Stanford Advisors

All Publications

  • Directed Evolution of a Bright Variant of mCherry: Suppression of Nonradiative Decay by Fluorescence Lifetime Selections. The journal of physical chemistry. B Mukherjee, S., Manna, P., Hung, S., Vietmeyer, F., Friis, P., Palmer, A. E., Jimenez, R. 2022


    The approximately linear scaling of fluorescence quantum yield (ϕ) with fluorescence lifetime (tau) in fluorescent proteins (FPs) has inspired engineering of brighter fluorophores based on screening for increased lifetimes. Several recently developed FPs such as mTurquoise2, mScarlet, and FusionRed-MQV which have become useful for live cell imaging are products of lifetime selection strategies. However, the underlying photophysical basis of the improved brightness has not been scrutinized. In this study, we focused on understanding the outcome of lifetime-based directed evolution of mCherry, which is a popular red-FP (RFP). We identified four positions (W143, I161, Q163, and I197) near the FP chromophore that can be mutated to create mCherry-XL (eXtended Lifetime: ϕ = 0.70; tau = 3.9 ns). The 3-fold higher quantum yield of mCherry-XL is on par with that of the brightest RFP to date, mScarlet. We examined selected variants within the evolution trajectory and found a near-linear scaling of lifetime with quantum yield and consistent blue-shifts of the absorption and emission spectra. We find that the improvement in brightness is primarily due to a decrease in the nonradiative decay of the excited state. In addition, our analysis revealed the decrease in nonradiative rate is not limited to the blue-shift of the energy gap and changes in the excited state reorganization energy. Our findings suggest that nonradiative mechanisms beyond the scope of energy-gap models such the Englman-Jortner model are suppressed in this lifetime evolution trajectory.

    View details for DOI 10.1021/acs.jpcb.2c01956

    View details for PubMedID 35709514

  • Characterizing dark state kinetics and single molecule fluorescence of FusionRed and FusionRed-MQ at low irradiances. Physical chemistry chemical physics : PCCP Mukherjee, S., Thomas, C., Wilson, R., Simmerman, E., Hung, S., Jimenez, R. 2022


    The presence of dark states causes fluorescence intermittency of single molecules due to transitions between "on" and "off" states. Genetically encodable markers such as fluorescent proteins (FPs) exhibit dark states that make several super-resolved single-molecule localization microscopy (SMLM) methods possible. However, studies quantifying the timescales and nature of dark state behavior for commonly used FPs under conditions typical of widefield and total internal reflection fluorescence (TIRF) microscopy remain scarce and pre-date many new SMLM techniques. FusionRed is a relatively bright red FP exhibiting fluorescence intermittency and has thus been identified as a potential candidate for SMLM. We herein characterize the rates for dark-state conversion and the subsequent ground-state recovery of FusionRed and its 2.5-fold brighter descendent FusionRed L175M M42Q (FusionRed-MQ) at low irradiances (1-10 W cm-2), which were previously unexplored experimental conditions. We characterized the kinetics of dark state transitions in these two FPs by using single molecule blinking and ensemble photobleaching experiments bridged with a dark state kinetic model. We find that at low irradiances, the recovery process to the ground state is minimally light-driven and FusionRed-MQ has a 1.3-fold longer ground state recovery time indicating a conformationally restricted dark-state chromophore in comparison to FusionRed. Our studies indicate that the brighter FusionRed-MQ variant exhibits higher dark state conversion rates with longer ground state recovery lifetimes, thus it is potentially a better candidate for SMLM applications than its progenitor FusionRed.

    View details for DOI 10.1039/d2cp00889k

    View details for PubMedID 35642612

  • Photophysical Engineering of Fluorescent Proteins: Accomplishments and Challenges of Physical Chemistry Strategies. The journal of physical chemistry. B Mukherjee, S., Jimenez, R. 2022; 126 (4): 735-750


    Fluorescent proteins (FPs) have become ubiquitous tools for biological research and concomitantly they are intriguing molecules that are amenable to study with a wide range of experimental and theoretical tools. This perspective explores the connection between the engineering of improved FPs and basic ideas from physical chemistry that explain their properties and drive the molecular design of brighter and more photostable variants. We highlight some of the progress and the many knowledge gaps in understanding the relationship between FP brightness and photostability. We also explore some of the pertinent remaining questions and suggest ways in which physical chemists might further examine the physical basis of brightness and photostability in these systems.

    View details for DOI 10.1021/acs.jpcb.1c05629

    View details for PubMedID 35075898

  • Engineering of a Brighter Variant of the FusionRed Fluorescent Protein Using Lifetime Flow Cytometry and Structure-Guided Mutations. Biochemistry Mukherjee, S., Hung, S. T., Douglas, N., Manna, P., Thomas, C., Ekrem, A., Palmer, A. E., Jimenez, R. 2020; 59 (39): 3669-3682


    The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and correct localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, "FR-MQV," which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (∼140 000 M-1 cm-1), increased radiative rate constant, and reduced nonradiative rate constant with respect to its precursor. The properties of FR-MQV derive from three mutations-M42Q, C159V, and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the para-hydroxyphenyl and the acylimine sites of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the para-hydroxyphenyl site of the chromophore were mutated. The M42Q mutation is located near the acylimine moiety of the chromophore and was discovered using site-directed mutagenesis guided by X-ray crystal structures. FR-MQV exhibits a 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells [based on fluorescence-activated cell sorting (FACS)] compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells. These properties make FR-MQV a promising template for further engineering into a new family of RFPs.

    View details for DOI 10.1021/acs.biochem.0c00484

    View details for PubMedID 32914619

  • Enrichment of rare events using a multi-parameter high throughput microfluidic droplet sorter. Lab on a chip Hung, S. T., Mukherjee, S., Jimenez, R. 2020; 20 (4): 834-843


    High information content analysis, enrichment, and selection of rare events from a large population are of great importance in biological and biomedical research. The fluorescence lifetime of a fluorophore, a photophysical property which is independent of and complementary to fluorescence intensity, has been incorporated into various imaging and sensing techniques through microscopy, flow cytometry and droplet microfluidics. However, the throughput of fluorescence lifetime activated droplet sorting is orders of magnitude lower than that of fluorescence activated cell sorting, making it unattractive for applications such as directed evolution of enzymes, despite its highly effective compartmentalization of library members. We developed a microfluidic sorter capable of selecting fluorophores based on fluorescence lifetime and brightness at two excitation and emission colors at a maximum droplet rate of 2.5 kHz. We also present a novel selection strategy for efficiently analyzing and/or enriching rare fluorescent members from a large population which capitalizes on the Poisson distribution of analyte encapsulation into droplets. The effectiveness of the droplet sorter and the new selection strategy are demonstrated by enriching rare populations from a ∼108-member site-directed mutagenesis library of fluorescent proteins expressed in bacteria. This selection strategy can in principle be employed on many droplet sorting platforms, and thus can potentially impact broad areas of science where analysis and enrichment of rare events is needed.

    View details for DOI 10.1039/c9lc00790c

    View details for PubMedID 31974539

    View details for PubMedCentralID PMC7135947

  • Fluorescence Enhancement of Tb-3(+) in the Tb3+-Trimesic Acid-Gd3+ Complex: Role of Polynuclear Structures CHEMISTRYSELECT Dubey, P., Mukherjee, S., Choudhury, A., Viswanathan, K. S. 2019; 4 (9): 2747-2752
  • Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter. Integrative biology : quantitative biosciences from nano to macro Manna, P., Hung, S. T., Mukherjee, S., Friis, P., Simpson, D. M., Lo, M. N., Palmer, A. E., Jimenez, R. 2018; 10 (9): 516-526


    Green fluorescent proteins (GFP) and their blue, cyan and red counterparts offer unprecedented advantages as biological markers owing to their genetic encodability and straightforward expression in different organisms. Although significant advancements have been made towards engineering the key photo-physical properties of red fluorescent proteins (RFPs), they continue to perform sub-optimally relative to GFP variants. Advanced engineering strategies are needed for further evolution of RFPs in the pursuit of improving their photo-physics. In this report, a microfluidic sorter that discriminates members of a cell-based library based on their excited state lifetime and fluorescence intensity is used for the directed evolution of the photo-physical properties of FusionRed. In-flow measurements of the fluorescence lifetime are performed in a frequency-domain approach with sub-millisecond sampling times. Promising clones are sorted by optical force trapping with an infrared laser. Using this microfluidic sorter, mutants are generated with longer lifetimes than their precursor, FusionRed. This improvement in the excited state lifetime of the mutants leads to an increase in their fluorescence quantum yield up to 1.8-fold. In the course of evolution, we also identified one key mutation (L177M), which generated a mutant (FusionRed-M) that displayed ∼2-fold higher brightness than its precursor upon expression in mammalian (HeLa) cells. Photo-physical and mutational analyses of clones isolated at the different stages of mutagenesis reveal the photo-physical evolution towards higher in vivo brightness.

    View details for DOI 10.1039/c8ib00103k

    View details for PubMedID 30094420

    View details for PubMedCentralID PMC6141309