Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution

Diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individuals 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 which 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 monitor 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. Further, 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 traps ability to sensitively monitor nanoscale biological objects, enabling new experiments on these systems. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/484789v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1fccfacorg.highwire.dtl.DTLVardef@a639b6org.highwire.dtl.DTLVardef@116fff6org.highwire.dtl.DTLVardef@fe95fd_HPS_FORMAT_FIGEXP M_FIG C_FIG