as an apparent lateral shift in its position, which causes the standard deviation (SD) of its lateral position to appear larger than the SD expected from photon shot noise. By correcting each localization based on an estimated orientation, we are able to improve SDs in lateral localization from ∼2× worse than photon-limited precision (48 vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation over different depths, we are able to improve from a lateral SD of 116 (∼4× worse than the photon-limited precision; 28 nm) to 34 nm (within 6 nm of the photon limit).
View details for DOI 10.1073/pnas.1216687109
View details for Web of Science ID 000311997200022
View details for PubMedID 23129640
View details for PubMedCentralID PMC3511094
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Fluorescence correlation spectroscopy at high concentrations using gold bowtie nanoantennas
CHEMICAL PHYSICS
2012; 406: 3-8
View details for DOI 10.1016/j.chemphys.2012.04.011
View details for Web of Science ID 000310569800002
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Enzymatic Activation of Nitro-Aryl Fluorogens in Live Cells for Turnover Activated Localization Microscopy
26th Annual Symposium of the Protein-Society
WILEY-BLACKWELL. 2012: 127–127
View details for Web of Science ID 000307019800188
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Fluorescent Saxitoxins for Live Cell Imaging of Single Voltage-Gated Sodium Ion Channels beyond the Optical Diffraction Limit
CHEMISTRY & BIOLOGY
2012; 19 (7): 902-912
Abstract
A desire to better understand the role of voltage-gated sodium channels (Na(V)s) in signal conduction and their dysregulation in specific disease states motivates the development of high precision tools for their study. Nature has evolved a collection of small molecule agents, including the shellfish poison (+)-saxitoxin, that bind to the extracellular pore of select Na(V) isoforms. As described in this report, de novo chemical synthesis has enabled the preparation of fluorescently labeled derivatives of (+)-saxitoxin, STX-Cy5, and STX-DCDHF, which display reversible binding to Na(V)s in live cells. Electrophysiology and confocal fluorescence microscopy studies confirm that these STX-based dyes function as potent and selective Na(V) labels. The utility of these probes is underscored in single-molecule and super-resolution imaging experiments, which reveal Na(V) distributions well beyond the optical diffraction limit in subcellular features such as neuritic spines and filopodia.
View details for DOI 10.1016/j.chembiol.2012.05.021
View details for Web of Science ID 000307261100016
View details for PubMedID 22840778
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STED Microscopy with Optimized Labeling Density Reveals 9-Fold Arrangement of a Centriole Protein
BIOPHYSICAL JOURNAL
2012; 102 (12): 2926-2935
Abstract
Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where super-resolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with a resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in nine clusters spaced around a ring of ∼300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size, and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.
View details for DOI 10.1016/j.bpj.2012.05.015
View details for Web of Science ID 000305546500027
View details for PubMedID 22735543
View details for PubMedCentralID PMC3379620
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Analytical Tools To Distinguish the Effects of Localization Error, Confinement, and Medium Elasticity on the Velocity Autocorrelation Function
BIOPHYSICAL JOURNAL
2012; 102 (11): 2443-2450
Abstract
Single particle tracking is a powerful technique for investigating the dynamic behavior of biological molecules. However, many of the analytical tools are prone to generate results that can lead to mistaken interpretations of the underlying transport process. Here, we explore the effects of localization error and confinement on the velocity autocorrelation function, Cυ. We show that calculation of Cυ across a range of discretizations can distinguish the effects of localization error, confinement, and medium elasticity. Thus, under certain regimes, Cυ can be used as a diagnostic tool to identify the underlying mechanism of anomalous diffusion. Finally, we apply our analysis to experimental data sets of chromosomal loci and RNA-protein particles in Escherichia coli.
View details for DOI 10.1016/j.bpj.2012.03.062
View details for Web of Science ID 000305003100006
View details for PubMedID 22713559
View details for PubMedCentralID PMC3368140
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Microscopy beyond the diffraction limit using actively controlled single molecules
JOURNAL OF MICROSCOPY
2012; 246 (3): 213-220
Abstract
In this short review, the general principles are described for obtaining microscopic images with resolution beyond the optical diffraction limit with single molecules. Although it has been known for several decades that single-molecule emitters can blink or turn on and off, in recent work the addition of on/off control of molecular emission to maintain concentrations at very low levels in each imaging frame combined with sequential imaging of sparse subsets has enabled the reconstruction of images with resolution far below the optical diffraction limit. Single-molecule active control microscopy provides a powerful window into information about nanoscale structures that was previously unavailable.
View details for DOI 10.1111/j.1365-2818.2012.03600.x
View details for Web of Science ID 000303993700001
View details for PubMedID 22582796
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The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision
APPLIED PHYSICS LETTERS
2012; 100 (15)
Abstract
The double-helix point spread function microscope encodes the axial (z) position information of single emitters in wide-field (x,y) images, thus enabling localization in three dimensions (3D) inside extended volumes. We experimentally determine the statistical localization precision σ of this approach using single emitters in a cell under typical background conditions, demonstrating σ < 20 nm laterally and <30 nm axially for N ≈ 1180 photons per localization. Combined with light-induced blinking of single-molecule labels, we present proof-of-concept imaging beyond the optical diffraction limit of microtubule network structures in fixed mammalian cells over a large axial range in three dimensions.
View details for DOI 10.1063/1.3700446
View details for Web of Science ID 000303128000085
View details for PubMedCentralID PMC3338582
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Watching dynamical processes for single biomolecules in solution
AMER CHEMICAL SOC. 2012
View details for Web of Science ID 000324503203259
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Three-Dimensional Super-Resolution Imaging of the Midplane Protein FtsZ in Live Caulobacter crescentus Cells Using Astigmatism
CHEMPHYSCHEM
2012; 13 (4): 1007-1012
Abstract
Single-molecule super-resolution imaging provides a non-invasive method for nanometer-scale imaging and is ideally suited to investigations of quasi-static structures within live cells. Here, we extend the ability to image subcellular features within bacteria cells to three dimensions based on the introduction of a cylindrical lens in the imaging pathway. We investigate the midplane protein FtsZ in Caulobacter crescentus with super-resolution imaging based on fluorescent-protein photoswitching and the natural polymerization/depolymerization dynamics of FtsZ associated with the Z-ring. We quantify these dynamics and determine the FtsZ depolymerization time to be <100 ms. We image the Z-ring in live and fixed C. crescentus cells at different stages of the cell cycle and find that the FtsZ superstructure is dynamic with the cell cycle, forming an open shape during the stalked stage and a dense focus during the pre-divisional stage.
View details for DOI 10.1002/cphc.201100686
View details for Web of Science ID 000301537300020
View details for PubMedID 22262316
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Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae
PLOS ONE
2012; 7 (2)
Abstract
Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe here a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches.
View details for DOI 10.1371/journal.pone.0031912
View details for Web of Science ID 000302796200110
View details for PubMedID 22359641
View details for PubMedCentralID PMC3281097
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Sensing Cooperativity in ATP Hydrolysis for Single Multisubunit Enzymes in Solution
CELL PRESS. 2012: 178A
View details for DOI 10.1016/j.bpj.2011.11.967
View details for Web of Science ID 000321561201188
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Single-Molecule Studies of Trapped Biomolecules in Solution with the ABEL Trap
CELL PRESS. 2012: 4A
View details for DOI 10.1016/j.bpj.2011.11.040
View details for Web of Science ID 000321561200023
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A Selenium Analogue of Firefly D-Luciferin with Red-Shifted Bioluminescence Emission
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
2012; 51 (14): 3350-3353
Abstract
A selenium analogue of amino-D-luciferin, aminoseleno-D-luciferin, is synthesized and shown to be a competent substrate for the firefly luciferase enzyme. It has a red-shifted bioluminescence emission maximum at 600 nm and is suitable for bioluminescence imaging studies in living subjects.
View details for DOI 10.1002/anie.201105653
View details for Web of Science ID 000302059400009
View details for PubMedID 22344705
View details for PubMedCentralID PMC3494413
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Two-color, 3D super-resolution imaging of bacterial protein ultrastructures with the double-helix point-spread function microscope.
AMER SOC CELL BIOLOGY. 2012
View details for Web of Science ID 000209348605067
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Enzymatic activation of nitro-aryl fluorogens in live bacterial cells for Enzymatic Turnover Activated Localization Microscopy beyond the diffraction limit.
AMER SOC CELL BIOLOGY. 2012
View details for Web of Science ID 000209348605069
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Resolving structural features in biological and biomedical imaging with STED super-resolution microscopy.
AMER SOC CELL BIOLOGY. 2012
View details for Web of Science ID 000209348605068
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STED super-resolution microscopy of multiciliated respiratory epithelial cells reveals structural organization of centriole and cilia components.
AMER SOC CELL BIOLOGY. 2012
View details for Web of Science ID 000209348601292
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Spectrally Resolved Anti-Brownian ELectrokinetic (ABEL) Trapping of Single Peridinin-Chlorophyll-Proteins in Solution
Conference on Biophotonics - Photonic Solutions for Better Health Care III
SPIE-INT SOC OPTICAL ENGINEERING. 2012
View details for DOI 10.1117/12.970546
View details for Web of Science ID 000305701200091
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Extending Microscopic Resolution with Single-Molecule Imaging and Active Control
ANNUAL REVIEW OF BIOPHYSICS, VOL 41
2012; 41: 321-342
Abstract
Superresolution imaging of biological structures provides information beyond the optical diffraction limit. One class of superresolution techniques uses the power of single fluorescent molecules as nanoscale emitters of light combined with emission control, variously described by the acronyms PALM/FPALM/STORM and many others. Even though the acronyms differ and refer mainly to different active-control mechanisms, the underlying fundamental principles behind these "pointillist" superresolution imaging techniques are the same. Circumventing the diffraction limit requires two key steps. The first step (superlocalization) is the detection and localization of spatially separated single molecules. The second step actively controls the emitting molecules to ensure a very low concentration of single emitters such that they do not overlap in any one imaging frame. The final image is reconstructed from time-sequential imaging and superlocalization of the single emitting labels decorating the structure of interest. The statistical, imaging, and active-control strategies for achieving superresolution imaging with single molecules are reviewed.
View details for DOI 10.1146/annurev-biophys-050511-102250
View details for Web of Science ID 000307955100016
View details for PubMedID 22577822
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Anti-Brownian ELectrokinetic (ABEL) Trapping of Single beta(2)-Adrenergic Receptors in the Absence and Presence of Agonist
Conference on Single Molecule Spectroscopy and Super-Resolution Imaging V
SPIE-INT SOC OPTICAL ENGINEERING. 2012
View details for DOI 10.1117/12.910018
View details for Web of Science ID 000305624900003
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Conformational dynamics of single G protein-coupled receptors in solution.
journal of physical chemistry. B
2011; 115 (45): 13328-13338
Abstract
G protein-coupled receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single β(2)-adrenergic receptors (β(2)ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently labeled β(2)AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single β(2)ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that the binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with a stretching exponent β ~ 0.5, suggesting protein dynamics over a range of time scales.
View details for DOI 10.1021/jp204843r
View details for PubMedID 21928818
View details for PubMedCentralID PMC3213290
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Conformational Dynamics of Single G Protein-Coupled Receptors in Solution
JOURNAL OF PHYSICAL CHEMISTRY B
2011; 115 (45): 13328-13338
Abstract
G protein-coupled receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single β(2)-adrenergic receptors (β(2)ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently labeled β(2)AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single β(2)ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that the binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with a stretching exponent β ~ 0.5, suggesting protein dynamics over a range of time scales.
View details for DOI 10.1021/jp204843r
View details for Web of Science ID 000296686000025
View details for PubMedCentralID PMC3213290
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Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (46): E1102-E1110
Abstract
Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40 nm in 3D) over a large field of view (12 12 μm).
View details for DOI 10.1073/pnas.1114444108
View details for PubMedID 22031697
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Modifications of DCDHF single molecule fluorophores to impart water solubility (vol 48, pg 3471, 2007)
TETRAHEDRON LETTERS
2011; 52 (43): 5711-5711
View details for DOI 10.1016/j.tetlet.2011.08.137
View details for Web of Science ID 000295742400041
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Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (42): 17269-17274
Abstract
Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism.
View details for DOI 10.1073/pnas.1113572108
View details for Web of Science ID 000295975300016
View details for PubMedID 21969548
View details for PubMedCentralID PMC3198337
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Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (41): 16962-16967
Abstract
In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperonin-bound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.
View details for DOI 10.1073/pnas.1112244108
View details for PubMedID 21896715
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Live-cell single-molecule and super-resolution imaging in bacteria
AMER CHEMICAL SOC. 2011
View details for Web of Science ID 000299378306327
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Single fluorescent molecules as nano-illuminators for biological structure and function
AMER CHEMICAL SOC. 2011
View details for Web of Science ID 000299378305771
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Analyzing 3D stochastic dynamics in live cells via new single particle tracking methods
AMER CHEMICAL SOC. 2011
View details for Web of Science ID 000299378305776
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Single-molecule studies of biomolecular dynamics in solution and fluorescence enhancements by metallic nanoantennas
AMER CHEMICAL SOC. 2011
View details for Web of Science ID 000299378306330
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An Adaptive Anti-Brownian Electrokinetic Trap with Real-Time Information on Single-Molecule Diffusivity and Mobility
ACS NANO
2011; 5 (7): 5792-5799
Abstract
We present the design and implementation of an adaptive Anti-Brownian ELectrokinetic (ABEL) trap capable of extracting estimates of the diffusion coefficient and mobility of single trapped fluorescent nanoscale objects such as biomolecules in solution. The system features rapid acousto-optic scanning of a confocal excitation spot on a 2D square lattice to encode position information on the arrival time of each detected photon, and Kalman filter-based signal processing unit for refined position estimation. We demonstrate stable trapping of multisubunit proteins (D ≈ 22 μm(2)/s) with a count rate of 6 kHz for as long as 15 s and small single-stranded DNA molecules (D ≈ 118 μm(2)/s) at a 15 kHz count rate for seconds. Moreover, we demonstrate real-time measurement of diffusion coefficient and electrokinetic mobility of trapped objects, using adaptive tuning of the Kalman filter parameters.
View details for DOI 10.1021/nn2014968
View details for Web of Science ID 000293035200057
View details for PubMedID 21612271
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Super-Resolution Imaging of the Nucleoid-Associated Protein HU in Caulobacter crescentus
BIOPHYSICAL JOURNAL
2011; 100 (7): L31-L33
Abstract
Little is known about the structure and function of most nucleoid-associated proteins (NAPs) in bacteria. One reason for this is that the distribution and structure of the proteins is obfuscated by the diffraction limit in standard wide-field and confocal fluorescence imaging. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. In this study, we investigate the distribution of HU in Caulobacter crescentus using a combination of super-resolution fluorescence imaging and spatial point statistics. By simply increasing the laser power, single molecules of the fluorescent protein fusion HU2-eYFP can be made to blink on and off to achieve super-resolution imaging with a single excitation source. Through quantification by Ripley's K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in predivisional cells. The methods presented in this letter should be of broad applicability in the future study of prokaryotic NAPs.
View details for DOI 10.1016/j.bpj.2011.02.022
View details for Web of Science ID 000289494200001
View details for PubMedID 21463569
View details for PubMedCentralID PMC3072666
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3D tracking of single mRNA particles in S. cerevisiae using a double-helix point spread function
AMER CHEMICAL SOC. 2011
View details for Web of Science ID 000291982801899
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STED Super-resolution Microscopy in Drosophila Tissue and in Mammalian Cells.
Proceedings of SPIE--the International Society for Optical Engineering
2011; 7910
Abstract
Far-field super-resolution microscopy is a rapidly emerging method that is opening up opportunities for biological imaging beyond the optical diffraction limit. We have implemented a Stimulated Emission Depletion (STED) microscope to image single dye, cell, and tissue samples with 50-80 nm resolution. First, we compare the STED performance imaging single molecules of several common dyes and report a novel STED dye. Then we apply STED to image planar cell polarity protein complexes in intact fixed Drosophila tissue for the first time. Finally, we present a preliminary study of the centrosomal protein Cep164 in mammalian cells. Our images suggest that Cep164 is arranged in a nine-fold symmetric pattern around the centriole, consistent with findings suggested by cryoelectron tomography. Our work demonstrates that STED microscopy can be used for superresolution imaging in intact tissue and provides ultrastructural information in biological samples as an alternative to immuno-electron microscopy.
View details for PubMedID 23447411
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Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects
OPTICS LETTERS
2011; 36 (2): 202-204
Abstract
We describe the corkscrew point spread function (PSF), which can localize objects in three dimensions throughout a 3.2 μm depth of field with nanometer precision. The corkscrew PSF rotates as a function of the axial (z) position of an emitter. Fisher information calculations show that the corkscrew PSF can achieve nanometer localization precision with limited numbers of photons. We demonstrate three-dimensional super-resolution microscopy with the corkscrew PSF by imaging beads on the surface of a triangular polydimethylsiloxane (PDMS) grating. With 99,000 photons detected, the corkscrew PSF achieves a localization precision of 2.7 nm in x, 2.1 nm in y, and 5.7 nm in z.
View details for Web of Science ID 000286188100036
View details for PubMedID 21263500
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Exploring protein superstructures and dynamics in live bacterial cells using single-molecule and superresolution imaging.
Methods in molecular biology (Clifton, N.J.)
2011; 783: 139-158
Abstract
Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the optical diffraction limit, and enables superresolution reconstruction of structures beyond the diffraction limit. This work summarizes how single-molecule and superresolution imaging can be applied to the study of protein dynamics and superstructures in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.
View details for DOI 10.1007/978-1-61779-282-3_8
View details for PubMedID 21909887
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HIGH-RESOLUTION SINGLE-MOLECULE SPECTROSCOPY IN CONDENSED MATTER
PHYSICS AND CHEMISTRY AT LOW TEMPERATURES
2011: 381-417
View details for Web of Science ID 000297316000013
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STED Super-resolution Microscopy in Drosophila Tissue and in Mammalian Cells
Conference on Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications III
SPIE-INT SOC OPTICAL ENGINEERING. 2011
View details for DOI 10.1117/12.881221
View details for Web of Science ID 000297729300032
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Live-cell single-molecule and superresolution imaging of proteins in bacteria
Conference on Single Molecule Spectroscopy and Imaging IV
SPIE-INT SOC OPTICAL ENGINEERING. 2011
View details for DOI 10.1117/12.873809
View details for Web of Science ID 000297674400014
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Azido Push-Pull Fluorogens Photoactivate to Produce Bright Fluorescent Labels
JOURNAL OF PHYSICAL CHEMISTRY B
2010; 114 (45): 14157-14167
Abstract
Dark azido push-pull chromophores have the ability to be photoactivated to produce bright fluorescent labels suitable for single-molecule imaging. Upon illumination, the aryl azide functionality in the fluorogens participates in a photochemical conversion to an aryl amine, thus restoring charge-transfer absorption and fluorescence. Previously, we reported that one compound, DCDHF-V-P-azide, was photoactivatable. Here, we demonstrate that the azide-to-amine photoactivation process is generally applicable to a variety of push-pull chromophores, and we characterize the photophysical parameters including photoconversion quantum yield, photostability, and turn-on ratio. Azido push-pull fluorogens provide a new class of photoactivatable single-molecule probes for fluorescent labeling and super-resolution microscopy. Lastly, we demonstrate that photoactivated push-pull dyes can insert into bonds of nearby biomolecules, simultaneously forming a covalent bond and becoming fluorescent (fluorogenic photoaffinity labeling).
View details for DOI 10.1021/jp907080r
View details for PubMedID 19860443
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Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2010; 132 (43): 15099-15101
Abstract
Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.
View details for DOI 10.1021/ja1044192
View details for PubMedID 20936809
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Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (42): 17864-17871
Abstract
Optical imaging of single biomolecules and complexes in living cells provides a useful window into cellular processes. However, the three-dimensional dynamics of most important biomolecules in living cells remains essentially uncharacterized. The precise subcellular localization of mRNA-protein complexes plays a critical role in the spatial and temporal control of gene expression, and a full understanding of the control of gene expression requires precise characterization of mRNA transport dynamics beyond the optical diffraction limit. In this paper, we describe three-dimensional tracking of single mRNA particles with 25-nm precision in the x and y dimensions and 50-nm precision in the z dimension in live budding yeast cells using a microscope with a double-helix point spread function. Two statistical methods to detect intermittently confined and directed transport were used to quantify the three-dimensional trajectories of mRNA for the first time, using ARG3 mRNA as a model. Measurements and analysis show that the dynamics of ARG3 mRNA molecules are mostly diffusive, although periods of non-Brownian confinement and directed transport are observed. The quantitative methods detailed in this paper can be broadly applied to the study of mRNA localization and the dynamics of diverse other biomolecules in a wide variety of cell types.
View details for DOI 10.1073/pnas.1012868107
View details for Web of Science ID 000283184800008
View details for PubMedID 20921361
View details for PubMedCentralID PMC2964242
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Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane
APPLIED PHYSICS LETTERS
2010; 97 (16)
Abstract
Wide-field microscopy with a double-helix point spread function (DH-PSF) provides three-dimensional (3D) position information beyond the optical diffraction limit. We compare the theoretical localization precision for an unbiased estimator of the DH-PSF to that for 3D localization by astigmatic and biplane imaging using Fisher information analysis including pixelation and varying levels of background. The DH-PSF results in almost constant localization precision in all three dimensions for a 2 μm thick depth of field while astigmatism and biplane improve the axial localization precision over smaller axial ranges. For high signal-to-background ratio, the DH-PSF on average achieves better localization precision.
View details for DOI 10.1063/1.3499652
View details for Web of Science ID 000283502100003
View details for PubMedCentralID PMC2980550
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Action of the Chaperonin GroEL/ES on a Non-native Substrate Observed with Single-Molecule FRET
JOURNAL OF MOLECULAR BIOLOGY
2010; 401 (4): 553-563
Abstract
The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Förster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.
View details for DOI 10.1016/j.jmb.2010.06.050
View details for Web of Science ID 000281262400001
View details for PubMedID 20600107
View details for PubMedCentralID PMC2927214
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Excited state dynamics of solution-phase photosynthetic antenna proteins studied one at a time
AMER CHEMICAL SOC. 2010
View details for Web of Science ID 000208164706013
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Molecules and methods for superresolution imaging in living cells
AMER CHEMICAL SOC. 2010
View details for Web of Science ID 000208164700294
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Single molecules as nanoscopic probes of 3D structure and metallic nanoantennas
AMER CHEMICAL SOC. 2010
View details for Web of Science ID 000208164701828
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A spindle-like apparatus guides bacterial chromosome segregation
NATURE CELL BIOLOGY
2010; 12 (8): 791-U46
Abstract
Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. We show that the C. crescentus ATPase ParA forms linear polymers in vitro and assembles into a narrow linear structure in vivo. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We propose that this ParB-stimulated ParA depolymerization activity moves the centromere to the opposite cell pole through a burnt bridge Brownian ratchet mechanism. Finally, we identify the pole-specific TipN protein as a new component of the Par system that is required to maintain the directionality of DNA transfer towards the new cell pole. Our results elucidate a bacterial chromosome segregation mechanism that features basic operating principles similar to eukaryotic mitotic machines, including a multivalent protein complex at the centromere that stimulates the dynamic disassembly of polymers to move chromosomes into daughter compartments.
View details for DOI 10.1038/ncb2083
View details for Web of Science ID 000280561600011
View details for PubMedID 20657594
View details for PubMedCentralID PMC3205914
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In vivo Three-Dimensional Superresolution Fluorescence Tracking using a Double-Helix Point Spread Function.
Proceedings - Society of Photo-Optical Instrumentation Engineers
2010; 7571: 75710Z
Abstract
The point spread function (PSF) of a widefield fluorescence microscope is not suitable for three-dimensional super-resolution imaging. We characterize the localization precision of a unique method for 3D superresolution imaging featuring a double-helix point spread function (DH-PSF). The DH-PSF is designed to have two lobes that rotate about their midpoint in any transverse plane as a function of the axial position of the emitter. In effect, the PSF appears as a double helix in three dimensions. By comparing the Cramer-Rao bound of the DH-PSF with the standard PSF as a function of the axial position, we show that the DH-PSF has a higher and more uniform localization precision than the standard PSF throughout a 2 μm depth of field. Comparisons between the DH-PSF and other methods for 3D super-resolution are briefly discussed. We also illustrate the applicability of the DH-PSF for imaging weak emitters in biological systems by tracking the movement of quantum dots in glycerol and in live cells.
View details for DOI 10.1117/12.842608
View details for PubMedID 20563317
View details for PubMedCentralID PMC2886306
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Optimal strategy for trapping single fluorescent molecules in solution using the ABEL trap
APPLIED PHYSICS B-LASERS AND OPTICS
2010; 99 (1-2): 23-30
Abstract
Trapping of 10-nm-sized single fluorescent bio-molecules in solution has been achieved using high-speed position sensing and electrokinetic feedback forces in the Anti-Brownian ELectrokinetic (ABEL) trap. The high diffusion coefficient of small objects in solution requires very fast, real-time sensing of position, and this has been previously achieved using a simple rotating beam, but improved strategies are needed for the smallest objects, such as single nanometer-sized fluorescent molecules. At the same time, single molecules are limited in photon emission rate and total number of photons, so each emitted photon must be used as efficiently as possible. We describe a new controller design for the ABEL trap which features fast, knight's tour scanning of an excitation beam on a 2D square lattice and a Kalman filter-based estimator for optimal position sensing. This strategy leads directly to a maximum-likelihood-based method to extract the diffusion coefficient of the object held in the trap. The effectiveness of the algorithms are demonstrated and compared to the simple rotating beam design through Monte Carlo simulations. Our new approach yields tighter trapping and a much improved ability to extract diffusion coefficients.
View details for DOI 10.1007/s00340-009-3843-y
View details for Web of Science ID 000275892200005
View details for PubMedCentralID PMC2850131
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Optimal strategy for trapping single fluorescent molecules in solution using the ABEL trap.
Applied physics. B, Lasers and optics
2010; 99 (1-2): 23-30
Abstract
Trapping of 10-nm-sized single fluorescent bio-molecules in solution has been achieved using high-speed position sensing and electrokinetic feedback forces in the Anti-Brownian ELectrokinetic (ABEL) trap. The high diffusion coefficient of small objects in solution requires very fast, real-time sensing of position, and this has been previously achieved using a simple rotating beam, but improved strategies are needed for the smallest objects, such as single nanometer-sized fluorescent molecules. At the same time, single molecules are limited in photon emission rate and total number of photons, so each emitted photon must be used as efficiently as possible. We describe a new controller design for the ABEL trap which features fast, knight's tour scanning of an excitation beam on a 2D square lattice and a Kalman filter-based estimator for optimal position sensing. This strategy leads directly to a maximum-likelihood-based method to extract the diffusion coefficient of the object held in the trap. The effectiveness of the algorithms are demonstrated and compared to the simple rotating beam design through Monte Carlo simulations. Our new approach yields tighter trapping and a much improved ability to extract diffusion coefficients.
View details for DOI 10.1007/s00340-009-3843-y
View details for PubMedID 20383275
View details for PubMedCentralID PMC2850131
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Photoactivatable azido push-pull fluorophores for single-molecule imaging in and out of cells
AMER CHEMICAL SOC. 2010
View details for Web of Science ID 000208189304193
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Watching conformational and photo-dynamics of single fluorescent proteins in solution
AMER CHEMICAL SOC. 2010
View details for Web of Science ID 000208189304187
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Single-Molecule Spectroscopy and Imaging of Biomolecules in Living Cells
ANALYTICAL CHEMISTRY
2010; 82 (6): 2192-2203
Abstract
The number of reports per year on single-molecule imaging experiments has grown roughly exponentially since the first successful efforts to optically detect a single molecule were completed over two decades ago. Single-molecule spectroscopy has developed into a field that includes a wealth of experiments at room temperature and inside living cells. The fast growth of single-molecule biophysics has resulted from its benefits in probing heterogeneous populations, one molecule at a time, as well as from advances in microscopes and detectors. This Perspective summarizes the field of live-cell imaging of single biomolecules.
View details for DOI 10.1021/ac9024889
View details for Web of Science ID 000275379300007
View details for PubMedID 20163145
View details for PubMedCentralID PMC2838489
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Single-Molecule and Superresolution Imaging in Live Bacteria Cells
COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY
2010; 2 (3)
Abstract
Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.
View details for DOI 10.1101/cshperspect.a000448
View details for Web of Science ID 000279881700002
View details for PubMedID 20300204
View details for PubMedCentralID PMC2829965
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Watching conformational- and photodynamics of single fluorescent proteins in solution
NATURE CHEMISTRY
2010; 2 (3): 179-186
Abstract
Observing the dynamics of single biomolecules over prolonged time periods is difficult to achieve without significantly altering the molecule through immobilization. It can, however, be accomplished using the Anti-Brownian ELectrokinetic (ABEL) Trap, which allows extended investigation of solution-phase biomolecules - without immobilization -through real-time electrokinetic feedback. Here we apply the ABEL trap to study an important photosynthetic antenna protein, Allophycocyanin (APC). The technique allows the observation of single molecules of solution-phase APC for more than one second. We observe a complex relationship between fluorescence intensity and lifetime that cannot be explained by simple static kinetic models. Light-induced conformational changes are shown to occur and evidence is obtained for fluctuations in the spontaneous emission lifetime, which is typically assumed to be constant. Our methods provide a new window into the dynamics of fluorescent proteins and the observations are relevant for the interpretation of in vivo single-molecule imaging experiments, bacterial photosynthetic regulation, and biomaterials for solar energy harvesting.
View details for DOI 10.1038/NCHEM.545
View details for Web of Science ID 000274648800012
View details for PubMedID 20625479
View details for PubMedCentralID PMC2899709
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In vivo Three-Dimensional Superresolution Fluorescence Tracking using a Double-Helix Point Spread Function
Conference on Single Molecule Spectroscopy and Imaging III
SPIE-INT SOC OPTICAL ENGINEERING. 2010
Abstract
The point spread function (PSF) of a widefield fluorescence microscope is not suitable for three-dimensional super-resolution imaging. We characterize the localization precision of a unique method for 3D superresolution imaging featuring a double-helix point spread function (DH-PSF). The DH-PSF is designed to have two lobes that rotate about their midpoint in any transverse plane as a function of the axial position of the emitter. In effect, the PSF appears as a double helix in three dimensions. By comparing the Cramer-Rao bound of the DH-PSF with the standard PSF as a function of the axial position, we show that the DH-PSF has a higher and more uniform localization precision than the standard PSF throughout a 2 μm depth of field. Comparisons between the DH-PSF and other methods for 3D super-resolution are briefly discussed. We also illustrate the applicability of the DH-PSF for imaging weak emitters in biological systems by tracking the movement of quantum dots in glycerol and in live cells.
View details for DOI 10.1117/12.842608
View details for Web of Science ID 000284353600015
View details for PubMedCentralID PMC2886306
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Watching Conformational and Photo-Dynamics of Single Fluorescent Proteins in Solution
CELL PRESS. 2010: 186A
View details for DOI 10.1016/j.bpj.2009.12.994
View details for Web of Science ID 000208762001431
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Counting Hydrolyzed ATP On Single Tric Nanomachines in Solution
CELL PRESS. 2010: 223A
View details for DOI 10.1016/j.bpj.2009.12.1203
View details for Web of Science ID 000208762002110
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Photoactivatable Azido Push-Pull Fluorophores for Single-Molecule Imaging in and out of Cells
CELL PRESS. 2010: 203A
View details for DOI 10.1016/j.bpj.2009.12.1082
View details for Web of Science ID 000208762002011
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Single-Molecule Approaches for Superresolution Imaging, Trapping, and Nanophotonics
Conference on Lasers and Electro-Optics (CLEO)/Quantum Electronics and Laser Science Conference (QELS)
IEEE. 2010
View details for Web of Science ID 000290513601017
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Single-Molecule Optical Spectroscopy and Imaging: From Early Steps to Recent Advances
Nobel Symposium 138: Single Molecule Spectroscopy in Chemistry, Physics and Biosciences
SPRINGER-VERLAG BERLIN. 2010: 25–60
View details for Web of Science ID 000285735900002
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Localizing and Tracking Single Emitters in Three Dimensions Using a Double-Helix Point Spread Function
Conference on Lasers and Electro-Optics (CLEO)/Quantum Electronics and Laser Science Conference (QELS)
IEEE. 2010
View details for Web of Science ID 000290513601019
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Localizing and Tracking Single Nanoscale Emitters in Three Dimensions with High Spatiotemporal Resolution Using a Double-Helix Point Spread Function
NANO LETTERS
2010; 10 (1): 211-218
Abstract
Three-dimensional nanoscale localization and tracking of dim single emitters can be obtained with a widefield fluorescence microscope exhibiting a double-helix point spread function (DH-PSF). We describe in detail how the localization precision quantitatively depends upon the number of photons detected and the z position of the nanoscale emitter, thereby showing a approximately 10 nm localization capability along x, y, and z in the limit of weak emitters. Experimental measurements are compared to Fisher information calculations of the ultimate localization precision inherent in the DH-PSF. The DH-PSF, for the first time, is used to track single quantum dots in aqueous solution and a quantum dot-labeled structure inside a living cell in three dimensions.
View details for DOI 10.1021/nl903295p
View details for Web of Science ID 000273428700036
View details for PubMedID 20000821
View details for PubMedCentralID PMC2806512
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MOLECULES AND METHODS FOR SUPER-RESOLUTION IMAGING
METHODS IN ENZYMOLOGY, VOL 475: SINGLE MOLECULE TOOLS, PT B
2010; 475: 27-59
Abstract
By looking at a fluorescently labeled structure one molecule at a time, it is possible to side-step the optical diffraction limit and obtain "super-resolution" images of small nanostructures. In the Moerner Lab, we seek to develop both molecules and methods to extend super-resolution fluorescence imaging. Methodologies and protocols for designing and characterizing fluorophores with switchable fluorescence required for super-resolution imaging are reported. These fluorophores include azido-DCDHF molecules, covalently linked Cy3-Cy5 dimers, and also the first example of a photoswitchable fluorescent protein, enhanced yellow fluorescent protein (EYFP). The imaging of protein superstructures in living Caulobacter crescentus bacteria is used as an example of the power of super-resolution imaging by single-molecule photoswitching to extract information beyond the diffraction limit. Finally, a new method is described for obtaining three-dimensional super-resolution information using a double-helix point-spread function.
View details for DOI 10.1016/S0076-6879(10)75002-3
View details for Web of Science ID 000280733800002
View details for PubMedID 20627152
View details for PubMedCentralID PMC3216693
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Micrometer-Sized DNA-Single-Fluorophore-DNA Supramolecule: Synthesis and Single-Molecule Characterization
SMALL
2009; 5 (21): 2418-2423
Abstract
The synthesis of single-fluorophore-bis(micrometer-sized DNA) triblock supramolecules and the optical and structural characterization of the construct at the single-molecule level is reported. A fluorophore-bis(oligodeoxynucleotide) triblock is synthesized via the amide-coupling reaction. Subsequent protocols of DNA hybridization/ligation are developed to form the supramolecular triblock structure with lambda-DNA fragments on the micrometer length scale. The successful synthesis of the micrometer-sized DNA-single-fluorophore-DNA supramolecule is confirmed by agarose gel electrophoresis with fluorescence imaging under UV excitation. Single triblock structures are directly imaged by combined scanning force microscopy and single-molecule fluorescence microscopy, and provide unambiguous confirmation of the existence of the single fluorophore inserted in the middle of the long DNA. This type of triblock structure is a step closer to providing a scaffold for single-molecule electronic devices after metallization of the DNAs.
View details for DOI 10.1002/smll.200900494
View details for Web of Science ID 000271791100011
View details for PubMedID 19517486
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Large single-molecule fluorescence enhancements produced by a bowtie nanoantenna
NATURE PHOTONICS
2009; 3 (11): 654-657
View details for DOI 10.1038/NPHOTON.2009.187
View details for Web of Science ID 000272302700012
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Lithographic positioning of fluorescent molecules on high-Q photonic crystal cavities
APPLIED PHYSICS LETTERS
2009; 95 (12)
View details for DOI 10.1063/1.3232233
View details for Web of Science ID 000270243800065
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Superresolution imaging of protein superstructures in live Caulobacter crescentus cells with EYFP
AMER CHEMICAL SOC. 2009
View details for Web of Science ID 000207861909276
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3D superresolution imaging with double helix photoactivated localization microscopy (DH-PALM)
AMER CHEMICAL SOC. 2009
View details for Web of Science ID 000207861909509
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Imaging beyond the diffraction limit in cells using single-molecule active control
AMER CHEMICAL SOC. 2009: 555–555
View details for Web of Science ID 000207857800509
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Bright, Red Single-Molecule Emitters: Synthesis and Properties of Environmentally Sensitive Dicyanomethylenedihydrofuran (DCDHF) Fluorophores with Bisaromatic Conjugation
CHEMISTRY OF MATERIALS
2009; 21 (5): 797-810
Abstract
A group of new fluorescent dye materials for single-molecule imaging applications comprised of an amine donor, a π-system comprised of phenyl and thiophene rings and a 2-dicyanomethylene-3-cyano-2,5-dihydrofuran acceptor group have been synthesized. Relative to comparable single-ring compounds these doubly aromatic conjugated fluorophores have red-shifted absorption and emission usually accompanied by significantly increased quantum yields. Solvatochromism studies indicate that the photophysical properties of these dyes are sensitive to the solvent polarity and environmental rigidity. Photophysical studies demonstrate that these DCDHF dye materials are strong single-molecule emitters and the total number of detected photons per molecule is among the highest thus far for this family of fluorophores.
View details for DOI 10.1021/cm801783f
View details for Web of Science ID 000263891700007
View details for PubMedCentralID PMC2731435
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Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (9): 2995-2999
Abstract
We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 microm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.
View details for DOI 10.1073/pnas.0900245106
View details for Web of Science ID 000263844100007
View details for PubMedID 19211795
View details for PubMedCentralID PMC2651341
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DCDHF Fluorophores for Single-Molecule Imaging in Cells
CHEMPHYSCHEM
2009; 10 (1): 55-65
Abstract
There is a persistent need for small-molecule fluorescent labels optimized for single-molecule imaging in the cellular environment. Application of these labels comes with a set of strict requirements: strong absorption, efficient and stable emission, water solubility and membrane permeability, low background emission, and red-shifted absorption to avoid cell autofluorescence. We have designed and characterized several fluorophores, termed "DCDHF" fluorophores, for use in live-cell imaging based on the push-pull design: an amine donor group and a 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) acceptor group, separated by a pi-rich conjugated network. In general, the DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. Especially valuable are fluorophores with sophisticated photophysics for applications requiring additional facets of control, such as photoactivation. For example, we have reengineered a red-emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. This molecule and its relatives provide a new class of bright photoactivatable small-molecule fluorophores, which are needed for super-resolution imaging schemes that require active control (here turning-on) of single-molecule emission.
View details for DOI 10.1002/cphc.200800581
View details for PubMedID 19025732
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Superresolution Imaging in Live Caulobacter Crescentus Cells Using Photoswitchable Enhanced Yellow Fluorescent Protein
Conference on Single Molecule Spectroscopy and Imaging II
SPIE-INT SOC OPTICAL ENGINEERING. 2009
View details for DOI 10.1117/12.809080
View details for Web of Science ID 000285712600011
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Probing High-Q Photonic Crystal Resonances With Fluorescent Molecules
Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science Conference (CLEO/QELS 2009)
IEEE. 2009: 2353–2354
View details for Web of Science ID 000274751302160
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Three-Dimensional Super-resolution Single-Molecule Fluorescence Imaging Using a Double-Helix Point Spread Function
Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science Conference (CLEO/QELS 2009)
IEEE. 2009: 1921–1922
View details for Web of Science ID 000274751301287
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Photoactivatable DCDHF fluorophores for single-molecule imaging
Conference on Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications
SPIE-INT SOC OPTICAL ENGINEERING. 2009
View details for DOI 10.1117/12.809257
View details for Web of Science ID 000285710200021
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Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP
NATURE METHODS
2008; 5 (11): 947-949
Abstract
The commonly used, monomeric EYFP enabled imaging of intracellular protein structures beyond the optical resolution limit ('super-resolution' imaging) in living cells. By combining photoinduced activation of single EYFP fusions and time-lapse imaging, we obtained sub-40 nm resolution images of the filamentous superstructure of the bacterial actin protein MreB in live Caulobacter crescentus cells. These studies demonstrated that EYFP is a useful emitter for in vivo super-resolution imaging.
View details for DOI 10.1038/nmeth.1258
View details for Web of Science ID 000260532500013
View details for PubMedID 18794860
View details for PubMedCentralID PMC2655310
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Visualization of Long Human Telomere Mimics by Single-Molecule Fluorescence Imaging
JOURNAL OF PHYSICAL CHEMISTRY B
2008; 112 (42): 13184-13187
Abstract
Study of long single-stranded telomeric DNA is important for a variety of basic science and biotechnological applications, yet few methods exist for synthesis and visualization of single copies of this DNA in solution at biologically relevant length scales necessary for assessment of heterogeneity in its structure and behavior. We have synthesized kilobase-long single-stranded human telomere mimics in situ by rolling circle replication (RCR) on a microscope coverslip surface and visualized individual strands by staining with SYBR Gold. Under buffer flow, differential extensibility and varying morphology of these long telomere-mimicking DNA sequences were observed at the single-molecule level in real time. Using this procedure, we detected striking differences in the extensibility of individual RCR products based on the human G-rich telomeric sequence in the presence and absence of short, complementary single-stranded oligonucleotides. We also apply this new mode of single-stranded DNA characterization to probe the interaction of kilobase-length telomere mimics with the small-molecule G-quadruplex-binding agent TMPyP4.
View details for DOI 10.1021/jp806696u
View details for Web of Science ID 000260100900005
View details for PubMedID 18817431
View details for PubMedCentralID PMC2688642
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Cy3-Cy5 covalent heterodimers for single-molecule photoswitching
JOURNAL OF PHYSICAL CHEMISTRY B
2008; 112 (38): 11878-11880
Abstract
Covalent heterodimers of the Cy3 and Cy5 fluorophores have been prepared from commercially available starting materials and characterized at the single-molecule level. This system behaves as a discrete molecular photoswitch, in which photoexcitation of the Cy5 results in fluorescence emission or, with a much lower probability, causes the Cy5 to enter into a long-lived, but metastable, dark state. Photoinduced recovery of the emissive Cy5 is achieved by very low intensity excitation (5 W cm(-2)) of the Cy3 fluorophore at a shorter wavelength. A similar system consisting of proximal, but not covalently linked, Cy3 and Cy5 has found application in stochastic optical reconstruction microscopy (STORM), a single-molecule localization-based technique for super-resolution imaging that requires photoswitching. The covalent Cy3-Cy5 heterodimers described herein eliminate the need for probabilistic methods of situating the Cy3 and Cy5 in close proximity to enable photoswitching. As proof of principle, these heterodimers have been applied to super-resolution imaging of the tubular stalk structures of live Caulobacter crescentus bacterial cells.
View details for DOI 10.1021/jp806698p
View details for Web of Science ID 000259342000004
View details for PubMedID 18754575
View details for PubMedCentralID PMC2715847
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A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole
CELL
2008; 134 (6): 945-955
Abstract
Bacterial replication origins move towards opposite ends of the cell during DNA segregation. We have identified a proline-rich polar protein, PopZ, required to anchor the separated Caulobacter crescentus chromosome origins at the cell poles, a function that is essential for maintaining chromosome organization and normal cell division. PopZ interacts directly with the ParB protein bound to specific DNA sequences near the replication origin. As the origin/ParB complex is being replicated and moved across the cell, PopZ accumulates at the cell pole and tethers the origin in place upon arrival. The polar accumulation of PopZ occurs by a diffusion/capture mechanism that requires the MreB cytoskeleton. High molecular weight oligomers of PopZ assemble in vitro into a filamentous network with trimer junctions, suggesting that the PopZ network and ParB-bound DNA interact in an adhesive complex, fixing the chromosome origin at the cell pole.
View details for DOI 10.1016/j.cell.2008.07.015
View details for Web of Science ID 000259318100015
View details for PubMedID 18805088
View details for PubMedCentralID PMC2745220
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Single-molecule motions of oligoarginine transporter conjugates on the plasma membrane of Chinese hamster ovary cells
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2008; 130 (29): 9364-9370
Abstract
To explore the real-time dynamic behavior of molecular transporters of the cell-penetrating-peptide (CPP) type on a biological membrane, single fluorescently labeled oligoarginine conjugates were imaged interacting with the plasma membrane of Chinese hamster ovary (CHO) cells. The diffusional motion on the membrane, characterized by single-molecule diffusion coefficient and residence time (tau R), defined as the time from the initial appearance of a single-molecule spot on the membrane (from the solution) to the time the single molecule disappears from the imaging focal plane, was observed for a fluorophore-labeled octaarginine (a model guanidinium-rich CPP) and compared with the corresponding values observed for a tetraarginine conjugate (negative control), a lipid analogue, and a fluorescently labeled protein conjugate (transferrin-Alexa594) known to enter the cell through endocytosis. Imaging of the oligoarginine conjugates was enabled by the use of a new high-contrast fluorophore in the dicyanomethylenedihydrofuran family, which brightens upon interaction with the membrane at normal oxygen concentrations. Taken as a whole, the motions of the octaarginine conjugate single molecules are highly heterogeneous and cannot be described as Brownian motion with a single diffusion coefficient. The observed behavior is also different from that of lipids, known to penetrate cellular membranes through passive diffusion, conventionally involving lateral diffusion followed by membrane bilayer flip-flop. Furthermore, while the octaarginine conjugate behavior shares some common features with transferrin uptake (endocytotic) processes, the two systems also exhibit dissimilar traits when diffusional motions and residence times of single constructs are compared. Additionally, pretreatment of cells with cytochalasin D, a known actin filament disruptor, produces no significant effect, which further rules out unimodal endocytosis as the mechanism of uptake. Also, the involvement of membrane potential in octaarginine-membrane interaction is supported by significant changes in the motion with high [K(+)] treatment. In sum, this first study of single transporter motion on the membrane of a living cell indicates that the mode by which the octaarginine transporter penetrates the cell membrane appears to either be a multimechanism uptake process or a mechanism different from unimodal passive diffusion or endocytosis.
View details for DOI 10.1021/ja710798b
View details for PubMedID 18578528
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A photoactivatable push-pull fluorophore for single-molecule imaging in live cells
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2008; 130 (29): 9204-?
Abstract
We have reengineered a red-emitting dicyanomethylenedihydrofuran push-pull fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. Photoactivation of the dark fluorogen leads to conversion of an azide to an amine, which shifts the absorption to long wavelengths. After photoactivation, the fluorophore is bright and photostable enough to be imaged on the single-molecule level in living cells. This proof-of-principle demonstration provides a new class of bright photoactivatable fluorophores, as are needed for super-resolution imaging schemes that require active control of single molecule emission.
View details for DOI 10.1021/ja802883k
View details for PubMedID 18572940
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Controlling Brownian motion of single protein molecules and single fluorophores in aqueous buffer
OPTICS EXPRESS
2008; 16 (10): 6941-6956
Abstract
We present an Anti-Brownian Electrokinetic trap (ABEL trap) capable of trapping individual fluorescently labeled protein molecules in aqueous buffer. The ABEL trap operates by tracking the Brownian motion of a single fluorescent particle in solution, and applying a time-dependent electric field designed to induce an electrokinetic drift that cancels the Brownian motion. The trapping strength of the ABEL trap is limited by the latency of the feedback loop. In previous versions of the trap, this latency was set by the finite frame rate of the camera used for video-tracking. In the present system, the motion of the particle is tracked entirely in hardware (without a camera or image-processing software) using a rapidly rotating laser focus and lock-in detection. The feedback latency is set by the finite rate of arrival of photons. We demonstrate trapping of individual molecules of the protein GroEL in buffer, and we show confinement of single fluorophores of the dye Cy3 in water.
View details for Web of Science ID 000256469800020
View details for PubMedID 18545398
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Superresolution Imaging in Live Bacterial Cells by Single-Molecule Active-Control Microscopy
Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science Conference (CLEO/QELS 2008)
IEEE. 2008: 246–247
View details for Web of Science ID 000260498400124
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Hardware-based anti-Brownian electrokinetic trap (ABEL trap) for single molecules: Control loop simulations and application to ATP binding stoichiometry in multi-subunit enzymes
Conference on Optical Trapped and Optical Micromanipulation V
SPIE-INT SOC OPTICAL ENGINEERING. 2008
View details for DOI 10.1117/12.798093
View details for Web of Science ID 000262711000004
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Interferometry of a single nanoparticle using the Gouy phase of a focused laser beam
OPTICS COMMUNICATIONS
2007; 280 (2): 487-491
View details for DOI 10.1016/j.optcom.2007.08.032
View details for Web of Science ID 000251077400043
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Gold bowtie nanoantennas for surface-enhanced Raman scattering under controlled electrochemical potential
CHEMICAL PHYSICS LETTERS
2007; 446 (4-6): 339-343
View details for DOI 10.1016/j.cplett.2007.08.074
View details for Web of Science ID 000250366100019
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Photophysical properties of acene DCDHF fluorophores: Long-wavelength single-molecule emitters designed for cellular Imaging
JOURNAL OF PHYSICAL CHEMISTRY A
2007; 111 (37): 8934-8941
Abstract
We report the solvatochromic, viscosity-sensitive, and single-molecule photophysics of the fluorophores DCDHF-N-6 and DCDHF-A-6. These molecules are members of the dicyanomethylenedihydrofuran (DCDHF) class of single-molecule emitters that contain an amine electron donor and a DCDHF acceptor linked by a conjugated unit; DCDHF-N-6 and DCDHF-A-6 have naphthalene- and anthracene-conjugated linkers, respectively. These molecules maintain the beneficial photophysics of the phenylene-linked DCDHF (i.e., photostability, emission wavelength dependence on solvent polarity, and quantum yield sensitivity to solvent viscosity), yet offer absorption and emission at longer wavelengths that are more appropriate for cellular imaging. We demonstrate that these new fluorophores are less photolabile in an aqueous environment than several other commonly used dyes (rhodamine 6G, Texas Red, and fluorescein). Finally, we image single copies of the acene DCDHFs diffusing in the plasma membrane of living cells.
View details for DOI 10.1021/jp0712598
View details for PubMedID 17718454
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PHYS 701-Using feedback to beat the Boltzmann distribution
AMER CHEMICAL SOC. 2007
View details for Web of Science ID 000207593907713
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PHYS 1-Recent progress in single-biomolecule fluorescence imaging
AMER CHEMICAL SOC. 2007
View details for Web of Science ID 000207593907421
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New directions in single-molecule imaging and analysis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (31): 12596-12602
Abstract
Optical imaging and analysis of single molecules continue to unfold as powerful ways to study the individual behavior of biological systems, unobscured by ensemble averaging. Current expansion of interest in this field is great, as evidenced by new meetings, journal special issues, and the large number of new investigators. Selected recent advances in biomolecular analysis are described, and two new research directions are summarized: superresolution imaging using single-molecule fluorescence and trapping of single molecules in solution by direct suppression of Brownian motion.
View details for DOI 10.1073/pnas.0610081104
View details for Web of Science ID 000248603900004
View details for PubMedID 17664434
View details for PubMedCentralID PMC1937512
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Principal-components analysis of shape fluctuations of single DNA molecules
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (31): 12622-12627
Abstract
Thermal fluctuations agitate molecules in solution over a broad range of times and distances. By passively watching the shape fluctuations of a thermally driven biomolecule, one can infer properties of the underlying interactions that determine the motion. We applied this concept to single molecules of fluorescently labeled lambda-DNA, a key model system for polymer physics. In contrast to most other single-molecule DNA experiments, we examined the unstretched, equilibrium state of DNA by using an anti-Brownian electrokinetic trap to confine the center of mass of the DNA without perturbing its internal dynamics. We analyze the long-wavelength conformational normal modes, calculate their spring constants, and measure linear and nonlinear couplings between modes. The modes show strong signs of nonlinear hydrodynamics, a feature of the underlying equations of polymer dynamics that has not previously been reported and is neglected in the widely used Rouse and Zimm approximations.
View details for DOI 10.1073/pnas.0610396104
View details for Web of Science ID 000248603900008
View details for PubMedID 17496147
View details for PubMedCentralID PMC1937516
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Bulk and single-molecule characterization of an improved molecular beacon utilizing H-dimer excitonic behavior
JOURNAL OF PHYSICAL CHEMISTRY B
2007; 111 (28): 7929-7931
Abstract
Pairs of fluorophores in close proximity often show self-quenching of fluorescence by the well-known H-dimer mechanism. We use a pair of fluorophores in the new dicyanomethylenedihydrofuran (DCDHF) dye family in the design and characterization of a new fluorescent probe for nucleic acid detection, which we refer to as a self-quenched intramolecular dimer (SQuID) molecular beacon (MB). We obtain a quenching efficiency of 97.2%, higher than the only other reported value for a MB employing fluorophore self-quenching by H-dimer formation. Furthermore, the excellent single-molecule (SM) emitter characteristics of the DCDHF dyes allow observation of individual SQuID MB-target complexes immobilized on a surface, where the doubled SM emission intensity of our target-bound beacon ensures a higher signal-to-background ratio than conventional fluorophore-quencher MBs. Additional advantages of the SQuID MB are single-pot labeling, visible colorimetric detection of the target, and intrinsic single-molecule two-step photobleaching behavior, which offers a specific means of discriminating between functional MBs and spurious fluorescence.
View details for DOI 10.1021/jp073310d
View details for Web of Science ID 000247966300001
View details for PubMedID 17583944
View details for PubMedCentralID PMC2663424
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Modifications of DCDHF single molecule fluorophores to impart water solubility
TETRAHEDRON LETTERS
2007; 48 (19): 3471-3474
Abstract
A series of dicyanomethylenedihydrofuran (DCDHF) fluorophores with different hydrophilic groups were synthesized and their photophysical properties and water solubilities were measured. Significant water solubility was achieved without compromising desirable photophysical properties, permitting applications of these fluorophores in biological systems.
View details for DOI 10.1016/j.tetlet.2007.03.026
View details for Web of Science ID 000246611400032
View details for PubMedCentralID PMC2744137
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Dicyanomethylenedihydrofuran (DCDHF) single molecule fluorophores for cellular applications
AMER CHEMICAL SOC. 2007
View details for Web of Science ID 000207722808431
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Internal mechanical response of a polymer in solution
PHYSICAL REVIEW LETTERS
2007; 98 (11)
Abstract
We observed single molecules of fluorescently labeled double-stranded (ds) lambda DNA held in an anti-Brownian electrokinetic trap. From the measured density fluctuations we extract the density-density response function of the molecule over times >4.5 ms and distances >250 nm, i.e., how a perturbation in density in one part of the molecule propagates through the rest of the molecule. We find a nonmonotonic radial dependence of the relaxation time. In contrast with earlier measurements on freely diffusing dsDNA, we observe clear signs of internal hydrodynamic interactions.
View details for DOI 10.1103/PhysRevLett.98.116001
View details for Web of Science ID 000244959300041
View details for PubMedID 17501066
View details for PubMedCentralID PMC2441641
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Single-molecule electron spin resonance
APPLIED MAGNETIC RESONANCE
2007; 31 (3-4): 665-676
View details for Web of Science ID 000247393500021
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OPTICAL FIELD ENHANCEMENT WITH PLASMON RESONANT BOWTIE NANOANTENNAS
SURFACE PLASMON NANOPHOTONICS
2007; 131: 125-137
View details for DOI 10.1007/978-1-4020-4333-8_9
View details for Web of Science ID 000288851900010
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Observing dynamics of individual biomolecules with single-molecule microscopy
BIOPHYSICAL SOCIETY. 2007: 363A
View details for Web of Science ID 000243972402247
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Both MHC class II and its GPI-anchored form undergo hop diffusion as observed by single-molecule tracking
BIOPHYSICAL SOCIETY. 2007: 527A
View details for Web of Science ID 000243972403316
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The influence of tetrahydroquinoline rings in dicyanomethylenedihydrofuran (DCDHF) single-molecule fluorophores
TETRAHEDRON
2007; 63 (1): 103-114
View details for DOI 10.1016/j.tet.2006.10.044
View details for Web of Science ID 000243365800009
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Single-molecule tracking.
Methods in molecular biology (Clifton, N.J.)
2007; 398: 193-219
Abstract
The current models of eukaryotic plasma membrane organization separate the plasma membrane nto different environments created by lipids and interactions between membrane proteins and the cytoskeleton, but characterization of their physical properties, such as their sizes, lifetimes, and the partitioning of membrane components into each environment, has not been accomplished. Single-moleule (fluorophore) tracking (SMT) experiments are well suited to the noninvasive study of membrane properties. In SMT experiments, the position of a single fluorescently labeled protein or lipid probe is followed optically as it moves within the membrane. If the motion of the probe is unhindered, then the atial trajectory of the molecule will follow two-dimensional Brownian motion. If the probe encounters a structure that in some way inhibits its movement, then the probe's trajectory will deviate from Brownian motion. It is likely that even if a certain type of lipid or protein partitions strongly into one nvironment, each individual lipid or protein will spend some fraction of its lifetime in the less favorable environment. Because SMT follows the motion of an individual probe over a large area (approximately 10 x 10 microm2), transitions between environments can be observed directly by monitoring the path of each protein or lipid. Additionally, heterogeneity owing to multiple populations of molecules permanently residng in different states may be distinguished from a single population of molecules transitioning between different states. By judicious choice of label, such that the motion of the labeled protein or lipid is unafected by the label itself, and through the use of probes with different affinities for each membrane environment, SMT measurements in principle can reveal the structure of the plasma membrane.
View details for DOI 10.1007/978-1-59745-513-8_14
View details for PubMedID 18214382
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Long-wavelength analogue of PRODAN: Synthesis and properties of Anthradan, a fluorophore with a 2,6-donor-acceptor anthracene structure
JOURNAL OF ORGANIC CHEMISTRY
2006; 71 (26): 9651-9657
Abstract
We have synthesized the environment-sensitive fluorophores 2-cyano-6-dihexylaminoanthracene and 2-propionyl-6-dihexylaminoanthracene (Anthradan) starting from 2,6-diaminoanthraquinone. Anthradan is the benzologue of the well-known family of naphthalene 2-propionyl-6-dimethylaminonaphthalene (PRODAN) fluorophores. The additional spectral red shift of the anthracene avoids the autofluorescence of many biological systems and provides for more favorable excitation wavelengths for fluorescence applications. Furthermore, Anthradan exhibits polarity-sensitive emission comparable to that of PRODAN and displays high quantum yields in a range of solvents. Single molecules of these anthracene-containing fluorophores have been imaged in polymer hosts as a proof-of-principle.
View details for DOI 10.1021/jo0616660
View details for Web of Science ID 000242845500012
View details for PubMedID 17168582
View details for PubMedCentralID PMC2663422
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Well-controlled living polymerization of perylene-labeled polyisoprenes and their use in single-molecule Imaging
MACROMOLECULES
2006; 39 (23): 8121-8127
View details for DOI 10.1021/ma0612475
View details for Web of Science ID 000241813200046
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Single-molecule mountains yield nanoscale cell images
NATURE METHODS
2006; 3 (10): 781-782
View details for DOI 10.1038/nmeth1006-781
View details for Web of Science ID 000240942600009
View details for PubMedID 16990808
View details for PubMedCentralID PMC2663419
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Single-molecule nanoprobes explore defects in spin-grown crystals
Festschrift in honor of the 65th Birthday of Robert J Silbey
AMER CHEMICAL SOC. 2006: 18939–44
Abstract
Thin, platelike single crystals of p-terphenyl (PT) doped with terrylene impurity molecules can be prepared by spin-coating from solution. Strikingly, individual terrylene molecules can be observed traveling inside the crystal over distances of several micrometers by using single-molecule fluorescence imaging at room temperature. Analysis of the motion by single-particle tracking and correlation methods indicates that the molecules act as nanoprobes by exploring long, thin crack-like defects with correlated orientations, defects that can be difficult to observe by other means. Apparently, the regions accessible to the moving molecules are in the interior of the crystal and hence are partially protected from oxidation. In addition to the traveling molecules, which photobleach in times on the order of 32 s under continuous irradiation at 2 kW/cm2, two other spatially fixed populations are observed: one with transition dipole oriented along the c-axis of the crystal with a characteristic photobleaching time greater than 32 h, and one with a characteristic photobleaching time of 18 min.
View details for DOI 10.1021/jp057570b
View details for Web of Science ID 000240654900027
View details for PubMedID 16986887
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COLL 60-Single-molecule fluorescence tracking probes membrane dynamics
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781603288
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COLL 101-Exploring surface-enhanced Raman scattering using gold bowtie nanoantennae
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781603230
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PHYS 504-Suppressing Brownian motion of individual molecules in solution
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781609110
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ORGN 54-Synthesis and properties of DCDHF chromophore dimers
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781608613
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PHYS 489-Direct observation of MreB treadmilling in Caulobacter by single-molecule fluorescence microscopy
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781609166
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PHYS 476-Progress towards real-time observation of T7 DNA polymerase activity by single-molecule fluorescence spectroscopy
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781609059
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PHYS 484-DCDHF dyes can probe environmental changes by protein binding in GroEL
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781609161
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PHYS 252-Single-molecule fluorescence imaging reports on biomolecular dynamics
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781609369
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PHYS 496-Lateral movements of single poly(arginine) peptides on the CHO plasma membrane before cellular entry
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781609163
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PHYS 665-An improved single-molecule molecular beacon utilizing H-dimer excitonic behavior
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781609422
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PHYS 488-DCDHF photophysics: Designing new single-molecule fluorophores for cellular imaging
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000207781609088
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Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (29): 10929-10934
Abstract
The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.
View details for DOI 10.1073/pnas.0604503103
View details for Web of Science ID 000239327200021
View details for PubMedID 16829583
View details for PubMedCentralID PMC1544151
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Diffusion of lipid-like single-molecule fluorophores in the cell membrane
JOURNAL OF PHYSICAL CHEMISTRY B
2006; 110 (15): 8151-8157
Abstract
The dicyanomethylenedihydrofuran (DCDHF) class of single-molecule fluorophores contains an amine donor and a dicyanomethylenedihydrofuran acceptor linked by a conjugated unit (benzene, naphthalene, or styrene). Molecules in this class have a number of useful properties in addition to those usually required for single-molecule studies (such as high fluorescence quantum yield and photostability), including second-order optical nonlinearity, large ground-state dipole moment, and sensitivity to local environment. Moreover, most DCDHF molecules have amphiphilic structures, with a polar dicyanomethylenedihydrofuran headgroup and nonpolar hydrocarbon tails on the amine or furan ring, and can be used as fluorescent lipid analogues for live cell imaging. Here we demonstrate that individual molecules of several different DCDHF lipid analogues can be observed diffusing in the plasma membrane of Chinese hamster ovary cells. The photophysical and diffusive behaviors of the DCDHF lipid analogues in membranes are described and are found to be competitive with the well-known lipid probe N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine.
View details for DOI 10.1021/jp0574145
View details for Web of Science ID 000236992100072
View details for PubMedID 16610918
View details for PubMedCentralID PMC1702323
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Synthesis and properties of the highly environment sensitive fluorophores 2-cyano and 2-propionyl-6-dihexylaminoanthracene (ANTHRADAN)
231st National Meeting of the American-Chemical-Society
AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000238125907016
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Suppressing Brownian motion of individual biomolecules in solution
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (12): 4362-4365
Abstract
Single biomolecules in free solution have long been of interest for detailed study by optical methods, but Brownian motion prevents the observation of one single molecule for extended periods. We have used an anti-Brownian electrokinetic (ABEL) trap to trap individual protein molecules in free solution, under ambient conditions, without requiring any attachment to beads or surfaces. We also demonstrate trapping and manipulation of single virus particles, lipid vesicles, and fluorescent semiconductor nanocrystals.
View details for DOI 10.1073/pnas.0509976103
View details for Web of Science ID 000236362600009
View details for PubMedID 16537418
View details for PubMedCentralID PMC1450176
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Toward nanometer-scale optical photolithography: Utilizing the near-field of bowtie optical nanoantennas
NANO LETTERS
2006; 6 (3): 355-360
Abstract
Optically resonant metallic bowtie nanoantennas are utilized as fabrication tools for the first time, resulting in the production of polymer resist nanostructures <30 nm in diameter at record low incident multiphoton energy densities. The nanofabrication is accomplished via nonlinear photopolymerization, which is initiated by the enhanced, confined optical fields surrounding the nanoantenna. The position, size, and shape of the resist nanostructures directly correlate with rigorous finite-difference time-domain computations of the field distribution, providing a nanometer-scale measurement of the actual field confinement offered by single optical nanoantennas. In addition, the size of the photoresist regions yields strong upper bounds on photoacid diffusion and resist resolution in SU-8, demonstrating a technique that can be generalized to the study of many current and yet-to-be-developed photoresist systems.
View details for DOI 10.1021/nl052322c
View details for Web of Science ID 000236049800004
View details for PubMedID 16522022
View details for PubMedCentralID PMC1447673
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Exploring the chemical enhancement for surface-enhanced Raman scattering with Au bowtie nanoantennas
JOURNAL OF CHEMICAL PHYSICS
2006; 124 (6)
Abstract
Single metallic bowtie nanoantennas provide a controllable environment for surface-enhanced Raman scattering (SERS) of adsorbed molecules. Bowties have experimentally measured electromagnetic enhancements, enabling estimation of chemical enhancement for both the bulk and the few-molecule regime. Strong fluctuations of selected Raman lines imply that a small number of p-mercaptoaniline molecules on a single bowtie show chemical enhancement >10(7), much larger than previously believed, likely due to charge transfer between the Au surface and the molecule. This chemical sensitivity of SERS has significant implications for ultra-sensitive detection of single molecules.
View details for DOI 10.1063/1.2167649
View details for Web of Science ID 000235309400001
View details for PubMedID 16483189
View details for PubMedCentralID PMC1513182
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Cholesterol depletion induces solid-like regions in the plasma membrane
BIOPHYSICAL JOURNAL
2006; 90 (3): 927-938
Abstract
Glycosylphosphatidylinositol-linked and transmembrane major histocompatibility complex (MHC) class II I-E(k) proteins, as well as N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Tritc-DHPE), are used as probes to determine the effect of cholesterol concentration on the organization of the plasma membrane at temperatures in the range 22 degrees C-42 degrees C. Cholesterol depletion caused a decrease in the diffusion coefficients for the MHC II proteins and also for a slow fraction of the Tritc-DHPE population. At 37 degrees C, reduction of the total cell cholesterol concentration results in a smaller suppression of the translational diffusion for I-E(k) proteins (twofold) than was observed in earlier work at 22 degrees C (five sevenfold) Vrljic, M., S. Y. Nishimura, W. E. Moerner, and H. M. McConnell. 2005. Biophys. J. 88:334-347. At 37 degrees C, the diffusion of both I-E(k) proteins is Brownian (0.9 < alpha-parameter < 1.1). More than 99% of the protein population diffuses homogeneously when imaged at 65 frames per s. As the temperature is raised from 22 degrees C to 42 degrees C, a change in activation energy is seen at approximately 35 degrees C in the Arrhenius plots. Cytoskeletal effects appear to be minimal. These results are consistent with a previously described model of solid-like domain formation in the plasma membrane.
View details for DOI 10.1529/biophysj.105.070524
View details for Web of Science ID 000234586200022
View details for PubMedID 16272447
View details for PubMedCentralID PMC1367117
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Scanning interferometric microscopy for the detection of ultrasmall phase shifts in condensed matter
PHYSICAL REVIEW A
2006; 73 (2)
View details for DOI 10.1103/PhysRevA.73.021802
View details for Web of Science ID 000235668100022
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Probing the sequence of conformationally induced polarity changes in the molecular chaperonin GroEL with fluorescence spectroscopy
JOURNAL OF PHYSICAL CHEMISTRY B
2005; 109 (51): 24517-24525
Abstract
Hydrophobic interactions play a major role in binding non-native substrate proteins in the central cavity of the bacterial chaperonin GroEL. The sequence of local conformational changes by which GroEL and its cofactor GroES assist protein folding can be explored using the polarity-sensitive fluorescence probe Nile Red. A specific single-cysteine mutant of GroEL (Cys261), whose cysteine is located inside the central cavity at the apical region of the protein, was covalently labeled with synthetically prepared Nile Red maleimide (NR). Bulk fluorescence spectra of Cys261-NR were measured to examine the effects of binding of the stringent substrate, malate dehydrogenase (MDH), GroES, and nucleotide on the local environment of the probe. After binding denatured substrate, the fluorescence intensity increased by 32 +/- 7%, suggesting enhanced hydrophobicity at the position of the label. On the other hand, in the presence of ATP, the fluorescence intensity decreased by 13 +/- 3%, implying increased local polarity. To explore the sequence of local polarity changes, substrate, GroES, and various nucleotides were added in different orders; the resulting changes in emission intensity provide insight into the sequence of conformational changes occurring during GroEL-mediated protein folding.
View details for DOI 10.1021/jp0534232
View details for Web of Science ID 000234259900043
View details for PubMedID 16375456
View details for PubMedCentralID PMC1414071
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A novel fluorophore for two-photon-excited single-molecule fluorescence
CHEMICAL PHYSICS
2005; 318 (1-2): 7-11
View details for DOI 10.1016/j.chemphys.2005.03.010
View details for Web of Science ID 000233353500003
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Distinct constrictive processes, separated in time and space, divide Caulobacter inner and outer membranes
JOURNAL OF BACTERIOLOGY
2005; 187 (20): 6874-6882
Abstract
Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed pre-fission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.
View details for DOI 10.1128/JB.187.20.6874-6882.2005
View details for Web of Science ID 000232509500003
View details for PubMedID 16199556
View details for PubMedCentralID PMC1251605
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Field enhancement and gap-dependent resonance in a system of two opposing tip-to-tip Au nanotriangles
PHYSICAL REVIEW B
2005; 72 (16)
View details for DOI 10.1103/PhysRevB.72.165409
View details for Web of Science ID 000232934900111
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Single-molecule biophysics, nanophotonics, and trapping
AMER CHEMICAL SOC. 2005: U336
View details for Web of Science ID 000236797300650
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Enhancement of the fluorescence of the blue fluorescent proteins by high pressure or low temperature
JOURNAL OF PHYSICAL CHEMISTRY B
2005; 109 (26): 12976-12981
Abstract
Green fluorescent proteins bearing the Y66H mutation exhibit strongly blue-shifted fluorescence excitation and emission spectra. However, these blue fluorescent proteins (BFPs) have lower quantum yields of fluorescence (Phi(f) approximately 0.20), which is believed to stem from the increased conformational freedom of the smaller chromophore. We demonstrate that suppression of chromophore mobility by increasing hydrostatic pressure or by decreasing temperature can enhance the fluorescence quantum yield of these proteins without significantly affecting their absorption properties or the shape of the fluorescence spectra. Analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes. Temperature-dependent fluorescence measurements reveal two barriers ( approximately 19 and 3 kJ/mol, respectively) for the transition into nonfluorescing states. These steps are probably linked with dissociation of the hydrogen bond between the chromophore and His148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state, respectively. The chromophore's hydrogen-bond equilibrium at room temperature is dominated by entropic effects, while below approximately 200 K the balance is enthalpy-driven.
View details for DOI 10.1021/jp0448595
View details for Web of Science ID 000230224700046
View details for PubMedID 16852610
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Nonlinear optical chromophores as nanoscale emitters for single-molecule spectroscopy
ACCOUNTS OF CHEMICAL RESEARCH
2005; 38 (7): 549-556
Abstract
Fluorescence imaging of single molecules at room temperature is a powerful technique for studying complex condensed phase systems and revealing structure and dynamics hidden by ensemble measurements. Successful single-molecule spectroscopic experiments rely upon strong emitters that can be detected at the level of individual copies above the relevant background signals. This Account discusses a class of nonlinear optical chromophores that not only are well-suited for single-molecule imaging but also offer additional beneficial properties such as a significant ground-state dipole moment, moderate hyperpolarizability, and sensitivity to local environment. An overview of the photophysical properties of several members of this class of molecules as well as a mechanism to help understand the environmental sensitivity is presented. Some preliminary applications of the chromophores as single-molecule reporters in cellular and polymer systems are discussed, along with detection of the emitters by two-photon fluorescence.
View details for DOI 10.1021/ar0401294
View details for Web of Science ID 000230688000007
View details for PubMedID 16028889
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Monolithically integrated semiconductor fluorescence sensor for microfluidic applications
SENSORS AND ACTUATORS B-CHEMICAL
2005; 105 (2): 393-399
View details for DOI 10.1016/j.snb.2004.06.028
View details for Web of Science ID 000227882000041
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Soliton-induced waveguides in an organic photorefractive glass
OPTICS LETTERS
2005; 30 (5): 519-521
Abstract
We demonstrate optical waveguiding of a probe beam at 980 nm by a soliton beam at 780 nm in an organic photorefractive monolithic glass. Both planar and circular waveguides induced by one- and two-dimensional spatial solitons formed as a result of orientationally enhanced photorefractive nonlinearity are produced in the organic glass. Possibilities for increasing the speed of waveguide formation are discussed.
View details for Web of Science ID 000227371800023
View details for PubMedID 15789722
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Method for trapping and manipulating nanoscale objects in solution
APPLIED PHYSICS LETTERS
2005; 86 (9)
View details for DOI 10.1063/1.1872220
View details for Web of Science ID 000228991600060
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Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas
PHYSICAL REVIEW LETTERS
2005; 94 (1)
Abstract
Metallic bowtie nanoantennas should provide optical fields that are confined to spatial scales far below the diffraction limit. To improve the mismatch between optical wavelengths and nanoscale objects, we have lithographically fabricated Au bowties with lengths approximately 75 nm and gaps of tens of nm. Using two-photon-excited photoluminescence of Au, the local intensity enhancement factor relative to that for the incident diffraction-limited beam has been experimentally determined for the first time. Enhancements >10(3) occur for 20 nm gap bowties, in good agreement with theoretical simulations.
View details for DOI 10.1103/PhysRevLett.94.017402
View details for Web of Science ID 000226308000082
View details for PubMedID 15698131
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Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane
BIOPHYSICAL JOURNAL
2005; 88 (1): 334-347
Abstract
Glycosylphosphatidylinositol (GPI)-linked and native major histocompatibility complex class II I-E(k) were used as probes to determine the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane. These proteins were imaged in Chinese hamster ovary cells using single-molecule fluorescence microscopy. Observed diffusion coefficients of both native and GPI-linked I-E(k) proteins were found to depend on cholesterol concentration. As the cholesterol concentration decreases the diffusion coefficients decrease by up to a factor of 7 for native and 5 for GPI-linked I-E(k). At low cholesterol concentrations, after sphingomyelinase treatment, the diffusion coefficients are reduced by up to a factor of 60 for native and 190 for GPI-linked I-E(k). The effect is reversible on cholesterol reintroduction. Diffusion at all studied cholesterol concentrations, for both proteins, appears to be predominantly Brownian for time lags up to 2.5 s when imaged at 10 Hz. A decrease in diffusion coefficients is observed for other membrane proteins and lipid probes, DiIC12 and DiIC18. Fluorescence recovery after photobleaching measurements shows that the fraction of immobile lipid probe increases from 8 to approximately 40% after cholesterol extraction. These results are consistent with the previous work on cholesterol-phospholipid interactions. That is, cholesterol extraction destroys liquid cholesterol-phospholipid complexes, leaving solid-like high melting phospholipid domains that inhibit the lateral diffusion of membrane components.
View details for Web of Science ID 000226090900031
View details for PubMedID 15516525
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The Anti-Brownian ELectrophoretic trap (ABEL trap): Fabrication and software
SPIE-INT SOC OPTICAL ENGINEERING. 2005: 296–305
View details for DOI 10.1117/12.598689
View details for Web of Science ID 000229039000036
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Synthesis, Properties and Applications of Dicyanomethylenedihydrofuran (DCDHF) Single Molecule Fluorophores
NONLINEAR OPTICS QUANTUM OPTICS-CONCEPTS IN MODERN OPTICS
2005; 34 (1-4): 241–46
View details for Web of Science ID 000213763100050
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Visualization of the movement of single histidine kinase molecules in live Caulobacter cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2004; 101 (45): 15921-15926
Abstract
The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle. This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle. Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds. In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane. By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 +/- 2 x 10(-3) microm(2)/s for the mobile copies of PleC not bound at the cell pole. This D value is maintained across all cell cycle stages. We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium. We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported.
View details for DOI 10.1073/pnas.0404200101
View details for Web of Science ID 000225196800020
View details for PubMedID 15522969
View details for PubMedCentralID PMC528753
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Visualizing single-molecule dynamics in cells.
AMER CHEMICAL SOC. 2004: U300
View details for Web of Science ID 000223713801621
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Cholesterol depletion suppresses the translational diffusion of class II MHC proteins in the plasma membrane.
AMER CHEMICAL SOC. 2004: U292
View details for Web of Science ID 000223713801565
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Single-photon sources based on single molecules in solids
NEW JOURNAL OF PHYSICS
2004; 6
View details for DOI 10.1088/1367-2630/6/1/088
View details for Web of Science ID 000222971800004
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Spectral analysis of strongly enhanced visible light transmission through single C-shaped nanoapertures
APPLIED PHYSICS LETTERS
2004; 85 (4): 648-650
View details for DOI 10.1063/1.1774270
View details for Web of Science ID 000222855400046
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Experimental and theoretical investigations of environmentally sensitive single-molecule fluorophores
JOURNAL OF PHYSICAL CHEMISTRY B
2004; 108 (29): 10465-10473
View details for DOI 10.1021/jp049684d
View details for Web of Science ID 000222763000031
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Organic photorefractives: Mechanisms, materials, and applications
CHEMICAL REVIEWS
2004; 104 (7): 3267-3314
View details for DOI 10.1021/cr960055c
View details for Web of Science ID 000222769100003
View details for PubMedID 15250742
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Gap-dependent optical coupling of single "Bowtie" nanoantennas resonant in the visible
NANO LETTERS
2004; 4 (5): 957-961
View details for DOI 10.1021/nl049951r
View details for Web of Science ID 000221410000039
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Integrated semiconductor vertical-cavity surface-emitting lasers and PIN photodetectors for biomedical fluorescence sensing
IEEE JOURNAL OF QUANTUM ELECTRONICS
2004; 40 (5): 491-498
View details for DOI 10.1109/JQE.2004.826440
View details for Web of Science ID 000221090900007
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Laser background characterization in a monolithically integrated biofluorescence sensor
Conference on Advanced Biomedical and Clinical Diagnostic Systems II
SPIE-INT SOC OPTICAL ENGINEERING. 2004: 59–65
View details for DOI 10.1117/12.525133
View details for Web of Science ID 000223125300007
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Single-molecule fluorescence spectroscopy and microscopy of biomolecular motors
ANNUAL REVIEW OF PHYSICAL CHEMISTRY
2004; 55: 79-96
Abstract
The methods of single-molecule fluorescence spectroscopy and microscopy have been recently utilized to explore the mechanism of action of several members of the kinesin and myosin biomolecular motor protein families. Whereas ensemble averaging is removed in single-molecule studies, heterogeneity in the behavior of individual motors can be directly observed, without synchronization. Observation of translocation by individual copies of motor proteins allows analysis of step size, rate, pausing, and other statistical properties of the process. Polarization microscopy as a function of nucleotide state has been particularly useful in revealing new and highly rotationally mobile forms of particular motors. These experiments complement X-ray and biochemical studies and provide a detailed view into the local dynamical behavior of motor proteins.
View details for DOI 10.1146/annurev.physchem.55.091602.094340
View details for Web of Science ID 000222766400004
View details for PubMedID 15117248
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Self-trapping of light in an organic photorefractive glass
OPTICS LETTERS
2003; 28 (24): 2509-2511
Abstract
We report the first observation, to our knowledge, of self-trapping of light as well as optically induced focusing-to-defocusing switching in an organic photorefractive glass, owing to the orientationally enhanced photorefractive nonlinearity of the material.
View details for Web of Science ID 000187075300025
View details for PubMedID 14690130
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Optical measurements of single molecules in cells
Workshop on Emerging Technologies for the Analysis of Endogenous Biomaterials and Single-Molecule Studies
ELSEVIER SCI LTD. 2003: 544–48
View details for DOI 10.1016/S0165-9936(03)00905-1
View details for Web of Science ID 000185578400021
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Methods of single-molecule fluorescence spectroscopy and microscopy
REVIEW OF SCIENTIFIC INSTRUMENTS
2003; 74 (8): 3597-3619
View details for DOI 10.1063/1.1589587
View details for Web of Science ID 000184346600001
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Role of temperature in controlling performance of photorefractive organic glasses
CHEMPHYSCHEM
2003; 4 (7): 732-744
Abstract
We present a detailed temperature-dependence study of dielectric, birefringent, conductive, and photorefractive (PR) properties of high-performance low-molecular weight organic glasses that contain 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) derivatives. DCDHF organic glasses sensitized with C60 exhibit high two-beam coupling gain coefficients in the red-wavelength region. However, in the best performing DCDHF glasses at room temperature the PR dynamics are limited by slow molecular reorientation in the electric field. While orientational and, therefore, PR speed can be significantly improved by increasing the temperature above the glass-transition temperature of the material, the steady-state performance may worsen. Comprehensive study of the temperature dependence of various processes, which contribute to the PR effect in DCDHF glasses, clarifies the limiting factors and allows for optimization of the overall PR performance.
View details for DOI 10.1002/cphc.200200633
View details for Web of Science ID 000184243600009
View details for PubMedID 12901305
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Fluorescence bleaching reveals asymmetric compartment formation prior to cell division in Caulobacter
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2003; 100 (14): 8235-8240
Abstract
Asymmetric cell division in Caulobacter crescentus yields daughter cells that have different cell fates. Compartmentalization of the predivisional cell is a critical event in the establishment of the differential distribution of regulatory factors that specify cell fate. To determine when during the cell cycle the cytoplasm is compartmentalized so that cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments, we designed a fluorescence loss in photobleaching assay. Individual cells containing enhanced GFP were exposed to a bleaching laser pulse tightly focused at one cell pole. In compartmentalized cells, fluorescence disappears only in the compartment receiving the bleaching beam; in noncompartmentalized cells, fluorescence disappears from the entire cell. In a 135-min cell cycle, the cells were compartmentalized 18 +/- 5 min before the progeny cells separated. Clearance of the 22000 CtrA master transcriptional regulator molecules from the stalked portion of the predivisional cell is a controlling element of Caulobacter asymmetry. Monitoring of a fluorescent marker for CtrA showed that the differential degradation of CtrA in the nascent stalk cell compartment occurs only after the cytoplasm is compartmentalized.
View details for DOI 10.1073/pnas.1433105100
View details for PubMedID 12824468
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High-performance photorefractive organic glass with near-infrared sensitivity
APPLIED PHYSICS LETTERS
2003; 82 (21): 3602-3604
View details for DOI 10.1063/1.1577214
View details for Web of Science ID 000182993700006
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Photorefractive properties of poly(siloxane)-triarylamine-based composites for high-speed applications
JOURNAL OF PHYSICAL CHEMISTRY B
2003; 107 (20): 4732-4737
View details for DOI 10.1021/jp027456i
View details for Web of Science ID 000182932900009
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Synthesis and photorefractive properties of multifunctional glasses
CHEMISTRY OF MATERIALS
2003; 15 (5): 1156-1164
View details for DOI 10.1021/cm020702i
View details for Web of Science ID 000181441100018
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Novel fluorophores for single-molecule imaging
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2003; 125 (5): 1174-1175
Abstract
Nonlinear optical chromophores based on dicyanodihydrofuran acceptors paired with amine donors have been found to exhibit sufficiently large fluorescence quantum yields and stability to enable single-molecule detection in polymeric hosts. To illustrate the breadth of this class, six fluorophores are presented, spanning the emission range from 505 to 646 nm. In contrast to conventional single-molecule fluorophores, the new molecules feature sensitivity to local rigidity, large ground-state dipole moments, and large polarizability anisotropies, properties that can be used to design new reporter experiments at the single-molecule level.
View details for DOI 10.1021/ja029100q
View details for Web of Science ID 000180713000033
View details for PubMedID 12553812
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The effect of varying cholesterol concentrations on the translational diffusion of individual class II MHC membrane proteins in cells
BIOPHYSICAL SOCIETY. 2003: 325A
View details for Web of Science ID 000183123801590
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Probing local polarity changes in GroEL/ES with fluorescence spectroscopy
BIOPHYSICAL SOCIETY. 2003: 27A
View details for Web of Science ID 000183123800128
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Optically induced focusing-to-defocusing switching and self-trapping of light in a photorefractive organic glass
9th International Conference on Photorefractive Effects, Materials, and Devices
OPTICAL SOC AMERICA. 2003: 425–429
View details for Web of Science ID 000231246200072
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Exploring novel methods of interferometric detection of ultrasmall phase shifts
Conference on Manipulation and Analysis of Biomolecules, Cells and Tissues
SPIE-INT SOC OPTICAL ENGINEERING. 2003: 110–120
View details for Web of Science ID 000184695100012
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Synthesis and properties of glassy organic multifunctional photorefractive materials
International Conference on Photo-Responsive Organics and Polymers (ICPOP 2001)
ELSEVIER SCIENCE BV. 2003: 353–57
View details for Web of Science ID 000180023100061
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Novel fluorophores for single-molecule imaging
Conference on Nanocrystals and Organic and Hybrid Nanomaterials
SPIE-INT SOC OPTICAL ENGINEERING. 2003: 150–157
View details for Web of Science ID 000188595800018
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Single-molecule optical spectroscopy of autofluorescent proteins
JOURNAL OF CHEMICAL PHYSICS
2002; 117 (24): 10925-10937
View details for DOI 10.1063/1.1521150
View details for Web of Science ID 000179774000003
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Translational diffusion of individual class II MHC membrane proteins in cells
BIOPHYSICAL JOURNAL
2002; 83 (5): 2681-2692
Abstract
Single-molecule epifluorescence microscopy was used to observe the translational motion of GPI-linked and native I-E(k) class II MHC membrane proteins in the plasma membrane of CHO cells. The purpose of the study was to look for deviations from Brownian diffusion that might arise from barriers to this motion. Detergent extraction had suggested that these proteins may be confined to lipid microdomains in the plasma membrane. The individual I-E(k) proteins were visualized with a Cy5-labeled peptide that binds to a specific extracytoplasmic site common to both proteins. Single-molecule trajectories were used to compute a radial distribution of displacements, yielding average diffusion coefficients equal to 0.22 (GPI-linked I-E(k)) and 0.18 microm(2)/s (native I-E(k)). The relative diffusion of pairs of proteins was also studied for intermolecular separations in the range 0.3-1.0 microm, to distinguish between free diffusion of a protein molecule and diffusion of proteins restricted to a rapidly diffusing small domain. Both analyses show that motion is predominantly Brownian. This study finds no strong evidence for significant confinement of either GPI-linked or native I-E(k) in the plasma membrane of CHO cells.
View details for Web of Science ID 000179024500032
View details for PubMedID 12414700
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Synthesis of fluorescently labeled polymers and their use in single-molecule imaging
MACROMOLECULES
2002; 35 (21): 8122-8125
View details for DOI 10.1021/ma020780r
View details for Web of Science ID 000178444400037
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Recent advances in understanding and development of photorefractive polymers and glasses
ADVANCED FUNCTIONAL MATERIALS
2002; 12 (9): 621-629
View details for Web of Science ID 000178295700010
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Photochromic polymers for the optical homodyne detection of ultrasonic surface displacements
OPTICS LETTERS
2002; 27 (5): 354-356
Abstract
We present a novel scheme with which to detect small ultrasonic surface displacements by use of a photochromic polymer instead of a photorefractive material as an adaptive beam combiner in a two-wave mixing geometry. Poly(methyl methacrylate) is doped with a derivative of zinc tetrabenzoporphyrin that possesses a long-lived triplet state that can be efficiently populated in a reversible manner. The resulting dynamic hologram consists of local absorption and refractive-index gratings, which can process speckled beams reflected from rough surfaces. We believe that this is the first use of a local nonlinear medium for adaptive homodyne detection of ultrasonic surface displacements.
View details for Web of Science ID 000174143400024
View details for PubMedID 18007801
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Monolithic photorefractive organic glasses with large coupling gain and strong beam fanning
ADVANCED MATERIALS
2002; 14 (4): 313-?
View details for Web of Science ID 000174056700017
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A dozen years of single-molecule spectroscopy in physics, chemistry, and biophysics
JOURNAL OF PHYSICAL CHEMISTRY B
2002; 106 (5): 910-927
View details for DOI 10.1021/jp012992g
View details for Web of Science ID 000173692100006
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Organic photorefractive material design strategies
Conference on Nonlinear Optical Transmission Processes and Organic Photorefractive Materials
SPIE-INT SOC OPTICAL ENGINEERING. 2002: 125–138
View details for Web of Science ID 000175310500014
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Uncorrelated diffusion of MHC class II proteins in the plasma membrane
BIOPHYSICAL SOCIETY. 2002: 523A
View details for Web of Science ID 000173252702574
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A comparison of through-the-objective Total Internal Reflection and epifluorescence microscopies for single-molece fluorescence experiments
BIOPHYSICAL SOCIETY. 2002: 45A-46A
View details for Web of Science ID 000173252700226
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Photophysics of DsRed, a red fluorescent protein, from the ensemble to the single-molecule level
BIOPHYSICAL SOCIETY. 2002: 46A–47A
View details for Web of Science ID 000173252700231
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Enhancement of the blue fluorescent protein's fluorescence by high pressure or low temperature
BIOPHYSICAL SOCIETY. 2002: 427A
View details for Web of Science ID 000173252702096
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Biomolecular applications of single-molecule measurements : Kinetics and dynamics of a single enzyme reaction
Conference on Methods for Ultrasensitive Detection II
SPIE-INT SOC OPTICAL ENGINEERING. 2002: 92–103
View details for Web of Science ID 000176067700010
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High-performance photorefractive organic glasses: understanding mechanisms and limitations
Conference on Organic Photorefractive and Photosensitive Materials for Holographic Applications
SPIE-INT SOC OPTICAL ENGINEERING. 2002: 21–32
View details for Web of Science ID 000179999100003
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High-performance photorefractive polymer composite with 2-dicyanomethylen-3-cyano-2,5-dihydrofuran chromophore
APPLIED PHYSICS LETTERS
2001; 79 (26): 4274-4276
View details for Web of Science ID 000172815100002
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Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules
BIOPHYSICAL JOURNAL
2001; 81 (5): 2851-2863
Abstract
Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.
View details for Web of Science ID 000171755200037
View details for PubMedID 11606296
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ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy
NATURE STRUCTURAL BIOLOGY
2001; 8 (6): 540-544
Abstract
Kinesin is an ATP-driven molecular motor protein that moves processively along microtubules. Despite considerable research, the detailed mechanism of kinesin motion remains elusive. We applied an enhanced suite of single- and multiple-molecule fluorescence polarization microscopy assays to report the orientation and mobility of kinesin molecules bound to microtubules as a function of nucleotide state. In the presence of analogs of ATP, ADP-Pi or in the absence of nucleotide, the kinesin head maintains a rigid orientation. In the presence of ADP, the motor domain of kinesin, still bound to the microtubule, adopts a previously undescribed, highly mobile state. This state may be general to the chemomechanical cycle of motor proteins; in the case of kinesin, the transition from a highly mobile to a rigid state after ADP release may contribute to the generation of the 8 nm step.
View details for Web of Science ID 000168924300019
View details for PubMedID 11373624
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Photophysics of DsRed, a red fluorescent protein, from the ensemble to the single-molecule level
JOURNAL OF PHYSICAL CHEMISTRY B
2001; 105 (21): 5048-5054
View details for Web of Science ID 000169036800036
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Single-molecule imaging in Caulobacter crescentus.
AMER CHEMICAL SOC. 2001: U285–U285
View details for Web of Science ID 000168824801666
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A comparison of through-the-objective total internal reflection microscopy and epifluorescence microscopy for single-molecule fluorescence Imaging
SINGLE MOLECULES
2001; 2 (3): 191-201
View details for Web of Science ID 000174074600005
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Orientation and dynamics of kinesin motors revealed by fluorescence polarization microscopy of many and single molecules.
BIOPHYSICAL SOCIETY. 2001: 572A
View details for Web of Science ID 000166692202608
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Photon antibunching in single CdSe/ZnS quantum dot fluorescence
CHEMICAL PHYSICS LETTERS
2000; 329 (5-6): 399-404
View details for Web of Science ID 000165164000012
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Single photons on demand from a single molecule at room temperature
NATURE
2000; 407 (6803): 491-493
Abstract
The generation of non-classical states of light is of fundamental scientific and technological interest. For example, 'squeezed' states enable measurements to be performed at lower noise levels than possible using classical light. Deterministic (or triggered) single-photon sources exhibit non-classical behaviour in that they emit, with a high degree of certainty, just one photon at a user-specified time. (In contrast, a classical source such as an attenuated pulsed laser emits photons according to Poisson statistics.) A deterministic source of single photons could find applications in quantum information processing, quantum cryptography and certain quantum computation problems. Here we realize a controllable source of single photons using optical pumping of a single molecule in a solid. Triggered single photons are produced at a high rate, whereas the probability of simultaneous emission of two photons is nearly zero--a useful property for secure quantum cryptography. Our approach is characterized by simplicity, room temperature operation and improved performance compared to other triggered sources of single photons.
View details for Web of Science ID 000089727400043
View details for PubMedID 11028995
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Image amplification and novelty filtering with a photorefractive polymer
APPLIED PHYSICS LETTERS
2000; 76 (23): 3358-3360
View details for Web of Science ID 000087554500008
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Single-molecule fluorescence resonant energy transfer in calcium concentration dependent cameleon
JOURNAL OF PHYSICAL CHEMISTRY B
2000; 104 (15): 3676-3682
View details for Web of Science ID 000086655600043
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Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2000; 97 (1): 151-156
Abstract
Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10(-6)-10(-3)-s time range, by using fluorescence correlation spectroscopy at 10(-8) M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over approximately 6 x 10(2) to 5 x 10(6) W/cm(2). These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of approximately 10(-3). Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-microseconds time range. The independence of these two bright-dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.
View details for Web of Science ID 000084624500029
View details for PubMedID 10618386
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Photorefractive polymers for laser-based ultrasound detection
Conference on Organic Photorefractives, Photoreceptors, and Nancomposites
SPIE-INT SOCIETY OPTICAL ENGINEERING. 2000: 110–117
View details for Web of Science ID 000167102400015
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The fluorescence dynamics of single molecules of green fluorescent protein
JOURNAL OF PHYSICAL CHEMISTRY A
1999; 103 (49): 10553-10560
View details for Web of Science ID 000084318700062
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Optical methods for exploring dynamics of single copies of green fluorescent protein
CYTOMETRY
1999; 36 (3): 232-238
Abstract
Single copies of four different phenolate ion mutants of the green fluorescent protein (GFP) exhibit a complex blinking and fluctuating behavior, a phenomenon that is hidden in measurements on large ensembles. Both total internal reflection microscopy and scanning confocal microscopy can be used to study the blinking dynamics, and autocorrelation analysis yields histograms of the correlation times for many individual molecules. While the total internal reflection method can follow several single molecules simultaneously, the confocal method offers higher time resolution at the expense of parallelism. We compare and contrast the two methods in terms of the ability to follow the complex dynamics of this system.
View details for Web of Science ID 000081024800013
View details for PubMedID 10404973
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Photorefractive properties of poly(N-vinyl carbazole)-based composites for high-speed applications
CHEMISTRY OF MATERIALS
1999; 11 (7): 1784-1791
View details for Web of Science ID 000081626800022
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Homodyne detection of ultrasonic surface displacements using two-wave mixing in photorefractive polymers
OPTICS COMMUNICATIONS
1999; 162 (1-3): 79-84
View details for Web of Science ID 000079947000016
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Illuminating single molecules in condensed matter
SCIENCE
1999; 283 (5408): 1670-?
Abstract
Efficient collection and detection of fluorescence coupled with careful minimization of background from impurities and Raman scattering now enable routine optical microscopy and study of single molecules in complex condensed matter environments. This ultimate method for unraveling ensemble averages leads to the observation of new effects and to direct measurements of stochastic fluctuations. Experiments at cryogenic temperatures open new directions in molecular spectroscopy, quantum optics, and solid-state dynamics. Room-temperature investigations apply several techniques (polarization microscopy, single-molecule imaging, emission time dependence, energy transfer, lifetime studies, and the like) to a growing array of biophysical problems where new insight may be gained from direct observations of hidden static and dynamic inhomogeneity.
View details for Web of Science ID 000079102800037
View details for PubMedID 10073924
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Design and optimization of chromophores for liquid crystal and photorefractive applications
Symposium F on Organic Nonlinear Optical Materials and Devices, at the 1999 MRS Spring Meeting
MATERIALS RESEARCH SOC. 1999: 119–130
View details for Web of Science ID 000082590800020
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Single-molecule studies of fluorescent proteins and enzymes
BIOPHYSICAL SOCIETY. 1999: A20
View details for Web of Science ID 000081085900114
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The fluorescence dynamics of single molecules of green fluorescent protein: Effect of mutations, ph, and matrix
BIOPHYSICAL SOCIETY. 1999: A445
View details for Web of Science ID 000081085902603
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Homodyne detection of ultrasonic surface displacements using two-wave mixing in photorefractive polymers
Conference on Process Control and Sensors for Manufacturing II
SPIE-INT SOC OPTICAL ENGINEERING. 1999: 22–29
View details for Web of Science ID 000079833700003
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Simultaneous imaging of individual molecules aligned both parallel and perpendicular to the optic axis
PHYSICAL REVIEW LETTERS
1998; 81 (24): 5322-5325
View details for Web of Science ID 000077511700017
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Synthesis of bifunctional photorefractive polymers with net gain: Design strategy amenable to combinatorial optimization
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1998; 120 (37): 9680-9681
View details for Web of Science ID 000076117200035
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High-speed photorefractive polymer composites
APPLIED PHYSICS LETTERS
1998; 73 (11): 1490-1492
View details for Web of Science ID 000075861100012
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Spectroscopic determination of trap density in C-60-sensitized photorefractive polymers
CHEMICAL PHYSICS LETTERS
1998; 291 (5-6): 553-561
View details for Web of Science ID 000075134400014
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Systematics of two-wave mixing in a photorefractive polymer
JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS
1998; 15 (2): 905-913
View details for Web of Science ID 000072015600047
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Amplified scattering in a high-gain photorefractive polymer
JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS
1998; 15 (2): 901-904
View details for Web of Science ID 000072015600046
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Gain enhancement by moving gratings in a photorefractive polymer
OPTICS COMMUNICATIONS
1998; 145 (1-6): 145-149
View details for Web of Science ID 000071260200030
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Probing single molecules in polyacrylamide gels
Conference on Laser Techniques for Condensed-Phase and Biological Systems
SPIE-INT SOC OPTICAL ENGINEERING. 1998: 165–173
View details for Web of Science ID 000074381300016
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Trapping studies on photorefractive polymers
Conference on Xerographic Photoreceptors and Organic Photorefractive Materials IV
SPIE - INT SOC OPTICAL ENGINEERING. 1998: 60–71
View details for Web of Science ID 000078396200007
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Single-molecule nanophotonics in solids
6th NEC Symposium on Quantum Optical Phenomena in Spatially Confined Materials - Fundamental Approaches to New Material Phases
ELSEVIER SCIENCE SA LAUSANNE. 1997: 169–74
View details for Web of Science ID A1997XY08600028
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Spontaneous oscillation and self-pumped phase conjugation in a photorefractive polymer optical amplifier
SCIENCE
1997; 277 (5325): 549-552
View details for Web of Science ID A1997XM86700047
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On/off blinking and switching behaviour of single molecules of green fluorescent protein
NATURE
1997; 388 (6640): 355-358
Abstract
Optical studies of individual molecules at low and room temperature can provide information about the dynamics of local environments in solids, liquids and biological systems unobscured by ensemble averaging. Here we present a study of the photophysical behaviour of single molecules of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria. Wild-type GFP and its mutant have attracted interest as fluorescent biological labels because the fluorophore may be formed in vivo. GFP mutants immobilized in aereated aqueous polymer gels and excited by 488-nm light undergo repeated cycles of fluorescent emission ('blinking') on a timescale of several seconds-behaviour that would be unobservable in bulk studies. Eventually the individual GFP molecules reach a long-lasting dark state, from which they can be switched back to the original emissive state by irradiation at 405 nm. This suggests the possibility of using these GFPs as fluorescent markers for time-dependent cell processes, and as molecular photonic switches or optical storage elements, addressable on the single-molecule level.
View details for Web of Science ID A1997XM52800043
View details for PubMedID 9237752
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Excitation of a single molecule on the surface of a spherical microcavity
APPLIED PHYSICS LETTERS
1997; 71 (3): 297-299
View details for Web of Science ID A1997XL48900001
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Measurement of the spatial phase shift in high-gain photorefractive materials
OPTICS LETTERS
1997; 22 (12): 874-876
Abstract
The correct determination of the spatial phase shift ø(p) in photorefractive materials is crucial to the proper characterization of novel materials. It is shown that the grating translation techniques commonly used for the measurement of ø(p) need to be reevaluated for high-gain materials. Strong energy and phase coupling leads to nonuniform slanted gratings, which result in an apparent dependence of the phase shift of the beam ratio and the optical polarization. A revised theory is presented, and analytical solutions are obtained for the special case of ø(p)?pi/2 . Numerical solutions for arbitrary ø(p) are in good agreement with measurements in a photorefractive polymer.
View details for Web of Science ID A1997XE10700010
View details for PubMedID 18185691
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High performance photorefractive polymer with improved stability
APPLIED PHYSICS LETTERS
1997; 70 (12): 1515-1517
View details for Web of Science ID A1997WP19900009
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Photorefractive polymers
ANNUAL REVIEW OF MATERIALS SCIENCE
1997; 27: 585-623
View details for Web of Science ID A1997XP62600020
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Recent advances in photorefractive polymer materials
Conference on Nonlinear Optical Properties of Organic Materials X
SPIE - INT SOC OPTICAL ENGINEERING. 1997: 84–94
View details for Web of Science ID A1997BJ78R00009
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Optical limiting in a photorefractive polymer
Symposium on Materials for Optical Limiting II, at 1997 MRS Spring Meeting
MATERIALS RESEARCH SOCIETY. 1997: 199–207
View details for Web of Science ID 000071653700027
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Large gain photorefractive polymers
Conference on Xerographic Photoreceptors and Organic Photorefractive Materials II
SPIE - INT SOC OPTICAL ENGINEERING. 1997: 216–226
View details for Web of Science ID A1997BJ78N00022
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Single-molecule spectroscopy and quantum optics in solids
ADVANCES IN ATOMIC, MOLECULAR, AND OPTICAL PHYSICS
1997; 38: 193-236
View details for Web of Science ID 000073110300006
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Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels
SCIENCE
1996; 274 (5289): 966-969
Abstract
Individual fluorescent molecules and individual singly labeled proteins were observed in the water-filled pores of poly(acrylamide) gels by far-field microscopy. Brownian motion was markedly reduced by the gel framework, thus enabling extended study of single fluorophores in aqueous environments. A highly axially dependent laser field was used both to excite the fluorophores and to image the molecules in three dimensions. Single molecules were followed as they moved within and through the porous gel structure. In contrast to dry polymeric hosts, these water-based gels may form a useful medium for single-molecule studies of biological systems in vitro.
View details for Web of Science ID A1996VR79200044
View details for PubMedID 8875935
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Holographic digital data storage in a photorefractive polymer
OPTICS LETTERS
1996; 21 (12): 890-892
Abstract
We report high-contrast storage of 64-kbit digital data pages in a photorefractive polymer material. Singlepage writing, reading, and erasing operations were demonstrated with a dual-function-dopant polymeric material having a dark lifetime of several days. Data were reconstructed without error by use of several simple readout algorithms.
View details for Web of Science ID A1996UP08500018
View details for PubMedID 19876193
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Mechanisms of photorefractivity in polymer composites
Conference on Organic Photorefractive Materials and Xerographic Photoreceptors
SPIE - INT SOC OPTICAL ENGINEERING. 1996: 2–13
View details for Web of Science ID A1996BG61K00001
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Single molecules solvated in pores of polyacrylamide gels
Proceedings of the Fifth International Meeting on Hole Burning and Related Spectroscopies (HBRS'96) - Science and Applications
GORDON BREACH PUBLISHING, TAYLOR & FRANCIS GROUP. 1996: 31–39
View details for Web of Science ID A1996WE58000006
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PHOTOREFRACTIVE POLYMERS BASED ON DUAL-FUNCTION DOPANTS
JOURNAL OF PHYSICAL CHEMISTRY
1995; 99 (12): 4096-4105
View details for Web of Science ID A1995QN63200034
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Photorefractivity in new organic polymeric materials
Conference on Xerographic Photoreceptors and Photorefractive Polymers
SPIE - INT SOC OPTICAL ENGINEERING. 1995: 82–93
View details for Web of Science ID A1995BD96Y00010
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PHOTOREFRACTIVE POLYMERS - A STATUS-REPORT
15th IUPAC Symposium on Photochemistry
INT UNION PURE APPLIED CHEMISTRY. 1995: 33–38
View details for Web of Science ID A1995QA39500006
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OPTICAL TRAP ACTIVATION IN A PHOTOREFRACTIVE POLYMER
OPTICS LETTERS
1994; 19 (22): 1822-1824
View details for Web of Science ID A1994PR09400008
View details for PubMedID 19855665
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CASCADING OF 2ND-ORDER PROCESSES IN QUADRATIC MOLECULAR MEDIA AT THE ORIGIN OF VERY LARGE CUBIC EFFECTS
Symposium D on Organic Materials for Electronics - Polymer Interfaces with Metals and Semiconductors, at the 1994 E-MRS Spring Conference
ELSEVIER SCIENCE SA. 1994: 303–7
View details for Web of Science ID A1994PW12000059
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QUASINONDESTRUCTIVE READOUT IN A PHOTOREFRACTIVE POLYMER
PHYSICAL REVIEW LETTERS
1994; 73 (15): 2047-2050
View details for Web of Science ID A1994PM77400010
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ELECTRIC-FIELD-SWITCHABLE STRATIFIED VOLUME HOLOGRAMS IN PHOTOREFRACTIVE POLYMERS
OPTICS LETTERS
1994; 19 (18): 1480-1482
Abstract
Two- and four-layer stratified volume holograms have been fabricated from the photorefractive polymeric material poly(methyl methacrylate): (1,3-dimethyl-2,2-tetramethylene-5-nitrobenzimidazoline):C(60) and characterized by holographic four-wave mixing experiments. Coherent addition of diffracted fields from the individual layers is observed, leading to a diffraction efficiency that increases with the square of the active layer thickness. Electricfield switching of the diffraction efficiencies of individual layers is demonstrated. The angular selectivity of the diffraction efficiency is also characterized for one, two, and four active layers. The angular width of the peaks narrows with increasing total structure thickness in agreement with theory.
View details for Web of Science ID A1994PJ26400035
View details for PubMedID 19855559
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2ND-ORDER CASCADING AS THE ORIGIN OF LARGE 3RD-ORDER EFFECTS IN ORGANIC SINGLE-CRYSTAL-CORE FIBERS
OPTICS LETTERS
1994; 19 (12): 868-870
Abstract
We have identified cascading of second-order nonlinear processes as the origin of previously reported, very large nonlinearities measured by self-phase-modulation experiments in organic single-crystal-core fibers of 4-(N,N-dimethylamino)-3-acetamidonitrobenzene.
View details for Web of Science ID A1994NQ38600006
View details for PubMedID 19844471
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OPTICAL-PROPERTIES OF POLY(N-VINYLCARBAZOLE)-BASED GUEST-HOST PHOTOREFRACTIVE POLYMER SYSTEMS
APPLIED OPTICS
1994; 33 (11): 2218-2222
Abstract
The photorefractive properties of poly(N-vinylcarbazole) doped with a variety of nonlinear optical chromophores and sensitizing agents are surveyed. Steady-state diffraction efficiencies of greater than 10(-3) and two-beam coupling gain exceeding the absorption loss are found in six materials combinations. The effect of the structure of the nonlinear optical chromophore on the photorefractive properties is discussed.
View details for Web of Science ID A1994NE30400020
View details for PubMedID 20885569
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ELECTRIC FIELD-DEPENDENT NONPHOTOREFRACTIVE GRATINGS IN A NONLINEAR PHOTOCONDUCTING POLYMER
APPLIED PHYSICS LETTERS
1994; 64 (6): 712-714
View details for Web of Science ID A1994MV50100015
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ORIENTATIONALLY ENHANCED PHOTOREFRACTIVE EFFECT IN POLYMERS
JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS
1994; 11 (2): 320-330
View details for Web of Science ID A1994MX45100009
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POLYMERIC PHOTOREFRACTIVE MATERIALS
CHEMICAL REVIEWS
1994; 94 (1): 127-155
View details for Web of Science ID A1994MX26600006
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RECENT PROGRESS IN PHOTOREFRACTIVE POLYMERS - MATERIALS AND STRUCTURES
Conference on Nonlinear Optical Properties of Organic Materials VII
SPIE-INT SOC OPTICAL ENGINEERING. 1994: 204–215
View details for Web of Science ID A1994BB57T00022
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POLY(SILANE)-BASED HIGH-MOBILITY PHOTOREFRACTIVE POLYMERS
JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS
1993; 10 (12): 2306-2312
View details for Web of Science ID A1993MP06300008
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NET 2-BEAM-COUPLING GAIN IN A POLYMERIC PHOTOREFRACTIVE MATERIAL
OPTICS LETTERS
1993; 18 (13): 1044-1046
View details for Web of Science ID A1993LJ22700004
View details for PubMedID 19823284
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MAGNETIC-RESONANCE OF A SINGLE MOLECULAR SPIN
NATURE
1993; 363 (6426): 242-244
View details for Web of Science ID A1993LC86600044
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OPTICAL SPECTROSCOPY OF SINGLE IMPURITY MOLECULES IN SOLIDS
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
1993; 32 (4): 457-476
View details for Web of Science ID A1993LC44000001
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PHOTOCONDUCTION AND PHOTOREFRACTION IN MOLECULARLY DOPED POLYMERS
SYNTHETIC METALS
1993; 54 (1-3): 9-19
View details for Web of Science ID A1993KT78600003
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NONLINEAR-OPTICAL PROPERTIES OF PHOTOREFRACTIVE POLYMERS
CONF ON NONLINEAR OPTICAL PROPERTIES OF ADVANCED MATERIALS
SPIE - INT SOC OPTICAL ENGINEERING. 1993: 253–265
View details for Web of Science ID A1993BY82W00020
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LIGHTWAVE TRANSMISSION OF MULTIPLE TELEVISION SIGNALS USING AN ORGANIC POLYMER ELECTROOPTIC PHASE MODULATOR
Conference on Nonlinear Optical Properties of Organic Materials VI
SPIE - INT SOC OPTICAL ENGINEERING. 1993: 499–506
View details for Web of Science ID A1993BZ78F00054
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C-60 SENSITIZATION OF A PHOTOREFRACTIVE POLYMER
APPLIED PHYSICS LETTERS
1992; 61 (25): 2967-2969
View details for Web of Science ID A1992KC86000006
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PHOTOCONDUCTIVITY STUDIES OF PHOTOREFRACTIVE POLYMERS
JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS
1992; 9 (11): 2059-2064
View details for Web of Science ID A1992JW72400017
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PHOTON ANTIBUNCHING IN THE FLUORESCENCE OF A SINGLE DYE MOLECULE TRAPPED IN A SOLID
PHYSICAL REVIEW LETTERS
1992; 69 (10): 1516-1519
View details for Web of Science ID A1992JL61000011
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2-BEAM COUPLING MEASUREMENTS OF GRATING PHASE IN A PHOTOREFRACTIVE POLYMER
JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS
1992; 9 (9): 1642-1647
View details for Web of Science ID A1992JM57400014
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SUBSECOND GRATING GROWTH IN A PHOTOREFRACTIVE POLYMER
OPTICS LETTERS
1992; 17 (16): 1107-1109
Abstract
We survey the dynamics of the photorefractive effect in a methyl methacrylate copolymer with the nonlinear chromophore p-nitroaniline in a pendant side group doped with a charge-transport agent, diethylaminobenzaldehyde diphenylhydrazone, a material that represents a new class of photorefractive polymer. The grating growth times are several orders of magnitude smaller than that for the previous epoxy-based photorefractive polymers and fall below 1 s at the highest intensities used. Grating competition and revelation effects suggest that charge carriers other than photogenerated holes are mobile. A sublinear dependence of growth rate on writing intensity implies that shallow traps may also be present.
View details for Web of Science ID A1992JG55500003
View details for PubMedID 19794733
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SINGLE MOLECULE SPECTRAL DIFFUSION IN A SOLID DETECTED VIA FLUORESCENCE SPECTROSCOPY
8TH INTERNATIONAL CONF ON DYNAMICAL PROCESSES IN EXCITED STATES OF SOLIDS ( DPC 91 )
ELSEVIER SCIENCE BV. 1992: 62–67
View details for Web of Science ID A1992JC50900016
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OPTICAL-SPECTRA AND KINETICS OF SINGLE IMPURITY MOLECULES IN A POLYMER - SPECTRAL DIFFUSION AND PERSISTENT SPECTRAL HOLE BURNING
TOPICAL MEETING ON PERSISTENT SPECTRAL HOLE-BURNING SCIENCE AND APPLICATIONS
OPTICAL SOC AMER. 1992: 829–36
View details for Web of Science ID A1992HT83400030
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OPTICAL MODIFICATION OF A SINGLE IMPURITY MOLECULE IN A SOLID
NATURE
1992; 355 (6358): 335-337
View details for Web of Science ID A1992HA59100061
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PHOTOCONDUCTIVITY OF PHOTOREFRACTIVE POLYMERS
SYMP ON MACROMOLECULAR HOST-GUEST COMPLEXES : OPTICAL AND OPTOELECTRONIC PROPERTIES AND APPLICATIONS, AT THE 1992 SPRING MEETING OF THE MATERIALS RESEARCH SOC
MATERIALS RESEARCH SOC. 1992: 135–144
View details for Web of Science ID A1992BW93U00016
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OPTICAL WAVE-GUIDING IN POLED NLO POLYMERS
SYMP ON MATERIALS FOR OPTICAL INFORMATION PROCESSING
MATERIALS RESEARCH SOC. 1992: 101–110
View details for Web of Science ID A1992BW94B00012
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NONLINEAR OPTICAL-PROPERTIES OF ORGANIC PHOTOREFRACTIVE POLYMERS
SYMP ON MACROMOLECULAR HOST-GUEST COMPLEXES : OPTICAL AND OPTOELECTRONIC PROPERTIES AND APPLICATIONS, AT THE 1992 SPRING MEETING OF THE MATERIALS RESEARCH SOC
MATERIALS RESEARCH SOC. 1992: 121–133
View details for Web of Science ID A1992BW93U00015
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NLO POLYMERIC WAVE-GUIDE ELECTROOPTIC PHASE MODULATOR
5TH CONF ON NONLINEAR OPTICAL PROPERTIES OF ORGANIC MATERIALS
SPIE - INT SOC OPTICAL ENGINEERING. 1992: 369–378
View details for Web of Science ID A1992BX88R00035
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DETECTION AND SPECTROSCOPY OF SINGLE PENTACENE MOLECULES IN A PARA-TERPHENYL CRYSTAL BY MEANS OF FLUORESCENCE EXCITATION
JOURNAL OF CHEMICAL PHYSICS
1991; 95 (10): 7150-7163
View details for Web of Science ID A1991GN53300014
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OBSERVATION OF THE PHOTOREFRACTIVE EFFECT IN A POLYMER
PHYSICAL REVIEW LETTERS
1991; 66 (14): 1846-1849
View details for Web of Science ID A1991FF23000012
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FLUORESCENCE SPECTROSCOPY AND SPECTRAL DIFFUSION OF SINGLE IMPURITY MOLECULES IN A CRYSTAL
NATURE
1991; 349 (6306): 225-227
View details for Web of Science ID A1991ET51900049
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PHASE-SENSITIVE OPTICAL-DETECTION OF BALLISTIC PHONON HEAT PULSES USING FREQUENCY-MODULATION SPECTROSCOPY AND PERSISTENT SPECTRAL HOLES
PHYSICAL REVIEW B
1991; 43 (2): 1743-1755
View details for Web of Science ID A1991ET21800049
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APPLICATIONS OF ORGANIC 2ND-ORDER NONLINEAR OPTICAL-MATERIALS
ACS SYMPOSIUM SERIES
1991; 455: 216-225
View details for Web of Science ID A1991FM04700013
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POLED EPOXY POLYMERS FOR OPTOELECTRONICS
NATO ADVANCED RESEARCH WORKSHOP ON ORGANIC MOLECULAR FOR NONLINEAR OPTICS AND PHOTONICS
KLUWER ACADEMIC PUBL. 1991: 433–445
View details for Web of Science ID A1991BT98C00031
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FINDING A SINGLE MOLECULE IN A HAYSTACK - LASER SPECTROSCOPY OF SOLIDS FROM SQUARE-ROOT-N TO N=1
CONF ON OPTICAL METHODS FOR ULTRASENSITIVE DETECTION AND ANALYSIS : TECHNIQUES AND APPLICATIONS
SPIE - INT SOC OPTICAL ENGINEERING. 1991: 244–251
View details for Web of Science ID A1991BT94G00024
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PHOTOREFRACTIVITY IN DOPED NONLINEAR ORGANIC POLYMERS
CONF ON NONLINEAR OPTICAL PROPERTIES OF ORGANIC MATERIALS 4
SPIE - INT SOC OPTICAL ENGINEERING. 1991: 278–289
View details for Web of Science ID A1991BU87X00028
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INTRACAVITY FREQUENCY DOUBLING OF A ND-YAG LASER WITH AN ORGANIC NONLINEAR OPTICAL-CRYSTAL
APPLIED PHYSICS LETTERS
1990; 57 (6): 537-539
View details for Web of Science ID A1990DR71200003
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TEMPERATURE-DEPENDENCE OF PHOTON-GATED PERSISTENT SPECTRAL HOLE-BURNING FOR THE MESO-TETRA-PARA-TOLYL-ZN-TETRABENZOPORPHYRIN CHLOROFORM SYSTEM IN POLY(METHYLMETHACRYLATE)
CHEMICAL PHYSICS
1990; 144 (1): 71-79
View details for Web of Science ID A1990DK95800006
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OPTICAL-DETECTION AND PROBING OF SINGLE DOPANT MOLECULES OF PENTACENE IN A P-TERPHENYL HOST CRYSTAL BY MEANS OF ABSORPTION-SPECTROSCOPY
JOURNAL OF PHYSICAL CHEMISTRY
1990; 94 (4): 1237-1248
View details for Web of Science ID A1990CP90300011
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ORGANIC NONLINEAR OPTICAL-MATERIALS AND THEIR DEVICE APPLICATIONS FOR FREQUENCY DOUBLING, MODULATION, AND SWITCHING
CONF ON NONLINEAR OPTICAL PROPERTIES OF ORGANIC MATERIALS 3
SPIE - INT SOC OPTICAL ENGINEERING. 1990: 2–11
View details for Web of Science ID A1990BS46Q00001
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ULTRASENSITIVE LASER SPECTROSCOPY IN SOLIDS - SINGLE-MOLECULE DETECTION
4TH INTERNATIONAL CONF ON UNCONVENTIONAL PHOTOACTIVE SOLIDS ( 4TH UPS )
GORDON BREACH SCI PUBL LTD. 1990: 47–57
View details for Web of Science ID A1990DJ22300006
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FINDING A SINGLE MOLECULE IN A HAYSTACK - OPTICAL-DETECTION AND SPECTROSCOPY OF SINGLE ABSORBERS IN SOLIDS
ANALYTICAL CHEMISTRY
1989; 61 (21): A1217-A1223
View details for Web of Science ID A1989AX15700003
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OPTICAL-DETECTION AND SPECTROSCOPY OF SINGLE MOLECULES IN A SOLID
PHYSICAL REVIEW LETTERS
1989; 62 (21): 2535-2538
View details for Web of Science ID A1989U666900029
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PSEUDO-STARK EFFECT AND FM/STARK DOUBLE-MODULATION SPECTROSCOPY FOR THE DETECTION OF STATISTICAL FINE-STRUCTURE IN ALEXANDRITE
CHEMICAL PHYSICS LETTERS
1988; 151 (1-2): 102-108
View details for Web of Science ID A1988Q468600021
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STATISTICAL FINE-STRUCTURE IN THE INHOMOGENEOUSLY BROADENED ELECTRONIC ORIGIN OF PENTACENE IN P-TERPHENYL
JOURNAL OF CHEMICAL PHYSICS
1988; 89 (4): 1768-1779
View details for Web of Science ID A1988P638800004
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STATISTICAL FINE-STRUCTURE OF INHOMOGENEOUSLY BROADENED ABSORPTION-LINES
PHYSICAL REVIEW LETTERS
1987; 59 (23): 2705-2708
View details for Web of Science ID A1987L084400023
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MECHANISM OF PHOTON-GATED PERSISTENT SPECTRAL HOLE BURNING IN METAL TETRABENZOPORPHYRIN HALOMETHANE SYSTEMS - DONOR-ACCEPTOR ELECTRON-TRANSFER
JOURNAL OF PHYSICAL CHEMISTRY
1987; 91 (15): 3998-4004
View details for Web of Science ID A1987J323600015
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PHOTON-GATED SPECTRAL HOLE BURNING BY DONOR-ACCEPTOR ELECTRON-TRANSFER
OPTICS LETTERS
1987; 12 (5): 370-372
Abstract
We have observed a new mechanism for photon-gated spectral hole burning, donor-acceptor electron transfer, in a material composed of a zinc-tetrabenzoporphyrin derivative (donor) with chloroform acceptors in poly(methyl methacrylate) thin films. Gated holes form when we simultaneously excite the donor 0-0 singlet absorption (630 nm) and the donor triplet-triplet absorption (350-550 nm), with the largest gating enhancement (>30) occurring for gating light near 480 nm. The gating action spectrum and the photoproduct spectrum confirm that the mechanism is electron transfer from an excited triplet of the porphyrin donor to the chloroform acceptor. This result opens up a new class of materials for photon gating that should be of interest for frequency-domain optical storage applications as well as high-resolution spectroscopy of electron transfer processes in solids.
View details for Web of Science ID A1987H017200024
View details for PubMedID 19738894
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FAST BURNING OF PERSISTENT SPECTRAL HOLES IN SMALL LASER SPOTS USING PHOTONGATED MATERIALS
APPLIED PHYSICS LETTERS
1987; 50 (8): 430-432
View details for Web of Science ID A1987G165100002
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PHASE SENSITIVE DETECTION OF PERSISTENT SPECTRAL HOLES USING SYNCHRONOUS ULTRASONIC MODULATION
APPLIED PHYSICS LETTERS
1986; 48 (18): 1181-1183
View details for Web of Science ID A1986C154600003
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PERSISTENT SPECTRAL HOLE BURNING FOR R' COLOR-CENTERS IN LIF CRYSTALS - STATICS, DYNAMICS, AND EXTERNAL-FIELD EFFECTS
PHYSICAL REVIEW B
1986; 33 (8): 5702-5716
View details for Web of Science ID A1986C014600069
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PHOTOCHEMICAL HOLE-BURNING IN A PROTONATED PHTHALOCYANINE WITH GAALAS DIODE-LASERS
CHEMICAL PHYSICS LETTERS
1985; 114 (5-6): 491-496
View details for Web of Science ID A1985ADU9100012
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2-COLOR, PHOTON-GATED SPECTRAL HOLE-BURNING IN AN ORGANIC MATERIAL
CHEMICAL PHYSICS LETTERS
1985; 118 (6): 611-616
View details for Web of Science ID A1985AQD6100016
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CAN SINGLE-PHOTON PROCESSES PROVIDE USEFUL MATERIALS FOR FREQUENCY-DOMAIN OPTICAL STORAGE
JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS
1985; 2 (6): 915-924
View details for Web of Science ID A1985AKC6100008
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HIGH-EFFICIENCY PHOTOCHEMICAL HOLE BURNING FOR AN INFRARED COLOR CENTER
PHYSICAL REVIEW B
1985; 32 (2): 1270-1277
View details for Web of Science ID A1985AMH6100086
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BEYOND THE BOTTLENECK - SUBMICROSECOND HOLE BURNING IN PHTHALOCYANINE
JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS
1984; 1 (3): 341-348
View details for Web of Science ID A1984SU90700001
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DETECTION OF PERSISTENT SPECTRAL HOLES USING ULTRASONIC MODULATION
JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS
1984; 1 (3): 349-353
View details for Web of Science ID A1984SU90700002
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READING AND WRITING OF PHOTOCHEMICAL HOLES USING GAALAS-DIODE LASERS
OPTICS LETTERS
1983; 8 (5): 280-282
Abstract
A current-tuned GaAlAs-diode laser is utilized both to burn and to detect narrow photochemical holes in the inhomogeneously broadened 833-nm zero-phonon line of the R' color center in LiF. Applications for reading and writing data into frequency-domain optical memories based on photochemical hole burning are discussed.
View details for Web of Science ID A1983QM43000014
View details for PubMedID 19718087
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FM SPECTROSCOPY DETECTION OF STIMULATED RAMAN GAIN
OPTICS LETTERS
1983; 8 (2): 108-110
Abstract
We have used FM spectroscopy to detect the stimulated Raman gain effect in deuterium, demonstrating for the first reported time the quantum-noise-limited performance of this technique.
View details for Web of Science ID A1983PZ95300015
View details for PubMedID 19714152