The granulomatous inflammatory response. A review.

Metal toxicity for rabbit alveolar macrophages in vitro

Several recent technological advances have increased the practicality and usefulness of the technique of electron microscopy of wet objects. (i) There have been gains in the effective penetration of high-voltage microscopes, scanning transmission microscopes, and high-voltage scanning microscopes. The extra effective penetration gives more scope for obtaining good images through film windows, gas, and liquid layers. (ii) Improved methods of obtaining contrast are available (especially dark field and inelastic filtering) that often make it possible to obtain sufficient contrast with wet unstained objects. (iii) Improved environmental chamber design makes it possible to insert and examine wet specimens as easily as dry specimens. The ultimate achievable resolution for wet objects in an environmental chamber will gradually become clear experimentally. Resolution is mainly a function of gas path, liquid and wet specimen thickness, specimen stage stability, acceleration voltage, and image mode (fixed or scanning beam) (13). Much depends on the development of the technique for controlling the thickness of extraneous water film around wet objects or the technique for depositing wet objects onto dry, hydrophobic support films. Although some loss of resolution due to water or gas scattering will always occur, an effective gain is anticipated in preserving the shape of individual molecules and preventing the partial collapse that usually occurs on drying or negative staining. The most basic question for biological electron microscopy is probably whether any living functions of cells can be observed so that the capabilities of the phase contrast and interference light microscopes can be extended. Investigators are now rapidly approaching a final answer to this question. The two limiting factors are (i) maintaining cell motility in spread cells immersed in thin layers of media and (ii) reducing beam radiation damage to an acceptable level. The use of sensitive emulsions and image intensifiers can bring the observation dose below that required to stop cell motility. Use of a timed, pulsed deflector system enables sufficiently short exposures to be obtained to eliminate blurring due to Brownian motion. Environmental chambers have enhanced the possibilities of electron diffraction analysis of minute crystals and ordered biological structures. High-resolution electron diffraction patterns (especially kinematic) of protein crystals can only be obtained in a wet environment. Hence, it may now be possible to obtain undistorted images of protein molecules. Moreover, by subjecting diffraction patterns to image-iterative techniques (56), it will be possible to phase the electron diffraction patterns to give a calculated image with a higher resolution than that which can be produced by electron microscope objective lenses. Environmental chambers offer exciting prospects for the determination of water structure and water and ice nucleation (atmospheric science). Nucleation data near the molecular level have been badly needed for some time. The application of environmental chambers in industrial chemistry, for example, in studies of polymerization, catalysis, and corrosion, are awaiting exploration. They offer an unusual approach to measurements of reaction kinetics through images that should be both sensitive and rapid.

https://science.sciencemag.org/content/sci/186/4162/407.full.pdf

Structure of Wet Specimens in Electron Microscopy.

The morphology of the polysaccharide coat of mammalian cells.

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

/pdf/the-high-voltage-electron-microscope-in-biology-k0l45mwoy9.pdf

The high voltage electron microscope in biology.

SUMMARY

Normal and stimulated peritoneal cells have been examined with the Stereo-scan scanning electron microscope. Normal lymphocytes were identified by their small size; some had numerous small stubby or pointed processes on their surfaces. Macrophages were larger and had ridge-like processes. Many of the stimulated cells showed no significant deviation from normal. Others, all macrophages, showed a marked deviation from the normal spherical shape. Ridge-like processes were more prominent on their surfaces, and large flange-like processes and a few finger-like processes were seen. This suggests that on stimulation peritoneal cells become more deformable and therefore irregular in shape, and that their surfaces become rougher.

The surface structure of mouse peritoneal cells—a study with the scanning electron microscope

SUMMARY

Red blood cells and bacteria have been viewed under the high voltage transmission electron microscope, using a controlled environment chamber. Red cell changes on introduction of an agglutinating serum into the environment and bacterial changes on addition of an antibiotic, were observed. The results are compared with those obtained by phase contrast light microscopy and the potentialities of the system discussed.

High voltage electron microscopy of cellular changes in the wet state.

SUMMARY

Preliminary results obtained by examining intact fixed human red and white blood cells containing inorganic particles under the high-voltage transmission electron microscope are described. Iron filings and ferritin ingested in vitro by granulocytes were observed as were inorganic particles in red cells from a case of lead poisoning. The nature of the particles in red cells is discussed. A faint intracellular network was seen in normal red cells and in cells from a case of lead poisoning. It was found possible to focus the electron microscope at different planes within the cell. The optimum accelerating voltage for red cells appeared to be around 750 kV, whereas a clear image of granulocytes was obtained at voltages between 750 and 1000 kV. The first results indicate that it is possible to examine intact blood cells under the high-voltage electron microscope. Further work is in progress to determine if more information can be acquired by this technique.

High-voltage electron microscopy of inorganic particles in intact blood cells.

SUMMARY

Examination of normal human red blood cells with a high (2.5 nm) resolution scanning electron microscope revealed a definite surface pattern. The pattern was slightly more pronounced in specimens dried in air after fixation than in those dried by the critical-point method. There was a radial arrangement of filament like elevations at the periphery of the red cell and a more reticular pattern to wards the centre of the cell. The appearances probably represented a network of elenin fibres lying just under the surface of the red cell.

J. A. Clarke

Papers

The surface structure of mouse peritoneal cells—a study with the scanning electron microscope

High voltage electron microscopy of cellular changes in the wet state.

High-voltage electron microscopy of inorganic particles in intact blood cells.

High-resolution scanning electron microscopy of the surface of red blood cells.