Vitreous cryo-sectioning of cells facilitated by a micromanipulator.
Sectioning vitrified cells and tissues for cryo-electron microscopy is more challenging than room-temperature sectioning of plastic-embedded samples. As the sample must be kept very cold (<-130 degrees C) and because there is no liquid upon which the sections can float as they are cut, transferring the sections from the knife edge to a grid is one of the more difficult steps in the process. We employed a micromanipulator to hold and control the cryo-sections as they come off the knife. This allows slower cutting speeds than are typically used in vitreous cryo-sectioning and contributes to better control during cutting, which facilitates repeatable placement of a ribbon of sections onto a grid. The ribbon is kept under tension during the entire cutting process, which may decrease folding and/or compression, features that are inherent to vitreous sections. Furthermore, the added control afforded by this technique makes it easier for multiple ribbons to be placed on a single grid, thereby increasing the number of sections that can be examined and imaged during a microscopy session. It even allows for serial cryo-electron microscopy. As such, this approach is an advance in the cryo-microtomy of vitreous sections.
Ladinsky
Mark S
MS
Boulder Laboratory for Three Dimensional Electron Microscopy of Cells, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA. marksl@bio3d.colorado.edu
Pierson
Jason M
JM
McIntosh
J Richard
JR
D020285:Cryoelectron Microscopy / Q000379:methods*; D005629:Frozen Sections / Q000379:methods; D008846:Micromanipulation; D008867:Microtomy / Q000295:instrumentation / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure*