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
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
Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution.
The journal of physical chemistry letters
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
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
Interferometric Scattering Enables Fluorescence-Free Electrokinetic Trapping of Single Nanoparticles in Free Solution.
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