Proceedings Article | 22 February 2019
KEYWORDS: Molecules, Fluorescence correlation spectroscopy, Fluorescence resonance energy transfer, Proteins, Imaging systems, Fluorescence lifetime imaging, Confocal microscopy, Single molecule spectroscopy, FRET and FLIM microscopy, Biophysics
Single molecule imaging techniques allow tracking dynamic behaviors of individual molecules, providing insight information of molecular processes that could be hidden by the ensemble average. Combining with the time-resolved imaging capability, the laser scanning confocal microscopy at the single molecule sensitivity allows quantitative multiparameter analyses of single molecule dynamics. Here, we describe the single molecule imaging tools in the ISS VistaVision 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 photophysical properties and behaviors of single molecules, such as diffusion rates, molecular brightness, triplet time, rotational relaxation time, fluorescence lifetime. By using donor-acceptor FRET pair-labeled proteins, we detect changes in protein conformation and dynamics by quantitatively measuring FRET efficiency, stoichiometry and lifetime. This quantitative multi-parameter analysis approach gives researchers more opportunities for a better understanding of single molecule dynamics.
