Capturing endocytic segregation events with HPF-CLEM

Edward Brown; Jan Van Weering; Thom Sharp; Judith Mantell; Paul Verkade

doi:10.1016/B978-0-12-416026-2.00010-8

Capturing endocytic segregation events with HPF-CLEM

Methods Cell Biol. 2012:111:175-201. doi: 10.1016/B978-0-12-416026-2.00010-8.

Authors

Edward Brown¹, Jan Van Weering, Thom Sharp, Judith Mantell, Paul Verkade

Affiliation

¹ Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK.

PMID: 22857929
DOI: 10.1016/B978-0-12-416026-2.00010-8

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.

Publication types

Review

MeSH terms

Animals
Cryoelectron Microscopy / instrumentation*
Cryoelectron Microscopy / methods
Cryopreservation / instrumentation*
Cryopreservation / methods
Electron Microscope Tomography / instrumentation*
Electron Microscope Tomography / methods
Endocytosis*
HeLa Cells
Humans
Imaging, Three-Dimensional
Microscopy, Fluorescence / instrumentation
Microscopy, Fluorescence / methods
Time-Lapse Imaging