PubMed · 4399199
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H Moor. 1971-05-27. Recent progress in the freeze-etching technique.. https://doi.org/10.1098/rstb.1971.0042
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Seawater, with its 3.5% salt content, freezes into hexagonal ice (Ih) that encloses concentrated brine within its matrix. When unsubmerged sea ice reaches a certain height and temperature, the brine drains downward through narrow channels. This mechanism was now modeled by frozen 2-3.5% saline as investigated by cryo-etch high-resolution secondary electron microscopy. Thus, saline was either plunge-frozen in liquid ethane at -183 degrees C or else high-pressure frozen to -105 degrees C in 5-6 ms. Ice from a freshly exposed surface was then subjected to a high-vacuum sublimation ("etching"), a procedure that removes pure bulk ice in preference to ice from frozen hydrated salt. After chromium-coating the etched surface with a 2-nm film, the sample was examined by cryo-HRSEM. Granular icy "fences" were seen surrounding empty areas where amorphous ice had originally resided. Since the fences, about 1-2 mum high, survived the etching, it is likely that they consist of frozen brine. The presence of such fences suggests that, during freezing, saline can purge itself of salt with remarkable speed (5-6 ms). Alternatively, channels (perhaps routed around submicroscopic crystallites of cubic ice (Ic) embedded in the amorphous ice at -105 degrees C) can guide the migration of salt to the periphery of ice patches. Macromolecules fail to form fences because they diffuse too slowly or because they are too large to pass through the channels.
Galactosylceramides (GalCers) containing nervonoyl (24:1(Delta15(cis))) acyl chains have the capacity to assemble into nanotubular microstructures in excess water (. Biophys. J. 69:1976-1986). To define the structural parameters that modulate nanotube formation, GalCer derivatives were synthesized that contained cis monounsaturated acyl chains with the formula X:1((X-9)). X indicates the total acyl carbon number (24, 22, 20, or 18), and 1 indicates a single cis double bond, the location of which is designated by the superscript (X-9). Deep etching of freeze-fractured 24:1(Delta15(cis)) GalCer dispersions followed by replica production and transmission electron microscopic analysis confirmed nanotube morphology (25-30-nm diameter). Control experiments revealed that tubule formation was promoted by cooling through the main enthalpic phase transition coupled with repetitive freeze-thaw cycling. Imparting a negative charge to the sugar headgroup of 24:1(Delta15)GalCer via sulfate dramatically altered mesomorpholgy and resulted in myelinic-like, multilamellar structures. Removal of the sugar headgroup (24:1(Delta15)Cer) resulted in flattened cylindrical structures with a cochleate appearance. Compared to these large-scale changes in morphology, more subtle changes were induced by structural changes in the acyl chain of 24:1(Delta15)GalCer. 22:1(Delta13)GalCer dispersions consisted of long, smooth tubules (35-40-nm diameters) with a strong tendency to self-align into bundle-like aggregates. In contrast, the microstructures formed by 20:1(Delta11)GalCer resembled helical ribbons with a right-handed twist. Ribbon widths averaged 30-35 nm, with helical pitches of 80-90 nm. 18:1(Delta9)GalCer displayed a variety of morphologies, including large-diameter multilamellar cylinders and liposome-like structures, as well as stacked, plate-like arrays. The results are discussed within the context of current theories of lipid tubule formation.
Triacylglycerol is accumulated by Streptomyces spp. when grown in submerged culture. Ultrastructural studies using transmission electron microscopy (TEM), staining and freeze-fracture/freeze-etch procedures, and light microscopy confirmed the accumulation of neutral lipid by S. lividans and S. coelicolor during the stationary phase and its storage within membrane-bound globular structures within the cytoplasm. These structures were of various sizes and occupied up to approximately 80% of the total cell volume at that time. There was no evidence of such material within cells examined during the early exponential phase of growth. The globules visualised by TEM were electron-transparent since they comprised lipids containing saturated fatty acids that did not react with osmium tetroxide. The globules appeared to be bounded by a single membrane.