High-pressure freezing: current state and future prospects.

In this chapter we discuss the latest developments in the field of high-pressure freezing (HPF). The Leica HPF machine EM HPM100 is discussed in detail due to significant changes compared to its predecessor model. Its centerpiece is a multipart polymer cartridge which holds the specimen carrier sandwich and guides it automatically through the freezing process until immersed in liquid nitrogen. The cartridge can be adapted to the specimen and carrier geometry to optimize the flow of liquid nitrogen and hence rapid cooling. Dedicated cartridges are available for a variety of different carriers, including carriers for samples of up to 5 mm in diameter. Cartridge-specific handling and carrier assemblies are described extensively for freezing samples in aluminum specimen carriers, cell cultures grown on Sapphire discs, suspensions for freeze-fracturing, and specimens for cryo-sectioning. Additionally, we include an advanced technique to freeze monolayer cell cultures on Sapphire discs with the Leica EM PACT2 HPF machine using a composite carrier.