Cryo-SuperResolution implementation for Cryo-CLEM workflows
Magali Mondin, William Magrini, Daniel Choquet, Mónica Fernández-Monreal.
Bordeaux Imaging Center, CNRS UMS3420
Abstract
Correlative super-resolution fluorescence and electron microscopy (SR-CLEM) has emerged as one of the most revolutionary imaging tools for biologist. It combines the specificity of fluorescence labeling at nanoscale level with the global cellular ultrastructure. However, chemical fixation has some limitations by introducing fluorescence aberrations and cellular artifacts that cryo-fixation undoubtedly improves.
Cryo-fixation methods will fundamentally depend on the thickness of the sample. Very thin samples, such as protein preparations, cellular fractions, bacteria and small pathogens, are suitable to plunge freezing methods, and may be observed directly in Cryo-electron tomography. To observe these samples by Cryo-EM, an optional pathway is to perform screening and localization of regions of interest by Cryo-fluorescence microscopy (FM). There are some commercial solutions for Cryo-FM, however the resolution of these methods, in the order of 300 nm, is often a limiting factor to obtain an accurate localization of the molecule of interest and to correlate with the cryo-EM data.
Cryo-superresolution techniques are relatively recent and they need advanced technical expertise on hardware, software and sample manipulation. This method was first developed by Grant Jensen’s laboratory in 2014. Since then, many groups have developed Cryo-SR to characterize fluorescence molecules in cryogenic conditions. In our facility, we have adapted the Leica EM Cryo-CLEM wide-field commercial setup to perform Cryo-PhotoActivated Localization Microscopy (Cryo-PALM). This modality of SR relays in the expression of photo-activable/switchable fluorescent proteins. We obtain with this method an increase on resolution of about 5-6 folds.