Cryo-immobilization is the optimum technique to preserve the ultrastructure of biological specimens or more generally, soft condensed matter. However, freezing of samples thicker than a few microns without the use of antifreezing agents and at ambient pressure leads to considerable ice crystal formation and distortion of the biological material. Such samples can only be frozen adequately if intracellular antifreezing agents are used or under high pressure. Intracellular antifreezing agents are normally not incorporated by living cells or organisms under physiological conditions or are problematic concerning normal physiology of the cell. High-pressure freezing (HPF) was developed to overcome these drawbacks and allows adequate freezing of native biological samples of up to 200 μm in thickness. The optimum pressure of 2,100 bar needs to be applied to the sample within a few milliseconds followed by immediate rapid cooling to prevent the introduction of artifacts caused by high pressure (e.g., changes of dissociation constants). HPF became a state-of-the-art technique to contribute to the elucidation of numerous scientific questions which cannot be answered using room temperature preparation techniques like chemical fixation.

Currently, four different HPF machines are available: EM HPM100 and EM PACT2 (Leica Microsystems, Vienna, Austria), HPM 010 (Boeckeler Instruments, Tucson, AZ, USA), and HPF Compact 02 (Engineering Office M. Wohlwend, Sennwald, Switzerland). EM HPM100, HPM 010, and HPF Compact 02 are systems using liquid nitrogen at high pressure to pressurize and to cool the specimen in a small container located in the pressure chamber. A small volume of alcohol at room temperature is usually introduced first to coordinate pressure buildup and cooling. In the EM PACT2 system, the specimen in the container is pressurized with a transmission fluid (methylcyclohexane) and subsequently cooled with a stream of liquid nitrogen at 10 bars [1–3].

Several articles about HPF have been published in the last years. In particular, they focus on specimen preparation and handling before freezing, which are the most important steps to make HPF a successful method to preserve the ultrastructure of biological specimens [4–16]. Specimen processing after freezing is also extensively described in the literature for a variety of different specimens and applications. This chapter will focus on the recent developments regarding HPF machines, specimen carriers and methods rather than providing a review of previous articles. The recently developed HPF machine EM HPM100 is described in detail. Handling and specimen preparation is discussed as far as different from the other systems (HPM 010 and HPF Compact 02). Additionally, an improved method for freezing cell culture monolayers grown on Sapphire discs with the EM PACT2 system is presented.

The EM HPM100 is a largely automated system with an integrated workstation and binocular microscope (see Fig. 1a). The centerpiece is a versatile cartridge, consisting of highly durable polymer pieces (see Fig. 1b). The assembled cartridge holds the specimen carrier sandwich (or other container) and guides it automatically through the freezing process, finally releasing cartridge, carriers, and sample in liquid nitrogen. This guarantees that the whole freezing sequence from pickup of the specimen to liquid nitrogen is consistent and reproducible.

A wide range of different specimen geometries can be frozen with the according set of cartridge pieces and specimen carriers. In particular, samples of up to 5 mm in diameter can be processed, which facilitates handling before freezing considerably. The cartridge is made of a thermally insulating polymer leading to cooling of the sample and carriers rather than the surrounding cartridge and to optimized heat extraction from the sample. Additionally, the volume of the pressure chamber was reduced compared to the previous HPM 010 model resulting in a quicker pressure buildup, higher flow rate of liquid nitrogen, and less nitrogen consumption.

Injection of alcohol at room temperature into the pressure chamber to coordinate pressure buildup and cooling can be switched off on demand. Application of this option will be discussed later in this chapter. A chamber to keep the temperature and humidity at desired values is available for the work station. The EM HPM100 is mobile and the remaining liquid nitrogen in the storage Dewar and pressure cylinder can be retrieved after a freezing session similar to the EM PACT2. Temperature and pressure courses are recorded and can be stored. The freezing quality and yield of well frozen samples are comparable to the results achieved with HPM 010 [6, 17–21].

The option of freezing specimens of a diameter up to 5 mm is appreciated by many scientists in the field and lead recently to adaptations also of HPM 010 and HPF Compact 02. Appropriate holders for up to 6 mm specimen carriers are now available for HPM 010 (per. comm. Bill Graham, Bibst Labs, Brookline, NH) and HPF Compact 02. An additional new feature of the HPF Compact 02 is that specimen holder containing the specimen carrier sandwich is now introduced horizontally into the pressure chamber compared to the vertical insertion with the predecessor model (per. comm. M. Wohlwend, Engineering Office M. Wohlwend, NH).

For optimal results, the following basic rules for HPF are essential: Always use a fresh sample and as thin as possible, preferably thinner than 200 μm. Remove extracellular medium containing high amounts of water and substitute with a filler (e.g., 1-hexadecene, yeast paste, Dextran solution). Make sure that no air is trapped in the specimen carriers.