Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells

Cryo-electron tomography (cryo-ET) allows for the visualization of biological material in a close-to-native state, in three dimensions and with nanometer scale resolution. However, due to the low signal-to-noise ratio inherent to imaging of the radiation-sensitive frozen-hydrated samples, it appears oftentimes impossible to localize structures within heterogeneous samples. Because a major potential for cryo-ET is thereby left unused, we set out to combine cryo-ET with cryo-fluorescence microscopy (cryo-FM), in order to facilitate the search for structures of interest. We describe a cryo-FM setup and workflow for correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) that can be easily implemented. Cells are grown on finder grids, vitally labeled with one or two fluorescent dyes, and vitrified. After a structure is located by cryo-FM (with 0.4 μm resolution), its image coordinates are translated to cryo-ET stage coordinates via a home-built software routine. We tested our workflow on whole mount primary human umbilical vein endothelial cells. The correlative routine enabled us to investigate mitochondrial ultrastructure for the first time on intact human mitochondria, and led us to find mitochondrial cristae that were connected to the intermembrane space via large slits, which challenges the current view that such connections are established exclusively via small circular pores. Taken together, this study emphasizes that cryo-CLEM can be a routinely used technique that opens up exciting new possibilities for cryo-ET.