Application of an ultrahigh-resolution scanning electron microscope (UHS-T1) to biological specimens

In 1985 we developed an ultrahigh-resolution scanning electron microscope with a resolution of 0.5 nm. It is equipped with a field emission gun and an objective lens with a very short focal length. In this study we report a survey of some different preparation techniques and biological specimens using the new scanning electron microscope. Intracellular structures such as cell organelles were observed surprisingly sharper than those observed by ordinary scanning electron microscopes. However, at magnifications over 250,000 X, platinum particles could be discerned as scattered pebbles on the surface of all structures in coated materials. Using an uncoated but conductively stained specimen, we successfully observed ribosomes on a rough endoplasmic reticulum at a direct magnification of 1 million. In these images some protrusions were recognized on the ribosomes. Ferritin and immunoglobulin G were used as samples of biological macromolecules. These samples were observed without metal coating and conductive staining. The ferritin particles appeared as rounded bodies without any substructure on the surface and immunoglobulin G as complexes of three-unit bodies. In the latter the central body might correspond to the Fc fragment and two side ones to Fab fragments. We assume that ultrahigh-resolution scanning electron microscopy is an effective means for observation of the cell fine structures and biological macromolecules. It will open a new research field in biomedicine.     

Simulation of transmission electron microscope images of biological specimens

We present a new approach to simulate electron cryo‐microscope images of biological specimens. The framework for simulation consists of two parts; the first is a phantom generator that generates a model of a specimen suitable for simulation, the second is a transmission electron microscope simulator. The phantom generator calculates the scattering potential of an atomic structure in aqueous buffer and allows the user to define the distribution of molecules in the simulated image. The simulator includes a well defined electron–specimen interaction model based on the scalar Schrödinger equation, the contrast transfer function for optics, and a noise model that includes shot noise as well as detector noise including detector blurring. To enable optimal performance, the simulation framework also includes a calibration protocol for setting simulation parameters. To test the accuracy of the new framework for simulation, we compare simulated images to experimental images recorded of the Tobacco Mosaic Virus (TMV) in vitreous ice. The simulated and experimental images show good agreement with respect to contrast variations depending on dose and defocus. Furthermore, random fluctuations present in experimental and simulated images exhibit similar statistical properties. The simulator has been designed to provide a platform for development of new instrumentation and image processing procedures in single particle electron microscopy, two‐dimensional crystallography and electron tomography with well documented protocols and an open source code into which new improvements and extensions are easily incorporated.     

Cleaning of scanning electron microscope specimens by puncture perfusion.

Puncture perfusion method was described for elimination of blood and tissue fluid in the scanning electron microscope specimens. It was also demonstrated that the puncture perfusion is of excellent use in cleaning the small samples including the human biopsy samples, which are hardly washed by the vascular cannulation.     

A gripping stub* for the examination of multilaminous biological specimens in the scanning electron microscope

A simple specimen holder has been developed which expedites study of multilaminous biological specimens in the scanning electron microscope. It has the interesting additional property of allowing examination of the top and bottom of a single sample.     

Splitting wood specimens for observation in the scanning electron microscope

This report deals with the principles of splitting wood specimens for observation in the scanning electron microscope and gives some examples in which the splitting is a crucial step in specimen preparation.     

Cutting wood specimens for observation in the scanning electron microscope

This report deals with the cutting of wood specimens for observation in the scanning electron microscope. Several cutting devices and types of knives are described and critically evaluated.     

A simple technique for examining fresh, frozen, biological specimens in the scanning electron microscope

A technique is described for the examination of frozen biological specimens on a modified stub, and a clamping device for examining freeze-fractured specimens.     

The selection of conditions for examination of specimens in a scanning electron microscope

In the past not much attention has been paid to the use of the most desirable conditions for examining a particular kind of specimen in a scanning electron microscope (SEM). The various factors affecting resolution in the SEM, namely those of beam diameter, beam penetration, contrast, signal-to-noise ratio and depth of focus, are examined. It is not possible in practice to eliminate all detrimental effects and compromises have to be made in instrumental settings, such as gun potential, lens currents, etc. in order to obtain the best image for a given specimen. This paper deals with the problems involved in examining textile fibres, metal surfaces and thin films with particular emphasis being made on the choice of gun potential and the amount of image detail observed. Examples are shown comparing micrographs taken using gun potentials of 5 kV and 20 kV, showing that due to electron-penetration effects much surface detail is lost at the high gun potential. It is, therefore, useful to examine specimens initially at different accelerating voltages to determine if any desirable information is being lost due to the penetration of the electron beam. Some operating conditions are also given for use as a guideline for working at low accelerating voltages.     

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.     

Scanning tunneling microscope combined with scanning electron microscope

A scanning tunneling microscope (STM) has been installed in a usual scanning electron microscope (SEM) with a vacuum of 10⁻⁶ Torr. The STM image is displayed on the cathode ray tube of the SEM, 512 × 512 pixels, with a scanning rate of 80 s/picture. The spatial resolution of the STM is about 1 Å, while that of the SEM is several tens of ångströms. The combined scanning microscope covers a wide magnification range from 10 to 10⁷, where STM covers the high magnification region from 10⁵ to 10⁷.     

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.     

Imaging thin and thick sections of biological tissue with the secondary electron detector in a field-emission scanning electron microscope

A field-emission scanning electron microscope (FESEM) equipped with the standard secondary electron (SE) detector was used to image thin (70–90 nm) and thick (1–3 μm) sections of biological materials that were chemically fixed, dehydrated, and embedded in resin. The preparation procedures, as well as subsequent staining of the sections, were identical to those commonly used to prepare thin sections of biological material for observation with the transmission electron microscope (TEM). The results suggested that the heavy metals, namely, osmium, uranium, and lead, that were used for postfixation and staining of the tissue provided an adequate SE signal that enabled imaging of the cells and organelles present in the sections. The FESEM was also used to image sections of tissues that were selectively stained using cytochemical and immunocytochemical techniques. Furthermore, thick sections could also be imaged in the SE mode. Stereo pairs of thick sections were easily recorded and provided images that approached those normally associated with high-voltage TEM.     

The fibrous components of bovine ligamentum nuchae observed in the scanning electron microscope

A combination of enzymes have been employed to remove certain components of the adult bovine ligament sequentially. The course of this sequential enzymic dissection has been followed by scanning electron microscopy. We have been able to define the organization of elastic fibres embedded in matrix of collagen fibrils. The elastic fibres themselves appear to be made up from an amorphous core of elastin surrounded by a knitted sheath; the latter probably contains a mixture of collagen together with an unidentified protein component.     

Tannin-osmium conductive staining of biological specimens for non-coated scanning electron microscopy

Osmication of biological specimens with a mixture of tannic acid, guanidine hydrochloride, arginine hydrochloride and glycine imparted sufficient electron conductivity to permit SEM observations of non-coated samples. No charging was noted at 25 kV acceleration voltage and 1 × 10^?10 A specimen current in rat kidney specimens. The foot-processes of the glomerular podocytes were clearly visible without metal coating. This tannin-osmium method even allowed block staining and enabled continuous observations during and after dissection in the SEM.     

Gigahertz stroboscopy with the scanning electron microscope

The application of the stroboscopic scanning electron microscope to gigahertz Gunn effect devices is discussed. Two modes of operation, the deflection mode and the bunching mode, are considered. In the bunching mode, using 1.5 ps beam pulses, two-dimensional voltage contrast in a Gunn effect device triggered at 1 GHz has been observed. A powerful technique for a device in pulsed operation is also presented. With this technique, the nonuniform domain propagation in the three-dimensional-structure Gunn device in pulsed operation has been clearly observed. The duty cycle of the pulsed operation has been 4x10(-3).     

Nanometric cutting in a scanning electron microscope

A nanometric cutting device under high vacuum conditions in a scanning electron microscope (SEM) was developed. The performance, tool-sample positioning, and processing capacity of the nanometric cutting platform were studied. The proposed device can be used to realize a displacement of 7 μm, with a closed-loop resolution of 0.6 nm in both the cutting direction and the depth direction. Using a diamond cutting tool with an edge radius of 43 nm formed by focused ion beam (FIB) processing, nanometric cutting experiments on monocrystalline silicon were performed on the developed cutting device under SEM online observation. Chips and machining results of different depths of cut were studied during the cutting process, and cutting depths of less than 10 nm could be obtained with high repeatability. Moreover, the cutting speed was found to exhibit a strong relationship with the brittle–ductile transition depth on brittle material. The experimental results of taper cutting and sinusoidal cutting indicated that the developed device has the ability to perform multiple degrees of freedom (DOFs) cutting and to study nanoscale material removal behaviour.     

Development of a scanning electron microscope with a two detector system

The authors consider the design feature of a prototype scanning electron microscope which they have constructed. The article serves as basic revision of the function of all the components in an SEM system, as well as illustrating the authors' choice of alternatives. A distinctive aspect of the present instrument is that the idea of incorporating two detector systems for SE was present from the beginning of the planning stages. The applicability of this system is illustrated by compositional (A + B) and topographic (A − B) SE images.     

A fluorescence scanning electron microscope

Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative fluorescence and electron microscopy.