Cryo-fixation and resin embedding of biological samples for electron microscopy and chemical imaging v1

Electron microscopy and chemical imaging are now essential in life science to access the interior of a cell and unveil its structural and chemical landscape at nanoscale. Because of the high vacuum conditions required to visualize subcellular structures, organisms need to be fixed, dehydrated, and sectioned/milled. For fixation, the use of aldehydes at room temperature alters the ultrastructure and the chemistry of the cell creating major osmotic changes. Therefore, rapid freezing methods such as high-pressure freezing superiors in preserving native-state cell structure and chemistry at a fast rate (a scale of milliseconds compared with minutes for chemical fixation). Here, we present the workflow starting from rapid freezing (high pressure freezing) and ending with resin embedding (using freeze substitution) in order to prepare biological samples (cells or tissues) for 2D or 3D electron microscopy (Transmission/Scanning Electron microscopy, and FIB-SEM: Focused Ion Beam Scanning Electron Microscope) and chemical imaging.