Cryoultramicrotomy: cryoelectron microscopy of vitreous sections.
Cryoultramicrotomy allows the sectioning of vitrified biological samples. These biological samples are preserved at the atomic level and represent the real structure at the moment of freezing. Cryoultramicrotomy produces ultra-thin cryosections that are investigated in a cryoelectron microscope. The necessity of working during the whole preparation at temperatures less than -140 degrees C results in some difficulties, including the cryosection transfer from the knife-edge to the electron microscropy grid; the grid handling in the cryochamber and the grid transfer into the cryoholder of the electron microscope. Furthermore, ice crystal contamination (from air humidity) can obscure the structures of interest in the sections. It is mainly know-how and experience that will prevent the contamination of ice crystals and the recrystallization of the sections during the manipulations. Here, we describe the tips, tricks, the tools, and methods that help to overcome these burdens and pave the path for successful cryoultramicrotomy.
Vanhecke
Dimitri
D
Institut für Anatomie, University of Bern, Switzerland.
Studer
Luca
L
Studer
Daniel
D
D000818:Animals; D002358:Cartilage, Articular / Q000648:ultrastructure; D002417:Cattle; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D016616:Cryoultramicrotomy / Q000295:instrumentation / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure