High voltage electron microscopy of environmental reactions

An incorrect caption to Fig. 11 was printed in this paper which was published in the Journal of Microscopy, Vol. 97, Pts 1/2, pp. 171–190. The correct caption should read ‘Unstained glutaraldehyde-fixed 3T3 cell (bright- and dark-field pair) showing mitochondria (arrowed). In wet specimens the mitochondria appear broader (possibly due to damage) and scatter electrons less strongly than the surrounding material.’     

The use of specimen tilt in transmission electron microscopy of the central nervous system

Thin sections of nervous tissue were viewed at different tilt angles using a transmission electron microscope equipped with a eucentric goniometer stage. In a comparison study of various degrees of tilt, one can observe additional morphological features within synaptic profiles, define subsynaptic structures such as Taxi-bodies, and clearly see the crystalline formation of cytochemical tracers. This study demonstrates the value of tilting thin-sections in the analysis of synapses and other biological material at the ultrastructural level.     

High voltage electron microscopy of cellular changes in the wet state

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 cell nucleus fractions: feasibility studies

Cell nucleus fractions were analysed in high voltage electron microscopes equipped with a hydration chamber and an energy filter, and the different filtering modes were tested. The best results were obtained when the energy filter was used as an analyser of atomic tracers specifically combined with RNA. Although the resolution thus obtained was relatively poor, the prospects of being able to observe hydrated organelles are encouraging.     

Low voltage scanning electron microscopy

The scanning electron microscope (SEM) is usually operated with a beam voltage, V₀, in the range of 10–30 kV, even though many early workers had suggested the use of lower voltages to increase topographic contrast and to reduce specimen charging and beam damage. The chief reason for this contradiction is poor instrumental performance when V₀=1–3 kV, The problems include low source brightness, greater defocusing due to chromatic aberration greater sensitivity to stray fields, and difficulty in collecting the secondary electron signal. Responding to the needs of the semiconductor industry, which uses low V₀ to reduce beam damage, considerable efforts have been made to overcome these problems. The resulting equipment has greatly improved performance at low kV and substantially removes the practical deterrents to operation in this mode. This paper reviews the advantages of low voltage operation, recent progress in instrumentation and describes a prototype instrument designed and built for optimum performance at 1 kV. Other limitations to high resolution topographic imaging such as surface contamination, the de-localized nature of the inelastic scattering event and radiation damage are also discussed.     

Resolution in low voltage scanning electron microscopy

As the energy of an electron beam is reduced, the range falls and the secondary electron yield rises. A low voltage scanning electron microscope can therefore, in principle, examine without damage or charging samples such as insulators, dielectrics or beam sensitive materials. This paper investigates the way in which the choice of beam energy affects the spatial resolution of a secondary electron image. It is shown that for samples which are thin compared to the electron range, the edge resolution and contrast in the image improve with increasing beam energy. In samples that are thicker than the electron range, the resolution can be optimized at either high or low energies, but low energy operation will produce images of higher contrast. At an energy of 2 keV or less beam interaction limited resolutions of the order of 3 nm should be possible.     

Current developments in high voltage electron microscopy

In respect of instrument design two main developments are taking place in high voltage electron microscopy: towards even higher operating voltages (3–5 MV) and towards higher resolving power at moderate voltages (250–600 kV). Applications of existing instruments (650 kV-1.2 MV) are still primarily in metallurgy, especially for radiation damage studies, but their usefulness for biological research is now being actively explored. Microchambers have been developed for observing specimens, both metallurgical and biological, in a controlled gaseous or liquid environment. The prospects for observing living material, except at low magnifications, remain very doubtful on account of ionization effects, but informative work with wet specimens should be possible.     

Very low voltage electron microscopy.

We conclude that a 150 V scanning microscope with a resolution of 10 A is quite feasible and could have considerable value. It might consist of a field emission source, an electron gun to decelerate the electrons, a condenser lens to produce a parallel beam, a multipole corrector and a short focal length objective lens. Electrons reflected from the specimen surface would pass through a spectrometer whose principal features would be a large collecting power and low (1/200) energy resolution. Finally, we should add that such a microscope presents a considerable challenge and new opportunities for the electron optician in both lens and spectrometer design. We cannot refrain from pointing out that the Scherzer theorem does not necessarily hold for such a lens since the constraints of the theorem do not apply to this case.     

Three-dimensional tomographic reconstruction in high voltage electron microscopy

The methodology is described, as developed at the Albany NIH Biotechnological High Voltage Electron Microscope Resource, for the three-dimensional reconstruction of objects in thick sections. The reconstructions are obtained from projection sets which are recorded by high voltage electron microscopy. The different steps of the procedure are illustrated in detail, using the cilium reconstruction as an example.     

Detectors for low voltage scanning electron microscopy

Two simple electron detectors (low and high take-off angles) for low-voltage scanning electron microscopy were built and tested. They contain large area scintillators with an applied high voltage and are able to detect backscattered electrons with high efficiency. These detectors also can record the sum of backscattered and secondary electrons. The primary beam of the microscope is screened from the scintillator high voltage by grids, which also permit switching from the BSE to the (SE + BSE) mode. The circular symmetry of the grids minimizes the influence of applied potentials on the primary beam. The use of the low and high take-off detectors permits the detection of back-scattered electrons emitted from the specimen surface into different ranges of take-off angles.     

Adrenomedullin in the central nervous system

Adrenomedullin (AM) is a novel vasodilator peptide first purified from human pheochromocytoma by tracing its capacity to stimulate cAMP production in platelets. AM immunoreactivity is widely distributed in the central nervous system (CNS) and in the rat has been demonstrated by immunohistochemical techniques to be present in many neurons throughout the brain and spinal cord, as well as in some vascular endothelial cells and perivascular glial cells. Electron microscopy shows that the immunoreactivity is located mainly in the neuronal cytoplasm, but also occurs in the cell nucleus in some cells of the caudate putamen and olfactory tubercle. Biochemical analyses suggest that higher molecular forms, presumably precursor forms, may predominate over fully processed AM in some brain areas. The expression of AM immunoreactivity is increased in cortical neurons, endothelial cells, and perivascular processes after a simulation of ischemia by oxygen and glucose deprivation. Immunohistochemical, electrophysiological, and pharmacological studies suggest that AM in the CNS can act as a neurotransmitter, neuromodulator, or neurohormone, or as a cytoprotective factor in ischemic/hypoxic conditions, in addition to its vasodilator role.     

High resolution dark-field electron microscopy

In the last few years some promising images of biological specimens have been obtained using the high contrast and resolution of dark-field electron microscopy. However, important problems of image interpretation and difficulties in specimen preparation limit at the present time, the usefulness of this mode of image formation. The destruction of the sample by the electron beam, is of utmost importance. Some possibilities of partly overcoming it are discussed.     

High resolution in-focus Lorentz electron microscopy

The Foucault in-focus method for viewing magnetic domains in a conventional electron microscope has been modified. The main feature of our modification is to introduce an aperture below the intermediate lens and to use it for stopping out one part of the split central spot instead of the objective aperture. This arrangement substantially reduces the axial astigmatism due to the objective aperture and makes it possible to reach the point resolution 5–6 nm in both light and dark domains. The image resolution is limited by the small image magnification achieved using the three-stage optical system of a Tesla BS 413 microscope.     

Application of channel electron multipliers in an electron detector for low‐voltage scanning electron microscopy

An electron detector containing channel electron multipliers was built and tested in the range of low‐voltage scanning electron microscopy as a detector of topographic contrast. The detector can detect backscattered electrons or the sum of backscattered electrons and secondary electrons, with different amount of secondary electrons. As a backscattered electron detector it collects backscattered electrons emitted in a specific range of take‐off angles and in a large range of azimuth angles enabling to obtain large solid collection angle and high collection efficiency. Two arrangements with different channel electron multipliers were studied theoretically with the use of the Monte Carlo method and one of them was built and tested experimentally. To shorten breaks in operation, a vacuum box preventing channel electron multipliers from an exposure to air during specimen exchanges was built and placed in the microscope chamber. The box is opened during microscope observations and is moved to the side of the scanning electron microscope chamber and closed during air admission and evacuation cycles enabling storing channel electron multipliers under vacuum for the whole time. Experimental tests of the detector included assessment of the type of detected electrons (secondary or backscattered), checking the tilt contrast, imaging the spatial collection efficiency, measuring the noise coefficient and recording images of different specimens.     

Characterization of polymer blends by low voltage scanning electron microscopy

A technique for characterization of polymer blends by low voltage scanning electron microscopy is described. The method allows observation of the distribution of phases in a blend due to good topographical and compositional contrast. This is possible because of lower beam penetration and high secondary emission coefficient (δ ? 1) at low accelerating voltages. Uncoated polymer samples are imaged with no charging or beam damage problems. The technique has a great advantage over conventional transmission electron microscopy techniques because the sample preparation is minimal and larger areas can be prepared for viewing.     

A charge coupled device camera with electron decelerator for intermediate voltage electron microscopy

Electron microscopists are increasingly turning to intermediate voltage electron microscopes (IVEMs) operating at 300-400 kV for a wide range of studies. They are also increasingly taking advantage of slow-scan charge coupled device (CCD) cameras, which have become widely used on electron microscopes. Under some conditions, CCDs provide an improvement in data quality over photographic film, as well as the many advantages of direct digital readout. However, CCD performance is seriously degraded on IVEMs compared to the more conventional 100 kV microscopes. In order to increase the efficiency and quality of data recording on IVEMs, we have developed a CCD camera system in which the electrons are decelerated to below 100 kV before impacting the camera, resulting in greatly improved performance in both signal quality and resolution compared to other CCDs used in electron microscopy. These improvements will allow high-quality image and diffraction data to be collected directly with the CCD, enabling improvements in data collection for applications including high-resolution electron crystallography, single particle reconstruction of protein structures, tomographic studies of cell ultrastructure, and remote microscope operation. This approach will enable us to use even larger format CCD chips that are being developed with smaller pixels.     

High resolution electron microscopy of carbon structure

An investigation is made of the inherent performance of a high-resolution transmission electron microscope applied to the study of developing graphitic-sheet structures in heat-treated, coal-tar pitch carbons. Image detail is shown to be highly dependant on instrumental defocus. It is not obvious which form will be assumed by artefacts in these images; consequently, anomalous features are illustrated by reference to a specific electron-optical case and a corresponding light-optical analogy. Despite the difficulties associated with locating an optimum level of focus, optical diffractometry confirms that adequate conditions of microscopy are attainable on a routine basis. In a quantitative analysis of morphology in coal-tar pitch carbons, the technique reveals the cause of incipient, thermally-induced, molecular distortions which evidently result in a regression in the improvement of order otherwise developing with progressive heat-treatment.