Cryo super-resolution CLEM imaging of mammalian cells

Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

Cryo super-resolution CLEM imaging of mammalian cells

CryoCLEM on USOS cells, transfected with rsEGFP2-MAP2


Cryo electron microscopy (cryoEM) provides in-situ structural information of biological samples at high resolution, but it is essential that samples are fixed in their near-native state by vitrifcation. Although cryoEM is a powerful technique, it is challenging to locate specific proteins and regions of interests at low magnification. Cryo-fluorescence light microscopy (cryoFLM) is compatible with cryoEM and can provide protein localisation of individually labelled biomolecules within a large field of view. We describe cryo super-resolution CLEM acquisition using reversibly switchable fluorescent proteins (RSFPs)  and subsequent cryoEM imaging without the addition of any cryoprotectants and exclusively using commercially available equipment and sample supports. We demonstrate the utility of this method by performing SR-cryoCLEM on RSFP-labelled lipid nanotubes and on intact mammalian cells to achieve a localisation precision of ca. 30nm, with subsequent high-resolution imaging and correlation using cryoEM.

Maarten W.Tuijtel, Abraham J. Koster, Stefan Jakobs, Frank G.A. Faas and Thomas H. Sharp. Scientific Reports (2019) 9:1369.


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