title: A novel method for high-pressure freezing of adherent cells for frozen hydrated sectioning and CEMOVIS.
abstract: With the development of Cryo Electron Microscopy Of Vitreous Sections (CEMOVIS), imaging cells in a close to native state has become a reality. However with the commonly used carriers for high-pressure freezing and cryo-sectioning, adherent grown cells either need to be detached from their substrate. Here a new method is presented for high-pressure freezing adherent growing cells for frozen-hydrated sectioning and CEMOVIS. Cells are cultured on golden grids, containing a carbon coated Formvar film, and frozen on a membrane carrier which provides the grids with the structural support needed to withstand the strain of trimming and cryo-sectioning. This method was successfully tested for the two different types of high-pressure freezers, those using a pressure chamber (HPM010, EMHPF, Wohlwend Compact 01/02, HPM100) and those directly pressurizing the sample (EMPact series).
authors.affiliation: Membrane Enzymology, Department of Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; Biomolecular Imaging, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Electronic address: R.Mesman@science.ru.nl.
authors.forename: R J
authors.initials: RJ
authors.lastname: Mesman
mesh_terms: D002478:Cells, Cultured; D020285:Cryoelectron Microscopy / Q000379:methods*; D015925:Cryopreservation; D005629:Frozen Sections; D056945:Hep G2 Cells; D006801:Humans; D011312:Pressure
keywords: Adherent cells; CEMOVIS; Cryo-sectioning; HPF; High-pressure freezing; MC; high-pressure freezer; membrane carrier