A technique for obtaining sequential ribbons of semithin sections suitable for three-dimensional reconstruction

Abstract

The purpose of this paper is to describe a simple, dependable, and efficient technique that provides sequential ribbons of uniform semithin (0.5-μm) sections for three-dimensional reconstruction of relatively large specimens at the light microscope level. This technique was developed using whole heads of adult Aedes aegypti (L.) mosquitoes (Diptera: Culicidae) for reconstruction of the central nervous system. Adult A. aegypti mosquito heads are fixed in Karnovsky's, postfixed in OsO4, and embedded in Spurr's low viscosity epoxy medium. Each block is trimmed to form a "mesa" and Tackiwax® is applied to its leading edges. Sections are cut at 0.5 μm with a diamond knife. Ribbons of sections are collected directly on pieces of coverslip and the embedding medium is partially removed using an NaOH–EtOH solution. The sections are stained with silver nitrate. The pieces of coverslip are then mounted on glass slides to facilitate examination and photography of sections. In this work each mosquito head yielded sequences of approximately 1300 sections which were obtained without loss of sections and with less than 0.5% damaged material. If required, the number of sections could be greater. Due to the nature of this technique it may be applied to a wide range of biological specimens and facilitates correlation of structures at both the light microscope and transmission electron microscope level.