Murray Vernon King

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).

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.

Asbestos-contaminated drinking water: its impact on household air

Asbestos contamination in excess of 10 billion fibers per liter was detected in a communitys drinking water. To assess the possibility of waterborne asbestos becoming airborne, air samples were collected from impacted houses receiving contaminated water from three control houses. Collected within each house were three samples on 0.6-micrometer-pore Nuclepore filters and three samples on 0.8-micrometer-pore Millipore filters. In addition, bulk samples of suspect material and water samples were collected. Mean waterborne asbestos concentrations were 24 million fibers per liter (MFL) in the impacted houses versus only 1.1 MFL in the control houses. Transmission electron microscopy revealed that airborne asbestos concentrations were highest in impacted houses, with airborne asbestos concentrations positively correlated with waterborne concentrations. For fiber and mass measurements on both filter types, airborne asbestos concentrations were significantly higher in the impacted houses: mean concentrations in impacted houses were 0.12 fibers/cm3 and 1.7 ng/m3 on Nuclepore filters and 0.053 fibers/cm3 and 2.3 ng/m3 on Millipore filters versus only 0.037 fibers/cm3 and 0.31 ng/m3 on Nuclepore filters and 0.0077 fibers/cm3 and 0.14 ng/m3 on Millipore filters from control houses. Also detected in the air samples from impacted houses were clusters of chrysotile, often with several hundreds of fibers. When estimates of these individual fibers were added to the total fiber count, the difference between the impacted and control houses became even greater. The increased concentrations in impacted houses were due primarily to short (less than 1 micrometer) fibers. Bulk samples did not reveal likely materials within the impacted houses to account for these differences. Thus high levels of waterborne asbestos were apparently the source of increased concentrations of airborne asbestos within these houses.

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.

A perspicuous technique for directly visualizing radiation-damage artefacts in biological electron microscopy.

Levels of impairment of electron‐microscopic images of biological specimens stemming from radiation damage are assessed in a rapid visual procedure that involves taking a pair of low‐fluence micrographs of a specimen area before and after a fraction of the picture area has been more seriously damaged by applying a measured electron fluence. The pair of micrographs is treated as a mock‐stereo pair and is given contrasting colours. Lateral displacements of specimen details appear as false relief and changes in electron lucency as false colour.

chapter 2 Theory of Stereopsis

Publisher Summary This chapter discusses the theory and mechanisms of stereopsis. The view of the stereoptic mechanism that has emerged is one of a cerebral system for analyzing spatial information by integrating a wide variety of both binocular and monocular cues existing in the images seen by the two eyes. Its most potent source of depth cues is the disparity of positions of details in the two images, although many other features such as perspective and motion of objects in the scene also contribute greatly. The role of left-right positional disparities will be central to much of discussion, both because of the intrinsically large part they play in stereopsis and because they constitute the only type of depth information present in many electron microscopic situations, especially in the viewing of micrographs of tissue sections taken in the transmission electron microscope. The limiting angular disparities for breakaway and refusion prove to depend critically on the character of the object being examined and on their direction relative to the vector joining the observers eyes, which is as the horizontal axis.

High-voltage electron microscopy and three-dimensional graphic study of R and T cells in head and neck carcinomas.

Individual head and neck carcinomas show extreme regional cellular differentiation. Some cells are rich in keratin filaments (T cells) and some have little keratin and a high density of free ribosomes (R or RT cells). We attempted to isolate these two cell types in order to test their relative invasiveness in an in vitro model. The high frequency of mitosis of hyperkeratinized cells showed that there was no constraint on the motility of cell division. High-voltage electron microscopy of serial thick sections and three-dimensional graphic reconstruction demonstrated that keratin cytoskeleton filaments were cross-linked into short, thick bundles. However, the keratin cytoskeleton was absent from some portions of the cytoplasm. In normal differentiated keratinized cells, a more uniform spanning of the whole cell by thin keratin intermediate-filament bundles was evident. The cytoplasm may be more mobile in the keratinized tumor cells. Even heavily keratinized T cells, like the less keratinized cell types, may have invasive motility.

Detection and Characterization of Circulating Rat Mammary Tumor Cells in Buffy Coat and Correlation with Metastasis

A new method for detecting bloodborne TMT-081 rat mammary tumor cells in buffy coat has revealed dose-dependent variations in the latency period after inoculation of tumor cells, the concentration of circulating tumor cells, and the incidence of metastases. Cells isolated from buffy coat of right ventricular blood were more tumorigenic than tryptically dispersed cells from solid tumors. With the new method circulating tumor cells can be detected at concentrations as low as 3 cells/microliter of buffy coat, or approximately 60 cells/ml of whole blood. The morphologic and ultrastructural features of the primary tumor were generally retained in both the circulating and tryptically dispersed cells, as shown by light and electron microscopy. A sparse distribution of intermediate filaments was revealed by high-voltage electron microscopy, although the filaments were not evident in conventional transmission electron micrographs. They were identified as keratin by immunofluorescence studies.

Ultrastructural Characterization of Isolated Human Head and Neck Squamous Carcinoma Cells. Assessment of Isopycnic Centrifugation

In an experiment to evaluate the merit of isopycnic centrifugation as a method of separating cell types in human head and neck squamous cell carcinomas, cells have been isolated from four specimens of these tumors and subjected to isopycnic centrifugation in continuous Percoll gradients. Cell types were identified by electron microscopy. The R- (ribosome-rich), T- (tonofilament-rich), and RT- (intermediate) cell types yielded broad bands overlapping extensively with one another, and partially with the bands of leukocytes. The pattern differed for each tumor studied, so that universal density levels separating given cell types could not be found. Isopycnic centrifugation proves less suitable in analyzing cells dispersed from solid tumors than for cells in suspended culture, blood, effusions, etc., probably because of heterogeneous growth conditions of cells in solid tumors.

chapter 8 Mock Stereo

Publisher Summary This chapter describes the definition and scope of mock stereo. A mock-stereo display is one that generates depth perception from parallax information arising from sources other than a shift of vantage point. Such displays have at times proved effective in various fields of study including electron microscopy and therefore it is interesting to examine the conditions under which such displays can prove advantageous. Mock-stereo displays can include before-and-after pictures of a scene in which some details have become shifted by motion or distortion of objects and comparisons to establish identity or equivalence of objects in two scenes. A criterion for selecting types of images as candidates for mock-stereo treatment is that this method should offer advantages over other modes of display in enhancing recognizability of features. Mock-stereo methods gain another advantage when one wishes to display two different types of disparities in an image pair simultaneously to the observer. One of the types can be assigned to the mock-stereo channel by displaying it as horizontal parallax, and the other.