Enhanced visualization of peripheral nerve and sensory receptors in the scanning electron microscope using cryofracture and osmium-thiocarbohydrazide-osmium impregnation

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Abstract
Two methods of specimen preparation for the scanning electron microscope (SEM) have been combined for the reliable exposure and examination of nervous system tissue. When the specimen is postfixed with OsO4 prior to aqueous cryofracturing, large internal surfaces of nervous tissue are exposed, with minimal distortion to the cytoarchitecture. All tissue surfaces and interstices are subsequently impregnated with a conductive, metallic layer of osmium using a modified osmium-thiocarbohydrazide-osmium technique (OTOTO). This OTOTO technique permits SEM examination without any additional vacuum evaporated or ion-sputtered metallic layers, and has been found to eliminate specimen charging reliably. Nervous tissue has been examined in the secondary electron mode of the SEM with unrestricted use of beam currents varying from 1.3 to 60 μA, at accelerating voltages ranging from 2.5 to 80 kV, and at both low (10 ×) and high (80 000 ×) magnifications. In addition, a differential deposition of osmium in the tissue after the OTOTO technique has been identified using both transmission electron microscopy and energy dispersive X-ray microanalysis. The enhanced mass-density of myelin resulting from the amplification of osmium's natural affinity for unsaturated lipids was best demonstrated by the backscatter electron mode of the SEM. This mode of imaging was found useful in the identification of myelin sheaths.