Micron, 1981, Vol.: 12, pp.159-160. 0047-72061811020159702502.00/0 @Pergamon Press Ltd. Printed in Great Britain.

HIGH VOLTAGE ELECTRON MICROSCOPY OF BIOLOGICAL MATERIALS

Chris Hawes Botany School, University of Oxford, South Parks Road, Oxford, OXI 3RA

Biologists have made comparatively little use of high voltage electron microscopes (H.V.E.M.s), a fact partly due to greatly reduced image contrast at high accelerating voltages (up to 1.2 MV on the Oxford AEI EM 7) coupled with difficulty in the interpretation of the mass of information in 'thick' specimens.

Penetration of an electron beam through a specimen is approximately three times greater at 1000KV than at 100KV, thus correspondingly thicker specimens can be observed with the H.V.E.M. without substantial loss in resolution. This, coupled with the use of stereoscopic techniques allows three dimensional information to be extracted from thick sections (Figs. 1 & 2) which otherwise would only be obtainable by extensive serial sectioning.

High voltase electron microscopy has been used in the study of thin sections in the dark field mode ~, of whole wet fixed and unfixed animal cells with the use of an environmental cell 2 and of whole critically point dried cells 3. However the major use lies with the observation of sections of material up to five microns in thickness 4. The greatly reduced image contrast at high accelerating voltages can partially be overcome by prolonged treatment with conventional EM stains such as uranyl acetate and lead citrate. More distinct images can be obtained by the selective staining of membranes and organelles in the tissue to be studied without staining the background cytoplasm 5. In plant tissue impregnation with a mixture of zinc iodide and osmium tetroxide results in the deposition of a layer of stain on the membranes of nuclear envelopes, chloroplast thylakoid lamellae, tonoplasts, endoplasmic reticulum and the formative face of golgi dictyosomes (Fig. i & 2).

Current work in Oxford concerns the selective staining of higher plant and fungal organelles to study the three dimensional relationship of membrane systems in cells using thick sectioned material.

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REFERENCES

G. Dupouy, Performance and applications of the Toulouse 3 million volts electron microscope, Journal of Microscopy (Oxf) 97, 3 (1973).

D. F. Parsons & l.Uydess & V. R. Matricarde, High voltage electron microscopy of whole wet cells, Effect of different wet cell preparation methods on visibility of structures, Journal of Microscopy (Oxf) i00, 153 (1974).

J. J. Wolosewick & K. H. Porter, Stereo high-voltage electron microscopy of whole cells of the human diploid line WI-38, American Journal of Anatomy 147, 303 (1976).

P. Favard & N. Carasso, The preparation and observation of thick biological specimens in the high voltage electron microscope, Journal of Microscopy (Oxf) 97, 59 (1973).

A. M. Glauert, The high voltage electron microscope in biology, Journal of Cell Biology 63, 717 (1973).

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Figure i. Stereo pair five day bean leaf palisade cell stained with osmium tetroxide/zinc iodide showing tubular (TER) and clsternal (CER) endoplasmlc reticulum and chloroplast (C) 1 ~ section, 5 ° tilt, IO00KV. Bar = 1 ~.

Figure 2. Stereo pair of bean leaf epidermal tonoplast membranes stained with osmium tetroxide/zinc iodide. 5 ~ section, i ° tilt, 1000KV. Bar = 0.5 ~.