Quantitative analysis of image contrast in electron micrographs of beam-sensitive crystals

The contrast in high resolution electron micrographs of three different thin crystals has been compared quantitatively with that predicted theoretically from separate measurements of thier electron diffraction patterns. The crystals were vermiculite, a mineral which is not greatly affected by the electron beam, and two organic specimens, n-paraffin and purple membrane, which are both destroyed by doses of about 1 electron/Ų. The results, all at 4.0 to 4.5 Å resolution, show that the absolute contrast in images of vermiculite is roughly 1/5th of that expected for a theoretically perfect microscope, whereas images of paraffin and purple membrane seldom reach more than 1/25th of theoretical contrast. Much of this loss of contrast can be explained on the basis of known microscope parameters in the case of the non-beam-sensitive specimens. However, for the images of paraffin and purple membrane, it is necessary to postulate that beam-induced specimen movement results in further substantial blurring of the image. The tendency for such movement to occur may be unavoidable since the molecular structure is being destroyed during the exposure. The magnitude of this movement must be reduced before the image contrast will be able to approach the theoretical limit.     

Automatic focus correction for spot-scan imaging of tilted specimens.

The variation in defocus within an image of a highly tilted specimen can be a serious source of artifact. Spot-scan imaging can be combined with dynamic focusing to greatly reduce this range of defocus. A protocol is described for determining the parameters required for the automatic focus compensation during the recording of a spot-scan image. Images of a gold test specimen demonstrate the efficacy of this procedure in extending the area of the image that contains high-quality data. In case the tilt angle or resolution is high enough that the height difference of the specimen within each small illuminated area is larger than the depth of field, the image must be treated to compensate for the focus variation. The same principle is used as was developed for compensation of conventional images of tilted specimens.     

An expeditious procedure for the accurate alignment and assembly of stereo micrographs

A simple procedure is described for the reproducible and convenient assembly of stereo micrographs, particularly as transparencies, with the help of an apparatus developed for this purpose. Technical features and applications are discussed.     

Three-dimensional reconstruction from electron micrographs of disordered specimens. I. Method

A method is presented for three-dimensional reconstruction from electron micrographs of a specimen containing a disordered collection of identical objects with unknown orientations. All the data from all the images are simultaneously used to obtain an approximately maximum likelihood estimate of the three-dimensional electron density, which is represented as a truncated expansion in a complete orthonormal set of basis functions. Anomalous objects can nevertheless be detected and eliminated. The method remains under statistical control, and a hypothesis test is used to choose the lowest resolution reconstruction that is consistent with the data. Error propagation is quantitatively traced from the micrograph to the reconstructed electron density. Random orientation is not necessary, and prior knowledge of preferred orientation can be used to advantage. Similarly, symmetry in the object is not necessary, but it can be imposed and exploited, if appropriate. Evidence is presented that useful reconstructions can be obtained with only one or two extra tilts from highly disordered specimens, even if the objects are asymmetric. The companion paper discusses in detail the implementation and verification of the method.     

A new procedure for handling impervious biological specimens.

A new application of techniques for preparing impervious biological specimens for light microscopy (LM) and transmission electron microscopy (TEM) has been developed. Microwave irradiation was used to facilitate fixation. A priming technique was used to increase the bonding of the outer surface of the specimens with the resin. Priming the waxy or cuticular surface with Z-6040 (gamma-glycidoxypropyl trimethoxysilane) solved the problem of specimen "pull out" from the resin. Insect specimens with various types of cuticles (waxy or chitinous) and seeds were successfully studied ultrastructurally using this technique.     

Three-dimensional reconstruction from electron micrographs of disordered specimens. II. Implementation and results

The computational procedures to implement the method described in the companion paper for three-dimensional reconstruction from projections of a disordered collection of single particles are presented. Computer simulations are used to demonstrate the way the method functions, and practical aspects are discussed in detail. Examples are given of how different symmetries can be exploited by imposing selection rules on the model equations. Applications to negatively stained 50S ribosomes and to cryo-electron micrographs of thin vitrified layers of unstained and unsupported tomato bushy stunt and Semliki Forest viruses are described, and the resulting reconstructions are presented.     

A procedure for increasing the contrast of biological specimens in edge-projection TEM

Rotary shadowing has been used to increase the image contrast of biological specimens during edge-projection imaging in the transmission electron microscope (TEM). In this imaging mode, biological specimens are adsorbed from aqueous solution onto a highly curved substrate and observed in a direction parallel to its surface. High contrast TEM images are obtained at 200 kV when a 1–3 nm layer of tungsten is thermally evaporated onto the substrate at an angle of about 9°. Individual adsorbates are clearly delineated by the smooth, continuous, and fine-grained tungsten layer that surrounds them. TEM images obtained with this technique can provide a unique view of biological adsorbates on metal, insulator or semiconductor substrates.     

Application of recording and processing of energy-filtered image sequences for the elemental mapping of biological specimens: Imaging-Spectrum

Computerized energy-filtered transmission electron microscope (EFTEM) permits the recording and the processing of energy-filtered images, allowing a part of an electron energy-loss spectrum for each picture element to be obtained. This method, called ‘Imaging-Spectrum’, uses a Zeiss CEM902 coupled to several image analysis systems. The actual configuration records sequences of 48 images, 256 × 256 pixels, in steps of the energy loss, ΔE. Processing these sequences results in part of a core-loss EELS-spectrum for each pixel. This approach produces elemental maps with a short processing time. We have implemented three kinds of background calculation for the image subtraction. The influence of the irradiation dose and of the energy selecting slit width on the quality of the spectra is investigated. The method is applied to the analysis of some biological specimens (pericellular coat behaviour during adhesion between macrophages and red blood cells and location of calcite microcrystals in dental pulp cells). The Imaging-Spectrum method appears to be suitable for the analysis of large areas.     

A minicomputer system for image processing of electron micrographs

This paper describes how a PDP 11/34A computer system was set up to handle research problems in computer image processing of electron micrographs. This system is referred to as CIPRES, for Computer Image Processing and Reconstruction System (pronounced the same as cypress, the name of an evergreen tree). The RSX-11M operating system is used. Various execution speeds are given. For instance, a disk-based fast Fourier transform or FFT (complex to complex, 128 X 128 pixels) runs in three minutes. This report may be useful to anyone wishing to set up a similar computer system.     

A method for photographic densitometry of scanning electron micrographs

A method is described for the study of the surface structure of mammalian chromosomes. Densitometric analysis of scanning electron micrographs permits the detection of regularities where overlying material obscures these in directly inspected micrographs. The method has been applied to the study of mammalian chromosomes.     

Intensity gradient techniques for orientation analysis of electron micrographs

The variation in intensity between neighbouring picture points on an electron micrograph may be used to define an intensity gradient vector. This vector is related to the orientation of the feature at that point, and may be compared with similar vectors at all other points within the micrograph to obtain a quantitative measure of anisotropy. Alternatively, the results may be displayed graphically as a rosette diagram. This paper compares the results from using a simple ‘5-point’, i.e. the nearest neighbours with those from alternative formulations for the intensity gradient vector. Generally, the increased number of points improves the shape of the rosette diagram by minimizing the likelihood of pronounced ‘spikes’ at certain critical angles which is a feature of the ‘5-point’ method. The improved analysis requires an extra 60% computing time, which may be of significance if direct on-line work is contemplated. Some examples of micrographs of soil microfabric are presented to illustrate the different methods.     

Use of tris-buffer-fortified thionine-SO2 for detection of DNA-aldehyde following Feulgen procedure

This communication presents informations on the use of tris-buffer along with N HCl and potassium metabisulphite for the preparation of thionine-SO2 in staining DNA-aldehyde molecules of acid hydrolysed mammalian liver sections. It has been found that thionine, containing tris-buffer, N HCl and potassium metabisulphite, stains DNA-aldehyde molecules with better result than is possible with the control dye-SO2 reagent that does not contain this buffer. The absorption spectra of nuclei stained with this dye-reagent prepared with tris-buffer have also been presented. Further, it has been found that nuclei stained with the freshly prepared dye-SO2 reagent is bluish-violet, whereas those stained with an old dye-reagent is sky blue in colour. The reason for the slightly enhanced nuclear colouration with the experimental dye-reagent over the control has been considered to be due to slightly increased pH in the former as compared with that of the latter. The mechanism of staining with thionine-SO2 has been considered to be of Feulgen type.     

A method for increasing the photographic contrast of electron micrographs

Emulsion-developer combinations used regularly in conventional electron micrography yield an approximately linear relationship between density (D) and exposure (E) up to quite high densities (> 2.0). This is due to the fact that over the corresponding exposure range the density yield per electron is approximately constant. A practical consequence is that the slope of the D/log₁₀ curve, termed the contrast, is a function of D only, i.e. it is independent of the emulsion-developer combination. To achieve higher than normal contrast requires that the density yield per electron should increase with exposure over some exposure range, at least. This effect may be obtained by using what are known commercially as ‘lith’ films and a system of infectious development. The high contrast of such films results from an acceleration of development rate when the developer oxidation products reach a critical concentration. Sensitometric data are presented for electron exposures which show that for average densities of ～ 1.0 in a micrograph a six-fold increase of contrast can be obtained with a ‘lith’ system. It is also shown that though the special development effect gives higher granularity than that obtained at the same exposure with a conventional system, the signal/noise ratio is not adversely affected. Practical demonstrations of the effectiveness of the ‘lith’ system are presented.     

Quantitative analysis of scanning electron micrographs

Scanning electron microscopy (SEM) provides a considerably greater depth of field than optical microscopy at the same magnification. It is therefore particularly suitable for the examination of specimens with irregular surfaces. This review of procedures for quantitative measurements on SEM images falls into two parts. The first deals with mensuration; that is, with the determination of the lengths, dihedral angles and other properties of specific features in the image. Particular emphasis is given to means of correcting for non-linearity and other distortions caused by instrumental defects. The second part considers the analysis for bulk properties such as volume fraction and boundary area per unit volume. In the case where observations are made on a planar section, these properties can be determined by use of the standard stereological relationships after making a computable correction (where necessary) for the tilt of the specimen and non-linearities in the scan. However, for irregular surfaces (for example, those produced by fracture) rigorous estimations are not possible without certain assumptions which, unfortunately, are in many cases unrealistic.     

The application of stereo-techniques to electron micrographs

It is demonstrated that the only requisite for a true three-dimensional image is the presence of parallax in the stereo-pair. This parallax is introduced into electron micrographs by tilting the specimen between exposures. After deriving the usual parallax equation several examples of the use of stereo-measurements in electron microscopy illustrate the extra information retrieved by its use.     

High-speed computation techniques for the simulation of high resolution electron micrographs

A survey will be given of the theoretical and practical progress that has been made thus far in the field of high-resolution image computations especially with respect to computational speed and memory requirements. Attention is paid to some problems that have not completely been solved yet and some future prospects are outlined.     

High-speed imaging for online micrographs of polymer composites in tribological investigation

Studying wear process by effective means avoids undesirable situation such as frequent replacement of components, breakdown-maintenance and catastrophic accidents. In tribological investigations, dimensional changes due to wear processes are assessed using offline and online techniques. However, traditional postmortem analysis cannot display intermediate wear mechanisms. Visible textures as a direct measure of wear is a valuable evidence to understand tribological characteristics. In recent years, online-monitoring through computer vision has been used to study surface roughness. Nevertheless, online-monitoring for topographical and microstructural changes during wear could provide a clear insight of wear process. In this work, attempts are made to monitor rolling/sliding contact using a high-speed camera in conjunction with a microscope. Optical and imaging parameters were optimised to obtain clear images. Tests were conducted using polymer composite for understanding the contact surface topography of the wear process. Micrographs of the composite contact surface were acquired online using 35,000 fps and 0.375 ns shutterspeed. Periodical changes in wear mechanism are evident with resin back transfer, surface cracks and partial exposure of fibres. Thus, effective characterisation through online-monitoring gives a clear insight of the fundamental mechanisms behind wear process. The newly developed high speed imaging system stands as an effective tool in tribological investigations.     

An assay for local quality in cryo-electron micrographs of single particles

High quality of the cryo-electron micrographs is of crucial importance for the success of single particle three-dimensional reconstruction methods. In analyzing some micrographs from cryo-electron microscopy specimens, we found an extraordinary variability, within the same micrograph, in the appearance of particles. We developed a method for analyzing the variability of local image quality, using correspondence analysis of local power spectra. With this technique, we discovered a strong systematic variation of the envelope modulating an otherwise unchanged contrast transfer function. The underlying causes may be uncontrollable effects, such as variations in the thickness of ice, instability of the holey carbon, and charging. The method of assaying, resulting in "local quality maps", may be useful as a general tool for screening micrographs used as input for reconstructions.