Vorträge und Posterpräsentationen (mit Tagungsband-Eintrag):
S. Reipert, E. Hollergschwandtner, H. Goldammer, M. Eckhard, J. Neumüller, U. Kaindl, M. Stöger-Pollach, T. Schwaha:
"Automatized freeze substitution under agitation: opportunities for studies of intracellular mesostructures";
Vortrag: 13th Multinational Congress on Microscopy,
- 29.09.2017; in: "13th Multinational Congress on Microscopy",
Cryopreparation based on high-pressure freezing and freeze substitution has been proven as superior for sample preservation if compared with chemical fixation and processing of cells and tissues at ambient temperature. Here, we address its potential for studies of paracrystalline cellular inclusions, with specific reference to our findings of novel intracellular mesostructures. Recently we applied our agitation module for accelerated automatized freeze substitution in a commercial freeze substitution unit to cryoimmobilized animal- and plant tissues for transmission electron microscopy (TEM). Besides other advantages, the initial tests indicated the usefulness of this technique for preservation of paracrystals in algal microbodies and polysaccharides in form of starch (1). We also generated data from oocytes of brine shrimp demonstrating that particular small, organelle-bound nanocrystals are preserved by this
method. Here we provide a clear example how high-pressure freezing in combination with rapid freeze substitution can prevent disintegration of crystal-like inclusions that are part of mesoscopic superstructures. If applied to epidermal cells of brine shrimp Artemia franciscana, we found novel, flake-like entities with electron-opaque and electron-lucent stripes (Figure 1a) in abundance both in the cytoplasm and nucleoplasm of these cells. Under conditions of chemical fixation and processing at room temperature the zebra-striped ´flakes´ disintegrated into individual crystal-like rhomboids (Figure 1b). These rhomboids resemble previous observations made in different context in other species, which have been interpreted as proteinaceous paracrystals. Confocal laser scanning microscopy of native tissues correlated with our data based on cryopreparation. Moreover, it revealed optical activity of the superstructured flakes and the full extent of their distribution in the tissue (Figure 1c). Electron tomography provided a 3D-impression of individual superstructures flakes and their edged interface with the cytoplasm, but selected area electron diffraction could not verify the status of crystallinity. We conclude that A. franciscana possess epidermal cells with crystal-like superstructured inclusions that resemble mesocrystals. Mesocrystals are known to self-assemble from inorganic crystalline components and biopolymers (2). However, energy dispersive X-ray spectroscopy detected no raise of inorganic elements potentially involved in crystallization, but an increase of nitrogen inside the inclusions. Accordingly, in the formation of mesostructures in Artemia tissue proteinaceous precursors seem to play the part of the inorganic ones. Our hypothesis is supported by TEM observations likely to be related to the process of self-assembly. Artemia, therefore, could serve as an excellent model for interdisciplinary studying of mesostructure formation in vivo. If correct, mesocrystals are more widespread and diverse than currently thought. Cryofixation in combination with rapid freeze substitution could possible bring more structures like those reported here to our attention, because of better preservation of both proteins and (poly) carbohydrates. The interest in this topic is explained by the fact that mesocrystals are subject of intense studies by biophysicist and material scientist because of their extraordinary physical properties (2).
Erstellt aus der Publikationsdatenbank der Technischen Universität Wien.