Capturing endocytic segregation events with HPF-CLEM.

We have advocated the use of high-pressure freezing (HPF) in specific types of Correlative Light Electron Microscopy (CLEM) experiments because the intracellular components such as the cytoskeleton and membrane tubules can only be adequately preserved via cryofixation. To allow fast transfer from the light microscope into a cryofixation device, we have developed the Rapid Transfer System (RTS) for the EMPACT2 high-pressure freezer. In this chapter, we will describe how to prepare and perform a CLEM experiment using this device and will highlight the latest changes made to the original system to optimize the workflow.