Point by Point: An Introductory Guide to Sample Preparation for Single‐Molecule, Super‐Resolution Fluorescence Microscopy

Abstract Single‐molecule, localization‐based, super resolution microscopy is able to reveal detailed subcellular structures and protein distributions below the classical ∼250‐nm diffraction limit of light, but utilizing this technique effectively requires a combination of careful sample preparation, data acquisition, and data analysis, which can be daunting to novice researchers. In this protocol, detailed instructions on preparation of robust reference samples for super‐resolution microscopy, including the cytoskeleton (microtubules), membrane‐bound organelles (peroxisomes), and scaffold proteins (clathrin), are provided. These samples also constitute a representative subset of imaging targets of interest to biological researchers and highlight the differences and similarities in sample preparation. © 2015 by John Wiley & Sons, Inc.