The high voltage electron microscope in biology.

Abstract

The first published micrograph of a biological specimen photographed in a high voltage electron microscope (HVEM) appeared in a paper by Van Dorsten et al. in 1947, and it is consequently surprising that the HVEM does not play a greater role in biology 27 yr later. There are various reasons for this. The first prototypes were developed with the aim of enabling the investigator to examine thicker specimens than could be viewed at 100 kV. At the same time, however, the thin-sectioning technique was becoming established and consequently none of these early prototypes led to a commercial instrument (Glauert, 1972). Subsequently, the development of the HVEM was determined more by the needs of metallurgists and materials scientists rather than the needs of biologists, although the 1.5-MV microscope in Toulouse was constructed in the hope that it would make possible the examination of living cells (Dupouy et al., 1960). Results in this field have proved disappointing; even the best micrographs obtained so far reveal very little more internal detail of cells than can be seen with an ultraviolet microscope, and damage to living organisms during irradiation in the electron microscope is so fatal that any process that is observed is more correctly described as 'dying' than 'living'. In spite of the apparently insuperable problems in obtaining electron micrographs of living cells, the HVEM still has great potentiality in biology, arising from its capability to produce high resolution images of thick specimens. This capability enables the examination of much thicker sections than can be viewed in conventional instrumerits operating up to 100 kV and, in combination with stereo viewing, has restored the third dimension in studies of a great range of biological material. Examining very thin sections over more than 25 yr has led microscopists to think of biological structures in terms of thin slices: the term 'mitochondrion' summons up the vision of the cross-sectional view rather than of a threedimensional cylinder composed of interconnected membrane systems. This restoration of the third dimension in the appreciation of biological organization has been the major contribution of the HVEM so far. The other promising line of investigation is the examination of hydrated (not living) specimens in the HVEM. Water is an essential constituent of all biological structures, and consequently it is of paramount importance to be able to examine specimens in the wet state, particularly when considering the molecular level of organization. Rapid progress has been made during the past few years in the development of suitable preparative and imaging techniques, and in the design of 'environmental cells', and there is no doubt that further advances in this field will soon be reported. At present the few biologists who are using the HVEM are not all bringing the most fruitful problems to the microscope. For example, there is no point in obtaining a stereo view of a thick section unless an understanding of the threedimensional structure of the embedded material will make a real contribution to the solution of a particular problem. The primary aim of this review is to indicate the capabilities of the HVEM and to