Recent advances in high-pressure freezing: equipment- and specimen-loading methods.

This chapter is an update of material first published by McDonald in the first volume of this book. Here, we discuss the improvements in the technology and the methodology of high-pressure freezing (HPF) since that article was published. First, we cover the latest innovation in HPF, the Leica EM PACT2. This machine differs significantly from the BAL-TEC HPM 010 high-pressure freezer, which was the main subject of the former chapter. The EM PACT2 is a smaller, portable machine and has an optional attachment, the Rapid Transfer System (RTS). This RTS permits easy and reproducible loading of the sample and allows one to do correlative light and electron microscopy with high time resolution. We also place more emphasis in this article on the details of specimen loading for HPF, which is considered the most critical phase of the whole process. Detailed procedures are described for how to high-pressure freeze cells in suspension, cells attached to substrates, tissue samples, or whole organisms smaller than 300 microm, and tissues or organisms greater than 300 microm in size. We finish the article with a brief discussion of freeze substitution and recommend some sample protocols for this procedure.