title: Capturing endocytic segregation events with HPF-CLEM.
abstract: 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.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
authors.forename: Edward
authors.initials: E
authors.lastname: Brown
authors.forename: Jan
authors.initials: J
authors.lastname: Van Weering
authors.forename: Thom
authors.initials: T
authors.lastname: Sharp
authors.forename: Judith
authors.initials: J
authors.lastname: Mantell
authors.forename: Paul
authors.initials: P
authors.lastname: Verkade
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods; D015925:Cryopreservation / Q000295:instrumentation* / Q000379:methods; D055032:Electron Microscope Tomography / Q000295:instrumentation* / Q000379:methods; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D059008:Time-Lapse Imaging