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bioRxiv · 10.1101/2024.01.25.577300

A practical guide to time-resolved fluorescence microscopy and spectroscopy

Clark, B. S.·Silvernail, I.·Gordon, K.·Morgan, A. N.·Rolband, L. A.·Castaneda, J. F.·LeBlanc, S. J.
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Abstract

Time-correlated single photon counting (TCSPC) coupled with confocal microscopy is a versatile biophysical tool that enables real-time monitoring of biomolecular dynamics across many timescales. With TCSPC, Fluorescence correlation spectroscopy (FCS) and pulsed interleaved excitation-Forster resonance energy transfer (PIE-FRET) are collected simultaneously on diffusing molecules to extract diffusion characteristics and proximity information. This article is a guide to calibrating FCS and PIE-FRET measurements with several biological samples including liposomes, streptavidin-coated quantum dots, proteins, and nucleic acids for reliable determination of diffusion coefficients and FRET efficiency. The FRET efficiency results are also compared to surface-attached single molecules using fluorescence lifetime imaging microscopy (FLIM-FRET). Combining the methods is a powerful approach to revealing mechanistic details of biological processes and pathways.

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Clark, B. S., Silvernail, I., Gordon, K., Morgan, A. N., Rolband, L. A., Castaneda, J. F., LeBlanc, S. J.. 2024-01-30. A practical guide to time-resolved fluorescence microscopy and spectroscopy. https://doi.org/10.1101/2024.01.25.577300

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