Electron microscopy critical-point drying

Fig. 2.2. Mycelium of Hebeloma crustuliniforme after colonization of a potassium feldspar surface for seven months. The sample was prepared by fixation and critical point drying followed by gold coating and analysis by scanning electron microscopy. Hyphae (H) and bacteria (B) are visible. Scale bar = 10 pm. The hyphal surface contact is mediated by a fdm of extracellular mucilage (arrow) and bacteria are seen in the mucilage.

A tissue section cut from a frozen specimen in this situation, ice is the supporting matrix. See Yamada, E. and Watanabe, H., High voltage electron microscopy of critical-point dried cryosection, J. Electron Microsc. 26 (SuppL), 339-342, 1977 Maddox, P.H., Tay, S.K., and Jenkins, D., A new fixed cryosection technique for the simultaneous immuuocytochem-ical demoustratiou of T6 and SlOO antigens, Histochem. J. 19, 35-38,... 

Fig. 8.5. Human RBC after radiolabeling, examined by scanning electron microscopy. No sign of morphological alteration is visible. Perfusion fixation, critical point drying, xl500...

Fig. 8.9. Human platelets after the radiolabeling procedure, examined by transmission electron microscopy. Almost no signs of platelet activation, manifested as pseudopodia formation. Perfusion fixation, critical point drying, x 40 500...

Critical point drying A method of drying delicate samples for electron microscopy by freeze-drying at the critical point of water. At the critical point of agiven substance, the densities and other physical properties of the liquid and gaseous states are identical. 

Scanning electron microscopy -- Leaf samples for scanning electron microscopy (SEM) were fixed in acrolein and dehydrated in ethanol. Samples were critical-point dried (Evans, Gmur, and Da Costa, 1977) and a layer of carbon or silver was applied to each sample in a vacuum evaporator with a rotating and processing stage. The scanning electron microscope (Model 700, Materials Analysis Co., Palo Alto, CA 94303) was operated at 5 kV or 10 kV. 

Use of the microanalysis attachment in conjimc-tion with electron microscopy can provide a visual representation of concentrations. When finked to nondestructive preparation techniques, such as critical point drying, this offers the potential of assessing specific soil processes. Adamo et al. used scanning electron microscopy in back-scattered electron mode to examine the soil-root interface of plants grown at a range of soil pH and soil phosphorus concentrations. Excellent spatial resolution is, however, covm-terbalanced by relatively poor detection limits due to poor spectral resolution resulting in severe peak overlap. 

In the case of emulsions, latexes, some adhesives and wet membranes, the specimen of interest is wet, generally with water and must be dried prior to electron microscopy. The deleterious effects of air drying result from the stress of surface tension forces. The methods used by biologists [334] to avoid this problem are (1) the replacement of water with an organic solvent of lower surface tension, (2) freeze drying or (3) critical point drying. Some latexes can be fixed in their... 

Figure 4.49. Scanning electron microscopy images show a comparison between fracturing of a wet membrane after immersion in liquid nitrogen (A) and after critical point drying (B). Fracturing after freezing results in a deformed ductile failure, whereas the fracture after critical point drying shows no deleterious effects of surface tension and the result is a brittle fracture with excellent detail of the internal morphology.

The miscibility of water and hquid carbon dioxide is very poor and an intermediate solvent has to be used to allow the replacement of water by carbon dioxide. In a procedure initially developed to prepare representative samples for electron microscopy, water is replaced by ethanol through exchanges with alcoholic solutions of increasing concentration. The alcogel prepared by a final exchange with absolute ethanol (Fig. 3c) is introduced in a pressure vessel in which liquid CO2 is admitted and replaces ethanol in the gel. The C02-impregnated gel is compressed and heated above the critical point of CO2 (31.05°C, 73.8 bar). Release of pressure above the critical temperature allows CO2 to be extracted without the formation of any liquid-vapor interface and a dried aerogel is formed (Fig. 3d). 

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