Chlamydomonas cryopreparation methods for the 3-D analysis of cellular organelles

Chlamydomonas reinhardtii is a popular model organism in modern cell biology. Historically, methods for preparing this cell for transmission electron microscopy have used conventional chemical fixation that can result in artifacts that affect the 3-D organization of the cell. We have developed improved methods of specimen preparation that involve high-pressure freezing followed by freeze-substitution that are particularly well suited for 3-D studies (O'Toole et al., 2003, 2007). In this chapter, we describe the details of our cryopreparation methods for the optimal preservation of whole cells for immunocytochemistry and electron tomography. Examples are presented that show the utility of this approach for studying the 3-D architecture of membrane systems and cytoskeletal arrays in intact cells.