Donald F. Parsons

Progress in applying the high-voltage electron microscope to biomedical research

This review attempts a physical definition of the technical problems and achievements in applying the high-voltage electron microscope (HVEM) to biological and medical research. It is hoped that the review will summarize for biologists, funding agencies, and institutions the achievements of the HVEM, its future prospects, and the main problem areas that still need to be explored.At present it is not known whether future HVEMs will favor the fixed beam or the scanning transmission electron microscopy (STEM) mode. The STEM mode offers reduced radiation damage as a result of more efficient electron detection and ease of manipulation of the collected signals by separating the elastic and inelastic signals. Energy filtration to remove the inelastic signal provides a means to enhance the contrast and improve the resolution for thick specimens. Several prototype STEM-mode HVEMs are now under development and it is expected that, in a few years, comparisons of fixed beam and STEM modes will be possible.The review discusses several HVEM instrument features that remain poorly developed. In the area of image recording a photographic emulsion has been designed to give optimized performance at an acceleration voltage of 1 MV. However, this remains unavailable commercially. Conversion of the HVEM electron image to a usable light image by phosphors etc., involves some difficulties, making it difficult to obtain good performance from TV systems. Since the HVEM is particularly useful for three-dimensional imaging, the further development of improved goniometers for stereo viewing and image reconstruction is important. The large volume available in the objective specimen volume and the increased penetration at high acceleration voltages make the HVEM particularly suitable for the application of environmental chambers in the microscopy and electron diffraction of thick wet specimens. An improved signal-to-noise ratio improves the prospects for elemental analysis at high acceleration voltages.When carefully carried out, improved resolution can be obtained in dark-field over that obtainable at 100 kV. Dark-field provides the easiest way to obtain high contrast on weakly stained or unstained objects. Its further improvement requires the use of specially thick and shaped beam stops and apertures that are not penetrated by the 1 MV beam.Recent HVEM studies of whole cells and microorganisms are reviewed. These studies already show that the former thin-section approach led to some incorrect ideas about the shape of some organelles and their three-dimensional relationships. This new information is proving important in helping to establish the function of fibrillar and membranous components of the cell.The most important limitation in examining thick sections is the large depth of field that causes excessive overlap of in-focus structures in stereo views of thick sections. In a few cases special specific heavy metal stains have been developed to overcome this problem, but an optical solution would be more generally applicable. Attempts are now being made to unscramble overlapped detail by applying the image reconstruction techniques of tomography and holography.It is concluded that even with existing techniques, the HVEM examination of thick sections provides a very useful improvement in sampling statistics and in three-dimensional imaging of cell structures over that obtainable by examining thin sections at a lower acceleration voltage (100 kV).

Shape, size, and distribution of cell structures by 3-D graphics reconstruction and stereology. I. The regulatory volume decrease of astroglial cells.

This report discusses fundamental limitations in attempting to derive cell size, shape, or distribution from the two-dimensional images provided by conventional electron microscopy. Morphometric or stereologic measurement of random thin sections is a convenient way to obtain some information of this type. However, it cannot provide complete, objective information about real size, shape, or connectivity of cells containing irregular or unevenly distributed structures or nonuniform populations of cells.Anisotropic structures require analysis of a complete set of serial sections. The analysis may utilize either stereo, mono, or tilted optical slices, and subsequent integration of this information into a single 3-D computer data set.In this study, we analyze stereo pairs of high-voltage electron micrographs of serial thick sections (0.5 μm) and critical-point-dried whole-cell mounts of rat brain astroglial cell cultures. The Z-axis resolution is increased by digitizing contours at discrete levels within each stereo view. This is accomplished with a new type of stereoscopic contouring device. We calculated area and volume changes accompanying hypo-osmolar swelling and spontaneous reversal of the swelling. (Regulatory Volume Decrease-RVD). An understanding of the mechanism of swelling of astroglial cells is important for improving the treatment of brain injury. The total cell-volume results are comparable with results previously obtained using nonmetabolized, radioactively tagged compounds that diffuse into various cell compartments. Our serial-section and whole-cell data also provide new information about the relative swelling of nucleus, cytoplasm, and individual organelles such as mitochondria.The basic biological problem being approached is whether homeo-stasis of cell function is accompanied by surface area and volume regulation of enzyme-rich membranes and organelles. Conversely, it is proposed to explore the possibility that abnormal organelle areas and volumes are indicators of perturbations of cell division, metabolism, or gene expression.

Design features of a photographic film optimized for the high-voltage electron microscope.

Calculations of beam spread for 1-MeV electrons within photographic emulsions based on a simplified model of multiple scattering are used to predict the performance of emulsions as a function of the silver-halide content and thickness of the emulsion layer. Experimental results are given for the sensitivity, resolution, figure of merit, and sensitivity per unit emulsion thickness for a range of photographic films exposed to 1-MeV electrons. Multiple scattering of 1-MeV electrons proves not to be the limiting factor that governs the resolution in the tested commercial films having conventional emulsion thicknesses and silver-halide contents. A new film design is proposed in order to mitigate the serious problem of the low sensitivity of the existing photographic films to 1-MeV electrons, which entails inordinate radiation damage to biological specimens during high-voltage micrography. This film would have an enhanced silver-halide content (as much as 65% AgBr by volume) and an increased emulsion thickness (up to 55 μm). As compared with a parent film having an emulsion 20% AgBr by volume and 25 μm thick, the proposed film should provide sensitivity enhanced sevenfold, with only about 17% loss in resolution. The new film should also show improved performance at conventional accelerating voltages (40–100 kV). Especial advantages are expected both in studying intrinsically radiation-sensitive objects and in studying those that require multiple exposures to the same specimen area, as in tilt series or time-lapse series.

Shape changes and polarization of cells migrating through tissue. A high-voltage electron microscope and computer graphics study of serial thick sections.

Structural changes of carcinoma cells and fibroblasts migrating through small spaces in the elastic-collagen reticulum of mouse peritoneum have been studied by high-voltage electron microscopy of serial thick sections and by computer graphics reconstruction of cell profiles. The change of shape profile of an individual cell, between serial sections is large and the distribution of organelles is very non-uniform and changes markedly between sections. Conclusions about adhesion, intercell contact, cell shape and polarization of cytoplasmic organelles could only be reached by assessing a complete set of serial sections. Our preliminary results suggest that interesting structural changes occur in both carcinoma cells and fibroblasts when migrating through this tissue.

Tumor Cell Interactions with Stromal Elastin and Type I Collagen: The Consequences of Specific Adhesion and Proteolysis

Elastin and collagen are abundant fibrous molecules in the stroma. Tumor cells invading the stroma are in contact with fibers of both types much of the time. Both may serve as footholds for the traction required for movement. Elastin has an additional role. Elastin peptides are known to stimulate receptor signaling and chemotaxis, which could explain the morphometric changes (membrane and organelle polarization and cell volume shrinkage) that we have reported for certain tumor cell lines invading elastic lamina. Elastin and its peptides emerge as possible invasion enhancers for some tumor cells. In ongoing work we are screening human tumors that contact elastin (e.g., breast carcinomas) to see if the presence of elastin receptors correlates with early dissemination of metastatic tumor cells.

Patterns of blood-vessel invasion by mammary tumor cells

Rat mammary tumor TMT-081 was employed as a model for blood vessel invasion because its mode of metastasis resembles that of human tumors. The invasive mechanism was studied with two methods of serial transplantation: transfer of enzymatically dispersed solid tumors, and transfer of buffy coat containing circulating tumor cells. The latter method produced greater invasion of blood vessels, including larger veins and occasionally arteries, perhaps by obviating damage to tumor cells during enzyme treatment. The course of migration was traced by three-dimensional examination in the high voltage electron microscope, as well as the light microscope. Two broad patterns were found for the course of invasion of small and large vessels respectively.

Immediate Ascites Conversion of Mammary Tumors Induced in NYLR/Nya Mice by 7,12-Dimethylbenz-[a] Anthracene and Urethane Feeding and by Forced Breeding

Intragastric feeding of dimethylbenz-[a]anthracene in corn oil together with urethane in the drinking water and forced breeding were successful in rapidly inducing (2-4 months) mammary tumors (adenoacanthomas) in inbred NYLR/Nya mice, which have a low incidence of spontaneous breast tumors and of other tumors. The tumors could be quickly and permanently transformed to an ascites form by intraperitoneal inoculation of enzyme-dissociated cells and subsequent serial passage of free cells. Only 10 (3 in some cases) serial passages were required, thus conveniently providing multiple isogeneic carcinoma cell lines in this strain of mice. Some tumor cell lines proliferated strongly in the abdominal cavity even on the first passage.

Inflammation with restricted lysosomal proteolysis during early ascites carcinoma invasion of mouse parietal periotoneum. A medium and high-voltage electron microscopic and cytochemical study

A carcinoma invasion system (Krebs-2 and Ehrlich tetraploid ascites tumors invading mouse peritoneum) was studied by high-voltage electron microscope (HVEM) stereoscopy, conventional (medium voltage) electron microscopy (MVEM), and cytochemistry. Tumor cells entered areas of peritoneum (mainly parietal) only where mesothelial cells were damaged and where there was inflammation of the underlying stroma. The initial invasion was different from that of most other invading carcinomas in that there was minimal breakdown of basal lamina and collagen. Neither tumor cells, inflammatory leukocytes nor peritoneal fibroblasts showed significant secondary lysosome production or release of intracellular or extracellular acid phosphatase. Morphological and cytochemical criteria suggest that in some invading carcinomas, as with non-tumor migrating cells such as leukocytes, widespread proteolysis due to diffusion of proteases is not a prerequisite for invasion of stromal connective tissue.

Use of fast X-ray film for low-fluence biological electron microscopy at 100 kV and 1000 kV

A user evaluation has been made by electron microscopists of an X‐ray film for routine electron microscopy. The recent improvements in mammographic X‐ray films, with the attempt to reduce the patient dose required to produce a high‐resolution mammogram, have resulted in some useful films for medium‐ and high‐voltage electron microscopy. They can yield essential cytological information with a reduction of the electron fluence (exposure) applied to the specimen of more than an order of magnitude compared with conventional electron‐microscope films. Their use is indicated in situations where beam damage is severe.

Basophilic “Dark Cells:” Optimal Conditions for Toluidine Blue Staining

Differential staining of basophilic “dark cells” of epithelia in thick epoxy sections with toluidine blue is critically affected by fixation and embedding conditions. Optimal conditions are described.