Single-Molecule Fluorescence Spectroscopy Approaches for Probing Fast Biomolecular Dynamics and Interactions.

Single-molecule fluorescence spectroscopy, and particularly its Forster resonance energy transfer implementation (SM-FRET), provides the opportunity to resolve the stochastic conformational fluctuations undergone by individual protein molecules while they fold-unfold, bind to their partners, or carry out catalysis. Such information is key to resolve the microscopic pathways and mechanisms underlying such processes, and cannot be obtained from bulk experiments. To fully resolve protein conformational dynamics, SM-FRET experiments need to reach microsecond, and even sub-microsecond, time resolutions. The key to reach such resolution lies in increasing the efficiency at which photons emitted by a single molecule are collected and detected by the instrument (photon count rates). In this chapter, we describe basic procedures that an end user can follow to optimize the confocal microscope optics in order to maximize the photon count rates. We also discuss the use of photoprotection cocktails specifically designed to reduce fluorophore triplet buildup at high irradiance (the major cause of limiting photon emission rates) while improving the mid-term photostability of the fluorophores. Complementary strategies based on the data analysis are discussed in depth by other authors in Chap. 14 .