Methods in Molecular Biology Volume 950, 2013, pp 131-151

Photoswitchable Fluorophores for Single-Molecule Localization Microscopy

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Abstract

Over the past decade, fluorescence microscopy has been revolutionized by the development of novel techniques that allow near-molecular resolution. Many such methods—collectively referred to as “single-molecule localization microscopy” (SMLM)—are based upon the repeated imaging of sparse stochastic subsets of the fluorophores in a sample. Active fluorophores are localized by finding the centers of their point spread functions, and a super-resolution image is constructed.

Key to this strategy is the use of fluorophores that can be switched “on” and “off” in a controllable manner. Here we review the strengths and weaknesses of the wide variety of SMLM-compatible photoswitchable fluorophores and labeling strategies currently available. We also discuss their suitability for live-cell and multicolor imaging, as well as molecular counting.