Histochem Cell Biol (2009) 132:253–262 257 Fig. 2 High pressure freezing/FS combined with ET reveals the rela- tion between cellular compartments. Images illustrating the relation between the ER and the apicoplast (a plastid acquired by secondary endosymbiosis) outermost membrane in the parasite Toxoplasma gon- dii. a Image from the aligned tilt stack acquired from a thick section (200 nm), at ¡35° tilt. The gold particles, used for also for the align- ment of the series, are speciWc labeling for the apicoplast speciWc Acyl carrier protein. The relation between the membranes of the ER and the apicoplast is pointed (black arrowhead). b Optical slice from the tomo- graphic reconstruction of the same apicoplast. The lamellar structure of all the membranes is visible. No additional contrasting was applied. The close alignment of the outermost apicoplast membrane and the ER is pointed (black arrowhead). c View of a model generated by the tomographic reconstructions sown in b. Additional observation in these images is the budding of vesicles from the nuclear envelope (white arrowhead). Sample substituted in 0.1% uranyl and embedded in Lowicryl HM20. Sections were not post-stained. The scale bars in a and b are 100 nm Fig. 3 Tomography of thin vitreous cryo-sections of the Saccharomy- ces cerevisiae nucleus–vacuole junction. a A 10-nm slice from a recon- struction that shows the consistent width of the nuclear membranes in relation to the vacuole. ‘N’ denotes the nucleus and ‘V’ the vacuole. Scale bars 50 nm. b A second reconstruction that shows more of an overview of the region of the NV junction. The inner and outer mem- branes of the bulk nuclear envelope (white arrows) are further apart compared to those of the nuclear envelope at the NV junction. Around and within the nucleus, macromolecular complexes can be observed, along with a mitochondrion at the bottom of the image membrane, which is associated with Nvj1 in the outer nuclear membrane. NV junctions are the site of piecemeal microautophagy of the nucleus, which is a type of autophagy that targets portions of the nucleus. Artifacts induced by the process of sectioning (knife marks, crevasses, and compression) have been described in detail and must be taken into consideration (Al-Amoudi et al. 2005). An example of a crevasse is shown in Fig. 3b, where crevasses are partially visible in the vacuole (V) as white stripes perpendicular to the cutting direction (parallel to the NV junction). In certain cases crevasses can penetrate into the depths of the cryo-section of a vitreous sample and aVect the underlying biological ultra-structure, however, in ultra-thin sections (< 50 nm) these artifacts appear to be non-intrusive to the biological ultra-structure. It is possible to minimize knife marks and crevasses, making compression the most problematic artifact. Com- pression is a deformation that makes the vitreous section shorter along the cutting direction without changing the overall volume. The result is an increase in overall section thickness. Thirty percent compression within vitreous cryo- sections is not unusual. If compression is evenly distributed, then a reverse transform (Al-Amoudi et al. 2005) can be applied to the entire volume post-reconstruction. However we 123