Critical point drying

The drying preparation method in greatest use among biologists today is critical point drying (CPD). The method was first described by Anderson [420-422] and then by Hayat and Zirkin [423]. A clear explanation of the method and applications are given by Anderson [422, 424] and Cohen [425], while a somewhat more current review by Cohen [426] is recommended to the interested reader. 

In ordinary drying, the liquid in a specimen evaporates, and the resulting surface (interfacial) tension can distort the structure. In critical point drying [425], heating a specimen in a fluid above the critical temperature to above the critical pressure permits the specimen to pass through the critical point (that temperature and pressure where the densities of the liquid and vapor phases are the same and they coexist and thus there is no surface tension). By definition, a gas cannot condense to a liquid at any pressure above the critical temperature. The critical pressure is the minimum pressure required to condense a liquid from the gas phase at just 

Critical point drying is conducted using transitional fluids which go from liquid to gas through the critical point. The critical temperature (more than 300°C) and pressure (above 21 MPa) of water are much too high for it to be used. Unfortunately, this requires the removal of water and its replacement by a transitional liquid. Water is removed and replaced by dehydration fluids which are replaced by the selected transitional fluid. A typical transition sequence is  

Graded series are combinations of fluids that are used to gradually replace the water with the dehydrating fluid, such as water/ethanol 90/10, 75/25, 50/50, 25/75, 0/100. Freon TF (134) is useful as an intermediate fluid, as it does not have to be fully flushed out of the system when 

Finally, for critical point drying of polymers a suitable series of fluids and conditions must be chosen that will not damage the specimen. Carbon dioxide dissolves in epoxies and in polystyrene. Apparently long term high pressure exposure and then a sudden release can turn these polymers into popcorn. Microscopic comparison of a material prepared by a variety of different preparation methods is the best way to uncover any possible artifacts caused by the specimen preparation method. 

Critical point drying is conducted using tran- 

Graded series are combinations of fluids that are used to gradually replace the water with the dehydrating fluid, such as water/ethanol 90/10, 75/25, 50/50, 25/75, 0/100. Freon TF (113) is useful as an intermediate fluid, as it does not have to be fully flushed out of the system when used prior to drying with CO2. Critical constants for Freon 13 (CCI3), Freon 116 (CF3 - CF3), and carbon dioxide (CO2) are in the range of 20 40°C and 3.5-7.5 MPa. The CPD preparation is conducted in a pressure vessel to control both the temperature and the pressure. 

Finally, for critical point drying of polymers a suitable series of fluids and conditions must be chosen that will not damage the specimen. Car- 

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Production of net-shape siUca (qv) components serves as an example of sol—gel processing methods. A siUca gel may be formed by network growth from an array of discrete coUoidal particles (method 1) or by formation of an intercoimected three-dimensional network by the simultaneous hydrolysis and polycondensation of a chemical precursor (methods 2 and 3). When the pore Hquid is removed as a gas phase from the intercoimected soHd gel network under supercritical conditions (critical-point drying, method 2), the soHd network does not coUapse and a low density aerogel is produced. Aerogels can have pore volumes as large as 98% and densities as low as 80 kg/m (12,19). 

Membrane Characterization The two important characteristics of a UF membrane are its permeability and its retention characteristics. Ultrafiltration membranes contain pores too small to be tested by bubble point. Direc t microscopic observation of the surface is difficult and unreliable. The pores, especially the smaller ones, usually close when samples are dried for the electron microscope. Critical-point drying of a membrane (replacing the water with a flmd which can be removed at its critical point) is utihzed even though this procedure has complications of its own it has been used to produce a Few good pictures. 

Ris, H. (1985). The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded section. J. Cell Biol. 100,1474-1487. 

Fang S-G, Wan Q-H, Fujihara N. Formalin removal from archival tissue by critical point drying. BioTechniques 2002 33 604-611. 

Liquid C02 from a siphon-tube tank is introduced into the chamber and used to replace 100% of the ethanol in the specimen. After the ethanol has been totally replaced by the C02, the critical point drying chamber (CPD) is brought above the critical point. The temperature is kept above the critical point, while the gaseous C02 is vented from the chamber. The process is finished when the CPD is returned to atmospheric pressure. After critical point drying, the specimen should be totally dry and it is ready to be introduced to the vacuum system of the sputter coater and SEM (Dykstra, 1993 Hayat, 1978). 

Fig. 2. Scanning electron micrograph showing a natural microbial biofilm developed on surface of immobilized surface when dimethylphthalate was used as the sole source of carbon and energy after dehydration and critical-point dried and coating with palladium and gold (unpublished results). 

Discs were critically point dried using liquid carbon dioxide. 

Dehydrate in a graded alcohol series series (30%, 50%, 70%, 90%, 100%), for 100% use in 200 proof alcohol Freon or amyl acetate (use a graded series, e.g., 1 3, 1 2, 1 1 Freon/alcohol and then pure Freon) Critically point dry using liquid C02 soaks and flushes Mount on aluminum stubs using silver paint or double-stick tape0 Sputter coat with gold/palladium in a sputter coater flushed with argon... 

Critical point dry with liquid carbon dioxide. 

Tanaka K, lino A. Critical point drying method using dry ice. Stain Techno 1974 49 203-206. 

Figure 2. Scanning electron micrograph of a mesophyll cell of a dormant cotyledon of Buffalo gourd (Cucurbita foetidissima). Tissue was fixed in aqueous glutaraldehyde, dehydrated with ethanol and critically point dried. Note cell wall (W) and intracellular components including protein bodies (P) and emptied spherosomes that appear as a cytoplasmic reticulum.

Fig. 6.12 Critical PS thickness above which cracking of the PS film during evaporation of water is expected, according to Eq. (6.9). Note that drying in pentane or ethanol increases the critical thickness by a factor of about 26 or 11, respectively. Inset Cracking can be avoided by freeze drying (1) or critical point drying (2), which removes the solvent without crossing the fluid-gas boundary in the phase diagram (3).

After fixation and labeling, silver-enhance the gold using a commercially available kit. Generally, the time needed for enhancement for SEM is around 5 min. Dehydrate the samples, and critically point-dry. 

The immunoreplica technique (14) is used when it is necessary to detect antigenic sites on the plasma membrane of cultured cells. The cells are cultured on coverslips, and are fixed as described above depending on the antibody in question, and immunolabeled in situ as described in Section 3.1.1.2., steps 3-9. After immunolabeling (Section 3.1.1.2., step 9), they are further fixed with 1% osmium tetroxide and are dehydrated in a graded series of ethanol (70, 90, 100%), critically point-dried, and replicated with a layer of carbon and platinum, The replicas are cleaned with sodium hypochlorite and chronic acid before examination with the transmission electron microscope. Large areas of the replicated plasma membrane remain intact for observation. Colloidal gold probes are probably the only probes of sufficient density that can be detected on these surfaces. 

Ciyo-SEM methodology also facilitates the observation of highly hydrated systems. Harker and Sutherland [69] used the ability of cryo-SEM to preserve the structural integrity of the aqueous phase to characterize differences between mealy and non-mealy nectarines. The presence of juice on the surface of cells in non-mealy nectarines was observed after tensile tests produced a fractured surface. Such observations would not have been possible with conventional methods where dehydration and critical point drying are essential steps. A strong point to this study was the extensive use of other physical and chemical methodologies to help correlate textural difference based on storage parameters for nectarines. 

Fig. 20 SEM (sample preparation critical point drying) of inner surface areas. 1 - BASYC in the middle region of the interposition four weeks after implantation in the carotid artery of the rat with endothelial cells, 2 - BASYC before incorporation. Reprinted with permission from [65]...

Figure 1. Scanning electron micrograph of a 10% WPC gel in distilled water. Chemical fixation was made with glutaraldehyde followed by critical-point drying.

The ethanol was exchanged by amylacetate by a series of amylace-tate-ethanol mixtures 50% amylacetate for 30 min 70% amylacetate for one hour, and 100% amylacetate for two hours. The samples were critical-point-dried and coated with gold. The scanning electron micrographs were taken in a Cambridge Stereoscan S4. 

Sachlos E, Wahl DA, Triffitt JT, Czernuszka JT. (2008) The impact of critical point drying with liquid carbon dioxide on collagen-hydroxyapatite composite scaffolds. Acta Biomater 4(5) 1322-1331. 

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.

F. Preparation of Specimens. For observation by SEM, tissue and cell samples. must be carefully dried under conditions which preserve the cell surface features. This Is generally accomplished by fixing, dehydrating, and critical point drying the specimens. 

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,... 

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