Bhaskar Ganguly Ph.D. Scholar (Vety. Biochemistry) Animal Biotechnology Center Deptt. Of Vety. Physiol. & Biochem. C.V.A.Sc.

Pantnagar. INDIA

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Cut material into approximately 2-4 mm pieces. Place sections in 2% glutaraldehyde/1% OsO4 (0.1M Cacodylate buffer), and fix in refrigerator for 1.5 hours. Rinse 2 times in 0.1M cacodylate buffer wash (hold in 70% ETOH at 40 C). Dehydrate samples as follows: 30%, 50%, 70%, 85%, 95% ETOH for 15 minutes each; 100% ETOH, 2 changes for 15 minutes each. Samples must never be allowed to prematurely dry out of solutions. Critical Point Drying (CPD) Samples will be mounted on aluminum stubs with the appropriate adhesive.

Critical Point (CP):

That combination of temperature and pressure where the densities of the vapor and liquid phases are the same so they intermingle and cannot e distinguished. As a liquid in equilibrium with its own vapor is heated in a closed container, the liquid expands and becomes less dense, but the vapor pressure increases, making the vapor more dense. At the CP, they are identical. The meniscus at the vapor-liquid interface becomes fainter and flatter as surface tension decreases, and at the critical point it vanishes, surface tension is zero, and there is not distinction between liquid and vapor phases.

Critical Temperature (TC): The temperature above which no amount

of pressure will force a gas to condense to a liquid; the temperature above which the liquid phase cannot exist. Below the critical temperature, pressure on a gas which is greater than the vapor pressure will force the gas to condense to a liquid. Critical Pressure (PC): The minimum pressure at (or just below) the critical temperature necessary to condense a liquid from the gas phase. Also, the pressure at the critical temperature great enough to make the vapor as dense as the liquid. Critical Density (DC): The density of a fluid at the critical point.

Critical Opalescence: At the critical point the fluid molecules form

clusters large enough to scatter light. This opalescent haze is the critical opalescence. It is best seen by heating a fluid to the critical point. Just below the critical temperature, both upper and lower phases become hazy. The haze is the critical opalescence. If the process is reversed, the opalescence occurs just above the critical temperature and the contents suddenly become momentarily opaque as phase separation takes place.

Critical Point Drying (CPD):

In ordinary drying the liquid in which an object is immersed and which may permeate its structure evaporates away. The interface between liquid and gas passes through the specimen, and the surface (or interfacial) tension can disrupt or at least distort the structure. By heating above the critical temperature in a pressure vessel so that the pressure goes above the critical pressure (Pc), the specimen passes through or near to the critical point. It has been brought from a liquid to a gaseous, dry environment without having been subjected to the distortion of surface tension. Now, if the temperature is kept above the critical temperature (Tc), the gas may be bled off and the specimen removed at atmospheric pressure for examination. It will generally retain the shape and size it had in the liquid without the shriveled, flattened or otherwise distorted appearance generally associated with evaporative drying.

 Dehydration, Intermediate, and Transitional Fluids: The ideal

situation would be one in which the specimen goes directly from water to the fluid which makes the transition from liquid to gas through the CO; the transitional fluid. Nitrous oxide is claimed to be completely miscible with water, but attempts to go directly to N2O have not been successful. Therefore, water must be removed and replaced by a dehydration fluid, usually ethanol or acetone. In the earlier work with CO2 as the transitional fluid, it was believed that an intermediate fluid was amyl acetate. It is now known that one can successfully dehydrate through a graded water-ethanol or wateracetone series and go from 100% ethanol or acetone to the transitional fluid, either a Freon (13 or 116) or CO2. The term intermediate fluid is often used for all liquids performing both the dehydration and intermediate functions. Freon TF (Freon 113) may be employed as an intermediate since it does not have to be flushed out of the system as does amyl acetate, ethanol or acetone. A small amount of Freon TF (up to approx. 5%) raises the Tc and the pressure slightly above those of the pure transitional Freon. It is a useful intermediate in the CO2 system.

Critical Constants: The intrinsic properties of a fluid at

the critical point. Important critical constants are given in the table below.

Mounting:

Specimen stubs are used for supporting sample material. It must be securely attached, so the samples will not dislodge; in addition, the conductive material of the stub will conduct current away from the sample. If x-ray analysis is to be done, carbon shields attached to the stubs or carbon stubs must be used. It is very important that most of the sample be adhered to the stub and that very little, if any, be allowed to protrude over the stub edge. Insufficient contact between sample and stub will cause charge buildup on the sample, image distortion, or total loss of sample.

Adhesives: Silver paint, silver paste, sticky tabs,

carbon paste, and carbon paint are all conductive adhesives used in SEM work. Samples which are being mounted with liquid adhesives must be allowed to dry thoroughly before sputter coating. All samples must be kept in a desiccator. Coating : The two types of coating in SEM are vacuum evaporation and sputter coating. In vacuum evaporation, the metals or carbon rods are easily vaporized, forming a thin and even film over the sample. Samples for x-ray analysis are vacuum coated. Sputter coating is where an appropriate target metal (gold/gold-palladium) is bombarded by energetic materials by the aid of a gas (Argon). Samples for routine SEM are coated by sputter coating.

Sputter Coater

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