VistaVision toolbox for quantitative multi-parameter analysis of single molecule dynamics

摘要

Single molecule imaging techniques allow tracking dynamic behaviors of individual molecules, providing insightinformation of molecular processes that could be hidden by the ensemble average. Combining with the time-resolvedimaging capability, the laser scanning confocal microscopy at the single molecule sensitivity allows quantitative multiparameteranalyses of single molecule dynamics. Here, we describe the single molecule imaging tools in the ISSVistaVision software, including FCS, FCCS, PIE-FCCS, FLCS, PCH, FLIM, steady-state and time-resolved FRET,steady-state and time-resolved polarization anisotropy, burst analysis for single molecule FRET and stoichiometry,antibunching. We demonstrate with measurement examples how these techniques are used for studying photophysicalproperties and behaviors of single molecules, such as diffusion rates, molecular brightness, triplet time, rotationalrelaxation time, fluorescence lifetime. By using donor-acceptor FRET pair-labeled proteins, we detect changes inprotein conformation and dynamics by quantitatively measuring FRET efficiency, stoichiometry and lifetime. Thisquantitative multi-parameter analysis approach gives researchers more opportunities for a better understanding of singlemolecule dynamics.