The quality and the speed of sample fixation is still a major challenge for electron microscopy preparation of biological specimens. Compared to chemical fixation, cryo-fixation is regarded as superior, since all biochemical, physiological, and dynamic processes are instantly arrested in their actual state by a massive temperature drop. To avoid any segregation of molecules by ice crystal growth, a cooling velocity up to 100.000 K/s is intended, resulting in a sample completely embedded in vitreous—or sometimes called amorphous ice [1]. Thin samples like purified proteins on a grid or very small cells can be vitrified by simple plunge freezing [2]. Reaching a proper vitrification of samples in the size of eukaryotic cells and tissues, the use of high-pressure freezing (HPF)—as introduced by Riehle and Moor [3]—is mandatory and nowadays performed through commercially available HPF machines.

Recently, self-pressurized rapid freezing (SPRF) has been established as novel and low-cost cryo-fixation method to freeze biological samples in clamp-sealed copper tubes. Instead of applying about 2,000 bar pressure and synchronous cooling in a HPF apparatus, the tubes were plunged directly into the cryogen. This relatively simple procedure—as compared to the usage of HPF machines—provided excellent results for freeze-substituted and plastic embedded specimens [4]. The authors hypothesized that high cooling speed by plunge freezing in conjunction with simultaneous formation of internal pressure by hexagonal ice and/or super-cooled water expansion (Le Chatelier and Braun principle) supports the vitrification of the sample, at least in parts of the tube. Others provided theoretical calculations on the cooling speed in these capillary tubes with the surprising result, that minimizing the amount of cryoprotectant would improve the preservation of cellular ultrastructure [5]. As pointed out in a subsequent comment to this particular reports, the heat transfer at the tubes outer surface, which is the key limiting factor during sample cooling, was neglected, proving these computations unrealistic [6]. Unfortunately, the thermo-conductive peculiarity and small geometry of the tubes prevent all attempts to install sensors for recording the internal pressure and temperature progression during fast cooling. Therefore, the time line and amount of pressure reached during freezing remains speculative. However, the fact that thinner tube walls are severely deformed during freezing shows that the atmospheric pressure inside tubes is far exceeded (unpublished results). In the same direction points the observations that open tubes always lead to sample damage by ice crystal formation [4].

Hence, the direct way to determine freezing quality is the diffraction analysis of frozen SPRF samples below devitrification temperatures, which was explored in follow-up studies. Typical diffraction patterns of vitrified ice were detected by X-ray diffraction of 10–30 μm areas, unfeasible to differentiate between cells and medium [7]. By cryo-electron microscopy and electron diffraction of much smaller areas in vitreous sections, it was determined that appropriate freezing conditions result in vitrified ice present in intracellular as well as in extracellular areas, as illustrated in Fig. 1. See also ref. 8.

Thus, the general capacity to produce vitreous samples qualifies SPRF as cryo-fixation method for electron microscopy protocols associated with freeze substitution or for direct cryo-imaging procedures like CEMOVIS. Highlighting all facets of sample preparation using freeze substitution would be beyond the scope of this chapter. Therefore, we refer to available protocols [9] and excellent reviews [10, 11]. Figures 2 and 3 give typical examples from prokaryotic bacteria (Escherichia coli), eukaryotic yeast cells (Saccharomyces cerevisiae) to whole organisms like nematodes (Caenorhabditis elegans)—all cryo-fixed by SPRF and further processed by freeze substitution. CEMOVIS was introduced in the 1980s at EMBL Heidelberg [12, 13], gradually developed to a stage, where it can be applied to various biological specimens [14, 15], and provided unprecedented views of different structures in their native cellular environment, such as microtubules [16], desmosomes [17], mitochondria [18], neuronal synapses [19], or pleomorphic membrane structures like mammalian Golgi apparatus [20]. Ultrastructural details of yeast (S. cerevisiae), bacteria (E. coli), and cultured mammalian (COS-7) and human cells (HeLa)—all fixed by SPRF and analyzed by CEMOVIS—are shown in Figs. 2c and 4. After some adjustments, SPRF frozen samples could potentially be used for other cryo-imaging methods like soft X-ray cryo-tomography [21] or for cryo-FIB (focussed ion beam) milling [22].

The main drawbacks of HPF are determined by the relatively small volume, which can be vitrified thoroughly. Here, SPRF samples in tubes are slightly more delicate to handle than samples for HPF apparatus, especially regarding correlative light and electron microscopy down to the cellular or molecular level—a problem, which is not solved yet for SPRF fixation. Severe deformations of cells can occur in parts of the tube due to growth of large ice crystals (see Fig. 5). But such areas are easily recognized and analysis can be continued elsewhere in the sample.

However, SPRF gives besides its extreme independence and reliability the complete experimental freedom in choosing cryogens and freezing conditions, which are more or less preassigned in HPF machines. Furthermore, due to the completely closed sample volume architecture, SPRF is predestined for infectious and/or biological safety applications, where the sample content or the environment has to be protected from each other. Compared to commercial HPF machines, SPRF is faster in preparation time, is much cheaper, consumes less liquid nitrogen, and is maintenance-free. The last but not the least, SPRF provides by its simplicity the option to perform cryo-fixation of samples out in the field or at expeditions to extreme habitats and environments, as the basic procedure can be operated without electricity.