Methods for improved light microscope microtomy.

Abstract

Experiments undertaken to improve the ease and quality of preparing tissue for light microscopy have resulted in methods for routine production of high quality serial paraffin and glycol methacrylate sections in the 1 to 4 micrometer range using a standard rotary microtome. These methods involve sectioning with mechanically-broken Ralph-type knives, inspected for quality before mounting with double-stick tape on a holder-fluid trough in which the ribbon floats over an immersed slide and sections are mounted by lowering the fluid level in the trough. Specimen return stroke retraction, necessary for quality sectioning of plastic or with a fluid trough, is inexpensively provided by an attachable specimen holder that fits into the rotary microtome's chuck and, actuated by the microtome mechanisms, retracts the specimen during each return stroke. Descriptions of four instruments: a mechanical breaker for Ralph knives, a knife edge inspection device, an attachable specimen retractor for rotary microtomes, and a section collecting-mounting device as well as details of their use are included. Differences in cutting speed and cutting cycle cadence critical to quality sectioning with different embedments are detailed. Factors important in choosing the best embedment and most appropriate section thickness as well as choosing between dry or wet sectioning methods are also considered.