A method of mesa trimming with glass knives for obtaining large series of ultrathin sections

Abstract

Ultrastructural analysis of tissue based on 3D reconstruction from serial ultrathin sections is one of the most adequate methods in studies of spatial organization of bio-objects. The sample preparation technique for 3D reconstruction includes the two most technically difficult procedures: an obtaining of stable ribbon of serial sections and mounting of this ribbon onto a slot grid coated with a support film. To mount the ribbon, special approaches and technical tools have been proposed and well evaluated. Much attention has also been paid to obtaining a large and stable ribbon, but this attention deals mainly with the choice of epoxy embedding media. The critical condition of obtaining the straight and stable ribbon is the precise parallelism of trailing and leading edges of mesa falling onto the knife cutting edge. The mesa trimming with dry diamond knife for cryoultratomy allows this condition to be maintained. In the present communication, the way of obtaining parallel sides of the mesa has been proposed with the aid of two forms of glass knives.