Cryogenic transmission electron microscopy (cryo-TEM) has become a powerful tool for probing the structures of soft-matter materials at nanometer or sub-nanometer scale. For many complex molecular fluids (CMFs), the limiting factor in such studies is the preparation of a cryo-TEM specimen that represents the native structure. Plunge freezing of electron-transparent thin films has been widely used, but it is often influenced or even dominated by surface effects and mechanical disturbances of the CMF. In this article, we use liquid crystals as model CMF systems and present the general procedures and typical results of cryo-ultramicrotomy of rapidly frozen “bulk” samples.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that develops asynchronously inside parasitophorous vacuoles. Spore differentiation involves the construction of a cell wall commonly divided into an outer layer (exospore) and a thicker, chitin-rich inner layer (endospore). The developmental patterns of protein deposition and mRNA expression for 2 different spore wall proteins were studied using immunocytochemical and in situ hybridization procedures with ultrathin frozen sections. The onset of deposition of an exospore-destined protein (SWP1) correlated with the formation of lamellar protuberances during meront-to-sporont conversion. No evidence for a release of SWP1 towards the parasitophorous vacuole lumen was obtained. An endospore-destined protein (EnP1) was detected early on the plasma membrane of meronts prior to extensive accumulation within the chitin-rich layer of sporoblasts. swp1 mRNA was preferentially synthesized in early sporogony while enp1 mRNA was transcribed during merogony and a large part of sporogony. The level of both mRNAs was reduced in mature spores. Considering the availability of the E. cuniculi genome sequence, the application of nucleic and/or protein probes to cryosections should facilitate the screening of various genes for stage-specific expression during microsporidian development.