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

Yuansheng Sun; Sunil Shah; Shih-Chu (Jeff) Liao; Ulas C. Coskun; Beniamino Barbieri

doi:10.1117/12.2511291

22 February 2019 VistaVision toolbox for quantitative multi-parameter analysis of single molecule dynamics

Yuansheng Sun, Sunil Shah, Shih-Chu (Jeff) Liao, Ulas C. Coskun, Beniamino Barbieri

Proceedings Volume 10882, Multiphoton Microscopy in the Biomedical Sciences XIX; 1088234 (2019) https://doi.org/10.1117/12.2511291
Event: SPIE BiOS, 2019, San Francisco, California, United States

Abstract

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.

Citation Download Citation

Yuansheng Sun, Sunil Shah, Shih-Chu (Jeff) Liao, Ulas C. Coskun, and Beniamino Barbieri "VistaVision toolbox for quantitative multi-parameter analysis of single molecule dynamics", Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 1088234 (22 February 2019); https://doi.org/10.1117/12.2511291

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KEYWORDS

Molecules

Fluorescence correlation spectroscopy

Fluorescence resonance energy transfer

Proteins

Imaging systems

Fluorescence lifetime imaging

Biophysics

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