Preserving the photoswitching ability of standard fluorescent proteins for correlative in-resin super-resolution and electron microscopy

Methods in Cell Biology
Errin Johnson, Rainer Kaufmann

Abstract

There are many different correlative light and electron microscopy (CLEM) techniques available. The use of super-resolution microscopy in CLEM is an emerging application and while offering the obvious advantages of improved resolution in the fluorescence image, and therefore more precise correlation to electron microscopy (EM) ultrastructure, it also presents new challenges. Choice of fluorophore, method of fixation, and timing of the fluorescence imaging are critical to the success of super-resolution CLEM and the relative importance, and technical difficulty, of each of these factors depends on the type of super-resolution microscopy being employed. This chapter details the method we developed for in-resin super-resolution CLEM using single molecule localization microscopy (SMLM) with standard fluorescent proteins (e.g., GFP and mVenus). The key to this approach is being able to preserve not only the fluorescence, but also, and more importantly, the photoswitching ability of the fluorescent proteins throughout the EM sample preparation procedure. Cells are cryofixed using high pressure freezing for optimal structural preservation and then freeze substituted in tannic acid, which preserves the photoswitching ability of the flu...Continue Reading

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