Low temperature scanning electron microscopy: a review.

Low temperature scanning electron microscopy is useful for morphological and analytical studies both in situations where low temperature techniques are used during specimen preparation and where low temperature stages are used for specimen examination and analysis. Examples are given of different low temperature specimen preparation techniques and how they may be applied to different types of specimen. There are still a number of problems associated with morphological identification in fully frozen-hydrated samples and it is important to carry out parallel studies using more conventional transmission electron microscopy and light microscopy preparation techniques. A number of criteria are presented, some or all of which may be used to establish the existence of the frozen-hydrated state.     

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.     

Detection of concanavalin A receptors by affinity to peroxidase and iron dextran by scanning and transmission electron microscopy and x-ray microanalysis

A method of cell surface mapping has been developed on spermatozoa. Concanavalin A binding sites have been simultaneously revelaed both by peroxidase DAB reaction and by iron dextran coupling. The areas are examined in scanning and transmission electron microscopes and submitted to electron probe X-ray microanalysis.     

Semiconductor material assessment by scanning electron microscopy*

Considerable strides have been made in the application of the cathodoluminescent and beam-induced current modes of operation of the scanning electron microscope to the high resolution characterization of semiconductor materials. These techniques can be used to measure parameters such as resistivity, carrier concentration, mobility, composition, diffusion length, minority carrier lifetime, luminescence efficiency, dislocation density and temperature from regions of semiconductors as small as one micron across. The theoretical and experimental factors which limit the range of values over which measurements are possible and the accuracy attainable are discussed in detail. Each technique is illustrated by recent experimental results.     

Topographic and material contrast in low-voltage scanning electron microscopy

Two scintillation backscattered electron (BSE) detectors with a high voltage applied to scintillators were built and tested in a field emission scanning electron microscope (SEM) at low primary beam energies. One detector collects BSE emitted at low take-off angles, the second at high takeoff angles. The low take-off detector gives good topographic tilt contrast, stronger than in the case of the secondary electron (SE) detection and less sensitive to the presence of contamination layers on the surface. The high take-off detector is less sensitive to the topography and can be used for detection of material contrast, but the contrast becomes equivocal at the beam energy of 1 keV or lower.     

Preparation of plant material for scanning electron microscopy*

A general method is described which reduces shrinkage in plant material and which avoids the need for metal coating. The method must be adapted to particular tissues and techniques of observation.     

Lateral beam stability of a computer assisted scanning electron microscope / x-ray microanalysis system

Digital beam control by an image analyzer has opened up new dimensions for scanning electron microscopy and x-ray microanalysis, particularly if the feature density in the sample is high. Prior to its application to aerodynamically classified particles in the micron and submicron size range, the performance of a given system was tested by means of a special object, consisting of a pattern of micron sized holes in a metallic foil. The lateral position of the electron beam was registrated using the centre of gravity values (x- and y-axis) as obtained by consecutively analyzing transmission scanning electron micrographs (bright field mode) of the special object at constant intervals. A major drift source (time dependent electrical interference field) could be detected and eliminated, thus reducing shift speeds exceeding 3 μm/h to values below 0.5 μm/h if thermally induced effects both in the electron source and related electronic circuitry were allowed to become nearly saturated. It is shown that remanence effects in the scanning coils resulting from magnification changes can produce severe sideways jumps in the beam position that must be borne in mind if unattended operation of the system is intended on features of corresponding size.     

Application of high-resolution scanning electron microscopy to biological macromolecules

The development of ultrahigh-resolution scanning electron microscopes (SEMs) has made the observation of biological macromolecules feasible, but adequate preparation methods have not yet been established. Although it has been possible to observe some molecules after they have been spread on a carbon substrate, this method has not proved suitable for other molecules which exhibit lower contrast, or are more susceptible to damage by the electron beam. In this study we have applied heavy-metal impregnation methods using phosphotungstic acid, uranyl acetate, or osmium tetroxide mordanted by tannic acid. In addition, contamination due to the electron beam was reduced by improving the vacuum in the specimen chamber, and by the use of a heated specimen stage. Using these measures, haemocyanin, ferritin, apoferritin, thyro-globulin and immunoglobulin M were successfully imaged. Ultrahigh-resolution SEM seems likely to become an important means for studying the morphology of biological macromolecules.     

Atmospheric scanning electron microscopy and its applications for biological specimens

Scanning electron microscopy in ambient conditions (Air‐SEM) was developed recently and has been used mainly for industrial applications. We assessed the potential application of Air‐SEM for the analysis of biological tissues by using rat brain, kidney, human tooth, and bone. Hard tissues prepared by grinding and frozen sections were observed. Basic cytoarchitecture of bone and tooth was identified in the without heavy metal staining. Kidney tissue prepared using routine SEM methodology yielded images comparable to those of field emission (FE)‐SEM. Sharpness was lower than that of FE‐SEM, but foot process of podocytes was observed at high magnification. Air‐SEM observation of semithin sections of kidney samples revealed glomerular basement membrane and podocyte processes, as seen using conventional SEM. Neuronal structures of soma, dendrites, axons, and synapses were clearly observed by Air‐SEM with STEM detector and were comparable to conventional transmission electron microscopy images. Correlative light and electron microscopy observation of zebrafish embryos based on fluorescence microscopy and Air‐SEM indicated the potential for a correlative approach. However, the image quality should be improved before becoming routine use in biomedical research.     

Manipulating biological samples for environmental scanning electron microscopy observation

Biological samples having different characteristics were observed by environmental scanning electron microscopy (ESEM). The environmental conditions for untreated biological samples was determined by optimizing sample temperature and chamber pressure. When the temperature was at 4 degrees - 6 degrees C and chamber pressure was 5.2-5.9 Torr, the relative humidity in the specimen chamber was about 85%. Under these conditions, the surface features of the sample were completely exposed and did not exhibit charging. The images obtained from the untreated samples at different ESEM conditions were also compared with fixed and coated samples observed under high vacuum.     

Staining of biological material for the scanning electron microscope

A histochemical technique has been used in scanning electron microscopy to localize specific areas in biological material. The specific contrast introduced by the staining procedure depends on the interaction of complex variables such as the coating material, the accelerating voltage and the use of a primary or secondary electron signal.     

Processing small delicate biological specimens for scanning electron microscopy

To overcome the problems of collecting and handling delicate cells while dehydrating them for scanning electron microscopy, a method is suggested whereby cells are collected on solvent resistant Millipore filters. The schedule used for successfully processing a wide range of algae, protozoa and some other organisms is outlined as is the construction of a simple carrier to hold the filter in the critical point drying apparatus.