A Beginner’s Guide to Dye Selection and Labeling Strategies for Single-Molecule Localization Microscopy

Abstract

AbstractSingle-molecule localization microscopy (SMLM) is an optical super-resolution technique that circumvents the diffraction limit of light by localizing the center point of each fluorophore’s recorded point spread function. SMLM requires the use of labeling strategies and specialized fluorophores and/or buffer conditions that enable most of the fluorophores in the sample to be “turned off” during the experiment so that a sparse subset of the fluorophores in the sample can be isolated from their nearest “on” neighbors and localized to build up an image of interest. Due to the nature of super-resolution single-molecule imaging, careful sample preparation, imaging buffer preparation, and optimized imaging conditions are fundamental requirements for obtaining quality data. This article discusses key factors to consider when preparing samples and acquiring data for SMLM to serve as a starting point for sample preparation and acquisition optimization.