Low-noise boron supports for high resolution electron microscopy

Very thin (～1.5 nm) amorphous boron films are useful as specimen supports for the study of biochemical molecules by dark-field transmission electron microscopy. The noise factor is significantly smaller than for carbon films, probably because of the tendency to bond in icosahedral B₁₂ subunits. Signal-to-noise ratio of about 2 to 3 have been obtained with bromine and strontium atoms in test molecules.     

A porous carbon film for the collection of atmospheric aerosol for transmission electron microscopy

This paper describes a method of direct sampling of fine urban aerosol using porous carbon films, to increase collection efficiency and retain the structural integrity of the aerosol. A nuclepore membrane filter was coated with carbon and washed with chloroform to leave a clean porous carbon film on a 200-mesh transmission electron microscope copper grid. The grid was attached to a clean nuclepore filter using carbon cement. Sampling was conducted for 1 h at 1.9 L min⁻¹ flow rate. The porous carbon grid with sample was then assayed using transmission electron microscopy, and negatives were suitable for image analysis. The collection of fine airborne particles using this method is appropriate for electron microscopy, with a minimum of interference to the structure of collected particles.     

Mica disc supports for particulate specimens for scanning electron microscopy

Readily prepared mica discs have been found useful as supports for particulate specimens for the scanning electron microscope. The principal advantage is that they can be observed in the light microscope prior to further preparation for the scanning electron microscope.     

An energy filter for biological electron microscopy

A prism-mirror-prism electron energy filter has been inserted into a fixed beam transmission electron microscope. The insertion of the filter has not degraded the spatial resolution of the microscope, while providing an energy resolution of 2.5 eV. The filter greatly reduces the chromatic aberration of the images, increasing resolution in dark field and contrast in bright field. Images of bacteriophage T4 and of polyoma virus indicate that thick unstained and unshadowed biological specimens can now be observed at high resolution in dark field. In addition, the increased contrast and definition observed in bright-field images of conventional liver sections suggest that it should be possible to obtain sufficient contrast from stains containing atoms of lower atomic numbers than those employed at present.     

Energy-filtered transmission electron microscopy of biological samples on highly transparent carbon nanomembranes

Ultrathin carbon nanomembranes (CNM) comprising crosslinked biphenyl precursors have been tested as support films for energy-filtered transmission electron microscopy (EFTEM) of biological specimens. Due to their high transparency CNM are ideal substrates for electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) of stained and unstained biological samples. Virtually background-free elemental maps of tobacco mosaic virus (TMV) and ferritin have been obtained from samples supported by ∼1 nm thin CNM. Furthermore, we have tested conductive carbon nanomembranes (cCNM) comprising nanocrystalline graphene, obtained by thermal treatment of CNM, as supports for cryoEM of ice-embedded biological samples. We imaged ice-embedded TMV on cCNM and compared the results with images of ice-embedded TMV on conventional carbon film (CC), thus analyzing the gain in contrast for TMV on cCNM in a quantitative manner. In addition we have developed a method for the preparation of vitrified specimens, suspended over the holes of a conventional holey carbon film, while backed by ultrathin cCNM.     

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.     

Surface crystallography via electron microscopy

The study of atomic structure of surfaces is fundamental to the understanding of electronic, chemical and mechanical properties of surfaces and numerous techniques have been developed to this end. Transmission Electron Microscopy techniques, namely transmission electron imaging (TEM) and diffraction (TED), due to their ability to provide structural information at very high resolutions, have emerged as powerful tools for the study of surface structure. In this article we review the experimental method alongside the various post-processing routines that are necessary to extract vital structural information from experimental data.     

MgO single crystals as specimen supports for high resolution electron microscopy

The advantage of MgO platelet crystals as specimen supports is shown. The contrast transfer of carbon foil granulation through the thin MgO platelet crystals was measured. The reduction of the contrast due to the crystal of 40 nm thickness was about 50%. Gold particles and ferritin particles were used as test specimens on MgO platelet crystals. The lattice images of both specimens were observed through the crystals. The protein shell of ferritin was recognizable. Some different problems arising with this specimen support are discussed.     

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.     

Phase contrast electron microscopy of biological materials

The properties of two electron microscope phase contrast imaging methods are compared. The first is the conventional bright-field method in which dark phase contrast is created by defocusing; the second is a phase plate method in which bright phase contrast is created by means of a suitably shaped electric field. Using some negatively stained biological specimens which have a well-known repeating structure as the test object, it is shown that the phase plate method has some important advantages over the bright-field method. Its contrast transfer characteristics are such that it can provide a more faithful representation of the high resolution detail in the object. Moreover, by producing bright, rather than the normal dark, phase contrast it is able to simultaneously enhance the detail in the specimen and weaken the detail in the stain; this latter property enables the method to display information about the specimen that it would not be possible to detect with the bright-field method.     

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.     

Surface structure imaging by electron microscopy

Reconstructed structures at monolayer level on ‘clean and well-defined’ surfaces can be imaged by transmission electron microscopy in fixed beam illumination mode. The specimens are cleaned in-situ in the electron microscope in ultra high vacuum. Transmission electron diffraction pattern intensities can give useful information for determining the surface unit cell size of the structure, and the atom positions (geometric arrangement of atoms in the unit cell) especially those with a large unit cell, since the diffraction intensities are interpreted kinematically. High resolution surface imaging which gives directly the atom positions is tested here for a single monolayer terrace on Ag (111) surface. The result shows the value of HREM for studies of surface crystallography.     

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.     

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.     

Wetting agents for biological electron microscopy. II. Shadowing

When preparing specimens for shadowing it is frequently necessary to add a wetting agent to aqueous suspensions to enable them to spread on hydrophobic support films. However high-resolution platinum/carbon shadowing is capable of visualizing wetting agent molecules, so the effect of added wetting agent on the final appearance of the specimen must be considered. To facilitate interpretation of the specimen fine detail the background should be smooth and flat. This is achieved by using the concentration of wetting agent which produces a close-packed monolayer of wetting agent molecules after drying. Bacitracin is a better wetting agent for use when shadowing than the commonly-used bovine serum albumin (BSA). Because its molecules are smaller than those of BSA, the size of the background granularity is smaller, which facilitates interpretation of the specimen, and the layer of wetting agent molecules is thinner, so small specimen details are less likely to be buried and rendered invisible to the shadowing process. These points are described in detail and illustrated with the help of models where necessary.     

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.     

High-resolution electron microscopy of high-modulus carbon fibres*

High-resolution electron-microscope images of high-modulus carbon-fibre fragments have been recorded under conditions which allow resolution of the individual 0.34 nm graphite layer planes. Extinction bands previously observed at low resolution are now seen to originate from several different structural features. Single bands are caused by lattice distortion due to non-basal edge dislocations and low-angle boundaries of tilt or twist character; periodic bands are the result of Moiré effects from overlapping or interlinked crystallites. Accurately controlled tilting experiments give further insight into the complex interlinked nature of the crystallites and the results are summarized in terms of a model which is also capable of explaining the partial recrystallization observed with boron doping.