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Advanced Histological Techniques

Sectioning Tissue for Electron Microscopy

The ultrathin sections required in TEM are cut with knives of glass, diamond or sapphire. These materials produce extremely hard, ultrasharp edges, but they are brittle and subject to damage. Glass knives are produced as needed by fracturing. Sapphire knives and diamond knives may be purchased. The high cost of diamond knives possibly makes resharpening economically advantageous. Sections are cut into a trough mounted on the back of the blade, which is filled with water or 10-20% ethanol to "float out" the sections as they are cut. The refractive index of the sections will depend on their thickness, and this gives rise to apparent colors which can be used as a guide as to which sections are of usable thickness. Gold, silver and gray sections (ca 110, 75, and <60 nm respectively) are all usable for most purposes. Ultramicrotomy is challenging, and usually must be learned from an expert.

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