Abstract
AbstractUnambiguous targeting of cellular structures forin situcryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a novel cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes in the cell by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery for cryo-ET. This reliable and robust workflow allows for fastin situanalyses without the requirement for cryo-focused ion beam milling. We have developed a protocol where cells can be frozen, imaged by cryo-fluorescence microscopy and ready for batch cryo-ET within a day.
Publisher
Cold Spring Harbor Laboratory