256 (Nicastro et al. 2006) or viruses] or relatively thin regions of larger cells (Medalia et al. 2002). The physical properties of water, namely its poor heat conductivity, are the reason that freezing is limited for large cells and tissues. Currently, the only method to vitrify thicker samples (up to 200 m) is by HPF. The high pressure introduced at the moment of freezing lowers the freezing point of water. Synchronized pressuri- zation and cooling of the sample takes place within 20 ms (Studer et al. 2008). The development of this technique has allowed samples of various thicknesses to be vitriWed, although vitriWcation of most mammalian cells in the absence of non-isotonic cryo-protectants has not yet been established. Cryo-Wxation has two distinct advantages over chemical Wxation. It is achieved within milliseconds and it ensures simultaneous immobilization of all macromolecular components. Many protein networks are very labile and fall apart with the slightest osmotic or temperature change and these unwanted eVects are minimized during cryo-Wxation. These techniques allow the study of biological samples with improved ultra-structural preservation, and can facilitate the study of dynamic processes. Freeze-substitution Achieving optimal preservation of the biological sample is undoubtedly critical, but still only the Wrst step in visualiz- ing it. Until it was demonstrated that vitreous water could be sectioned and cellular structure visualized directly in the cryo-EM (Dubochet et al. 1988), the only way of examin- ing the cryo-Wxed sample was by embedding it in resin for sectioning. For this the vitriWed water in the HPF frozen specimen must be replaced by an organic solvent in a pro- cess known as freeze-substitution (FS). Freeze-substitution is a process of dehydration, per- formed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging eVects observed after ambient-temperature dehydration. Aggrega- tion of macromolecules in organic solvents and changes of the hydration shell surrounding the biological molecules can occur even at very low temperatures, but it is reason- able to assume that FS at temperatures below a speciWc threshold preserves the hydration shell (Hobot et al. 1985; Kellenberger 1991). Further, the total or partial loss of the hydration shell can be reduced during the cryo-embedding procedure, so minimizing aggregation and the redistribu- tion of diVusible elements (Edelman 1991; Quintana et al. 1991). Freeze-substitution combines instant physical immobi- lization of the cell constituents and resin embedding. Once substitution is complete, samples are gradually warmed-up and processed further as for conventionally prepared samples. Successful cryo-Wxation followed by FS shows superior 123 Histochem Cell Biol (2009) 132:253–262 preservation of Wne structure compared to chemical Wxa- tion techniques (Müller 1992; Steinbrecht and Müller 1987). This technique also gives the possibility of examin- ing thick (200–300 nm sections) samples by ET, so that relatively large cellular volumes can be studied in 3D. This approach is very beneWcial for an understanding of the complex relation between diVerent cellular organelles and randomly occurring events. Membrane contact sites between the ER and the outer-most membrane of the api- coplast (a plastid found in most parasites from Apicom- plexa Toxoplasma gondii and Plasmodium species) can be seen in Fig. 2 as an example of a sample that has been freeze-substituted in 0.1% uranyl and embedded in Lowic- ryl HM20 (Tomova et al. 2009). In addition, this image shows the potential that HPF/FS has in combining optimal structural preservation with protein detection and for applying ET to thick sections. There are numerous exam- ples where HPF/FS combined with ET have changed our understanding of cellular structure and dynamics (Marsh et al. 2001; Murk et al. 2003; Perkins et al. 2001; Perktold et al. 2007; Tomova et al. 2006; Zeuschner et al. 2005). Recently, it was shown that cellular Wbrils connect protoWlaments directly to the inner kinetochore (McIntosh et al. 2008), which nicely illustrates a mechanism for directly harnessing microtubule dynamics for chromosome movement. Even though numerous diVerent substitution protocols exist, there are only a few methodological investigations concerning the process of FS, such as the replacement of ice by an organic solvent at temperatures of ¡90 to 0°C (Müller et al. 1980), the substitution capacities of diVerent organic solvents in the presence of water (Humbel and Müller 1986), and the inXuence of the diVerent solvents on the ultra-structural preservation of high-pressure frozen, freeze-substituted samples (Studer et al. 1995). It is still arguable what alterations of the cellular structure are induced and to what extent the information obtained can be considered as an accurate representation of the living cell, especially in the case of the 3D structure of macro- molecules. Vitreous cryo-sectioning The expansion and development of cryo-ET and cryo-sec- tioning of native vitreous samples such as yeast and bacteria has made it possible to visualize cells directly in the absence of chemicals, heavy metals and hypertonic cryo-protectants. Vitreous cryo-sections of Saccharomyces cerevisiae (Fig. 3) showed that the nuclear envelope forms a unique membrane contact site with the vacuole, called the nucleus–vacuole (NV) junction (Millen et al. 2008). ‘Velcro’-like protein–protein interactions hold the junction in place. The proteins involved are Vac8 in the vacuole