Serial Cryomicrotomy of Saccharomyces cerevisiae for Serial Electron Cryotomography

Abstract

Electron cryotomography (cryo-ET) is an increasingly popular technique to study cellular structures and macromolecules in situ. Due to poor penetration of electrons through thick biological samples, the vitreously frozen samples for cryo-ET need to be thin. For frozen-hydrated cells, such samples can be produced either by cryomicrotomy or cryo-FIB-milling. As a result, a tomogram of such a sample contains information of a small fraction of the entire cell volume, making it challenging to image rare structures in the cell or to determine the distribution of scattered structures. Here, we describe the tools and workflow that we designed to facilitate serial cryomicrotomy, which makes possible the exploration of a larger volume of individual cells at molecular resolution. We successfully used serial cryomicrotomy to locate and image the Dam1/DASH complex located at microtubule plus ends inside mitotic Saccharomyces cerevisiae cells.