Critical-point dry technique

Boyd, A. Tarmarin, A. (1984) Improvement to critical point drying technique for SEM. Scanning 6, 30-35. 

Figure 7. Albumin adsorption by critical-point dry technique. Key a, sintered Teflon, 250 mg/dL solution and b, Fluorofilm Teflon, 25 mg/dL solution. Bar...

Figure 8. Summary of fluid shear dependence of protein adsorption on Fluorofilm Teflon, using critical-point dry technique for sample preparation...

To prepare scanning electron microscopic specimens, the cells were fixed in 2.5% glu-taraldehyde for 30 min and in 1% osmium tetraoxide for 30 min. After dehydration with serial ethanol, the cells were treated with the carbon dioxide critical point drying technique followed by a gold-platinum coating in vacuum evaporation and examined by a scanning electron microscope (Japan Electric Co., Model TSM 840). 

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. 

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,... 

Figure 4. Bovine y-globulin on sintered Teflon (3 mg/dL solution critical-point dried, partial gold decoration technique). Key bar equals 0.11xm.

Figure 5. Cohn I fibrinogen, 3 mg/dL, adsorbed on Teflon (partial gold decoration technique). Key a, air dried, sintered Teflon b, critical-point dried, sintered Teflon and c, critical-point dried, Fluorofilm Teflon. Bar equals 0.1 p,mfor b and c.

Figure 9. Albumin, 2500 mg/dL solution, preadsorped on sintered Teflon followed by Cohn I fibrinogen, 300 mg/dL, lh each (critical-point dry and PGDTEM techniques). Key bar equals 0.1 pm.

Figure 2. Fluid shear dependence of protein sorption on Teflon. Critical-point drying and partial gold decoration TEM techniques were employed. (Reproduced with permission from Ref. 5. Copyright 1982 American Chemical Society.)...

The air-dry preparation technique is inherently prone to artifacts [1,12]. Other dehydration procedures, including lyophilization, critical point drying, and freeze-drying, provide better results on biological specimens for SEM and AFM observation [2]. 

Trieu and Qutubuddin also investigated the structure of fieeze-thaw PVA gels obtained from aqueous DMSO solutions [74, 75], The authors characterized the gels by using freeze-etching and critical point drying SEM techniques. A higher porosity was observed at the surface than in the bulk of the gel. 

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. 

For uniform thick films, slow etching using V02 or with low H2 production is preferred. Critical point drying is a must (see chapter Drying Techniques Applied to Porous Silicon ). V2O5 dissolves in concentrated HF(aq) but not water. To make diluted solutions, always dissolve first in concentrated HF then add water. 

The SEM is used for study of the surface and bulk structures of membranes. Membranes are prepared by attaching them to the specimen stub and applying a conductive surface coating. Bulk structures are observed for membranes fractured in air or liquid nitrogen, sectioned or critical point dried. Figures 5.24 and 5.25 are examples of the varied structures of typical membranes which can be imaged by this technique. An experimental HDPE microporous membrane has... 

An alternative of critical point drying or subfimation is liquid substitution, in which a low-vapor-pressure liquid, such as glycerol or triethylene glycol, is used to infiltrate the sample and the sample can be examined by SEM without drying and coating. This technique has been successfully applied to different biological samples. ... 

The main problems and difficulties of the microscopic observation by both TEM and SEM are how to prepare a membrane sample without any artefact. The first step of the preparation is a careful drying of the sample, and in order to avoid collapse of the original structure, the freeze-dry technique using liquid nitrogen or the critical-point drying method with carbon dioxide is usually employed. 

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