After fixation and dehydration, the sample must pass through a critical point dryer to achieve complete dehydration. (a) Dehydrated samples preserve the natural appearance of tissue. Nevertheless, certain tissue retraction is unavoidable. In the critical point dryer, substitution of CO₂ for acetone at low temperature under vacuum conditions takes place. Once the substitution is complete, the temperature is raised to convert liquid CO₂ into gas, maintaining the shape of the tissue. (b) Sample subjected to critical point drying. Samples are mounted on microscope holders, on double tapered carbon tapes. Once fixed on the holder, the pieces are covered by gold or by a double coating of carbon and gold

Coal evaporator. (a) Samples are covered by graphite to allow conduction. Modern microscopes may not require sample coating. Evaporators may be necessary to remove traces of water from samples. (b) Process of sample evaporation in model using graphite thread

Gold sputter. (a) Device used to cover samples with a thin gold layer (10 nm). Graphite evaporators work under vacuum conditions. Different metals (such as palladium or platinum) are used for sample coating in SEM techniques. The overall depth of layers after double coating is about 18 nm (8 nm graphite). (b) Detail of samples after graphite and gold coating. Right, Rat lung. Left, Same human sciatic nerve shown in previous images

Scanning electron microscope, model JEOL JEM 6400, used in studies of human, animal, and vegetal tissues. The following images have been taken at 20 kilovolts (Kv) and 15 mm working distance (WD). Newer models allow the use of freeze techniques, under reduced kilovoltage or working at lower pressures. Preparation of samples in these models differ slightly, requiring fewer steps