Precision ultramicrotomy of narrow specimen embedments for electron microscopy—theory and practice

SYNOPSIS Ultramicrotomy of narrow specimen layers (< 15 μ) requires special attention to the mechanics of block alignment. A systematic approach to levelling of the block face can increase the percentage of full face (“isometric”) sections and minimize specimen loss. The method proposed has been derived from geometric equations and provides a rational basis for the instruction of technical personnel. The significance of an initial angular displacement with respect to the ideal cutting plane is gauged from the percentage of block face obtained with the first ultratome stroke, and the maximum thickness of the first section. The latter is determined from reflection colours. The magnitude of angular displacement may be directly calculated or estimated graphically. Present means of microtome chuck or knife stage control permit approximate mechanical correction of alignment errors, but chief reliance must remain upon technical expertise. Experiments indicate that visual training can result in a relatively consistent alignment of the block face in the horizontal (knife edge) plane (< 0.2° error), but that preliminary vertical alignment is usually less accurate. For a given percentage of isometric sections, the tolerable degree of alignment error is inversely proportional to the length of the block face.