Correlative Super-Resolution Fluorescence and Electron Cryo-Microscopy

Abstract:
Electron microscopy (EM) is particularly powerful for imaging biological structures at highest resolution, but direct identification of specific proteins and structures in the crowded cellular environment can be extremely challenging. On the contrary, fluorescence microscopy (FM) is ideally suited for localizing particular proteins in cellular samples, but lacks resolution power. Correlative light and electron microscopy is combining both imaging modalities in the same sample in a highly complementary way. Super-resolution FM methods present a true game changer for the field of correlative microscopy. They allow bridging the big resolution gap between conventional FM and EM. However, resolution improvement is only one side of the story. Equally important is structural preservation of the sample. On the EM side, vitrification (fast-freezing) of the sample has evolved into a routine method and allows cryo-EM to achieve Angstrom resolution. On the contrary, super-resolution FM under cryo-conditions is still at a very early and experimental stage. The key for super-resolution imaging in general is photo-switching of the fluorophores, but this is only poorly studied and understood under cryo-conditions. One of our major aims is to gain a deeper insight into the photo-physical mechanisms in different molecules at a temperature range suitable for vitrified biological samples. Furthermore, the setup for super-resolution cryo-FM presents various optical and mechanical challenges. Nevertheless, the combination of super-resolution cryo-FM and cryo-EM has great potential to open up a wide range of new application possibilities in structural and cellular biology.

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