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

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The high voltage electron microscope in biology.

SUMMARY

Techniques for the preparation and investigation of thick biological sections in the high voltage electron microscope (HVEM) have been studied using the Toulouse 1.5 and 3 MV microscopes.



Sections several micrometres thick can be made using conventional ultra-microtomes, but, in order to know their actual thickness, it is necessary to resection them perpendicularly to their surface after embedding them in Araldite.



When successive thick sections are made with glass knives, damage may be observed on their upper surface. This can be avoided by alternating several thin sections between each thick section. Such damage is further reduced if thick sections are made using diamond knives.



The maximum thickness that can be observed at a voltage of 2.5 MV is not limited by the penetrating power of the beam but by the damage suffered by irradiated Araldite. The limit is approximately 10 μm. Below 10 μm a general uniform loss in thickness is observed in irradiated areas; above 10 μm, that loss in thickness is irregular and craters are formed under the beam.



Contrast can be obtained by staining tissues before or after embedding them in Araldite. The cellular structures can be uniformly stained using uranium or lead salts, or selected parts of them can be stained using colloidal tracers, Gomori's lead method, osmium or silver impregnations. The latter techniques are the more convenient as far as sections several micrometres thick are concerned.



The resolving power depends on section thickness and accelerating voltage. At 2.8 MV, by using selective staining techniques, sections several micrometres thick can be investigated with a sufficient resolving power, especially with regard to understanding the spatial organization.



Stereoscopic viewing allows a better interpretation by removing the inconveniences resulting from overlapping structures, but the depth of field must then be considered. Its value, for a given resolving power, taking into account the focal length of the objective lens, depends on the objective aperture: by using a 5 μm aperture one can get a depth of field of 4 μm at a resolving power of 2.0 nm and 12 μm at a resolving power of 6.0 nm, the focal length of the Toulouse 3 MV electron microscope being 10 mm. At this voltage aberrations due to diffracting phenomena are not very disturbing.

The preparation and observation of thick biological sections in the high voltage electron microscope.

The high energy electron microscope has been used mainly in the field of material science and of minerology. Especially in minerology, the advantage of the higher penetrating power of electrons at higher accelarating voltage which enables the observation of material in bulk is enormous and the high voltage device becomes indispensable.

High Voltage Electron Microscopy

In order to investigate the geometry of the interactions which myosin molecules make with actin filaments we have studied thick (0.2–0.5 µm) transverse sections of striated muscles in the 1 MeV electron microscope at Imperial College. Sections obtained from fixed relaxed frog sartorius muscle and both fixed relaxed and fixed rigor insect flight muscles, show regular electron opaque features between the thick and thin filament profiles. These are thought to be the overlapping images of the many levels of myosin heads that occur in such sections. From the appearances of these images, together with studies of thin transverse sections, it appears that of the possible interactions which one myosin molecule can make, namely that its two component heads interact with the same thin filament or with two different thin filaments, it is the former interaction (both heads on the same filament) which is predominant. Nevertheless appearances have been seen similar to those expected if an interaction of one molecule with two thin filaments occurs. It is concluded that both single filament and two filament interactions can occur depending on the steric convenience of the available actin subunits, but that the single filament interaction occurs in the majority of cases in the muscle states we have studied.

Joan Moses

Papers

Contrast and Resolution of Thick Sections of Muscle in the HVEM