Automatic selection of molecular images from electron micrographs

Images of single molecules may be selected automatically from a digitized electron micrograph. The algorithm proposed facilitates single-particle averaging and related image procedures aimed at analyzing projections of single molecules. The automatic selection procedure has been applied to images of molecules of lipoyl transsuccinylase.     

Automatic selection of macromolecules from electron micrographs by component labelling and symbolic processing

A new solution to the problem of extracting images of individual biological macromolecules from electron micrographs is described. There are three distinct steps in the process. The initial stage of low-level image processing consists of noise suppression and edge detection. An intermediate stage of component labelling and feature computation bridges the gap between the iconic (low-level) processing and the final phase of symbolic (high-level) processing. Simple symbolic objects (bounding boxes) are derived from the edges, and are easily represented and manipulated in the decision-making process. The efficacy of the algorithm is demonstrated using electron micrographs of ribosomes and ribosomal subunits. The hierarchical nature of the analysis embodies a reduction in the amount of data and a change in its nature. Initially, thousands of pixels of continuous gray levels must be dealt with. After component labelling, there are fewer than a hundred bounding boxes whose manipulation can easily be defined and articulated by an expert. The software package that has been written can thus serve as a basis for applying artificial intelligence methodologies to analysis of electron micrographs.     

Signal to noise enhancement in dark field electron micrographs of vasopressin: filtering of arrays of images in reciprocal space

A technique for filtering arrays of images of dispersed molecules is presented which takes advantage of the crystallographic properties of regular arrays and the rapidity of optical treatment. A filter in reciprocal space consisting of perforations in a square or rectangular lattice, as determined by the image arrangement, reduces noise and averages the images simultaneously by transmitting only regularly recurring image structure. The filter is universal for all image contents and introduces no additional biases. The signal to noise improvement approaches √N for N independent images of the same molecule in the same configuration. The procedure, which is simple, rapid and inexpensive, is demonstrated with the aid of dark field electron micrographs of the protein vasopressin and combined with iodination of that molecule to elucidate its structure.     

Invariant classification of molecular views in electron micrographs

Biological macromolecules can exhibit many different orientations in electron microscopical preparations. In particular in vitreous-ice-embedded specimens, the number of different views can be high. Existing techniques of analysis require the alignment of the molecular views relative to one or more reference images with cross-correlation ("matched filtering") techniques and are somewhat unsatisfactory because of the high noise level and the large number of different views in such images. We here propose a method in which first rotation-, translation- and mirror-invariant functions are derived from the large set of input images. These functions are subsequently classified automatically using multivariate statistical classification techniques. The different molecular views in the images can therewith be found without bias, provided that a statistically significant number of copies of the views are present in the data set. The basic ideas are exemplified with realistic model data.     

A new experimental procedure to quantify annular dark field images in scanning transmission electron microscopy

A new procedure to quantify the contrast in annular dark field images recorded without lattice resolution in a scanning transmission electron microscope is proposed. The method relies on the use of an in‐column energy filter prior to the annular dark field detector and the acquisition of a series of energy‐filtered images as a function of the inner detection angle. When the image contrast of an interface between two materials in such energy‐filtered annular dark field images is plotted vs. camera length and extrapolated to zero (i.e. infinite scattering angle), the contrast is shown to behave exactly as predicted by Rutherford's scattering formula (i.e. intensity scales ∝Z²). This can then be used to determine the local chemistry at and the effective chemical widths of interfaces or thin films without any additional spectroscopy method for calibration, provided the global chemical composition is known. As examples, the systems SiGe/Si and InGaAs/Ge are considered in detail.     

Three-dimensional reconstruction of microtubule-containing structures from electron micrographs

Microtubules and associated structures are among the more difficult samples studied by 3D reconstruction techniques because of their size, complexity, and lability. Nevertheless, their importance in many cellular functions often justifies efforts to acquire 3D information up to the resolution limit of negatively stained specimens or beyond. A combined approach which utilizes methods appropriate to both helical symmetry and 2D crystal lattices seems to provide the surest route to 3D reconstruction. Images of unstained specimens obtained by newer techniques of microscopy present challenging problems in data analysis, but potentially offer higher-resolution information.     

A method for preparing colored scanning electron micrographs using SE and BSE images

Biological specimens impregnated with heavy metal are observed in an SEM equipped with a sensitive BSE detector. The SE image and the BSE image are successively recorded under the same conditions. Both pictures are displayed coincidentally on a screen, using two projectors of the same type, but with different color filters. The resulting color picture is photographed with color film. Specific regions of the specimen which were impregnated with heavy metal are clearly demonstrated.     

Low-contrast development of electron micrographs

From the fact that the response of photographic materials conforms to the ‘single-hit’ expression for exposure to electrons of the energies used in electron microscopy, it follows that the density log exposure curves have a shape substantially independent of the degree of development in conventional solutions. The only way in which the density range corresponding to a given ratio of intensities in the image can be reduced is by reduction of the exposure. The visibility of low-intensity detail in the image is adversely affected, however. A developer formula is presented with which the density contribution per electron decreases with increasing exposure so that contrast is less than if determined solely by the nature of the exposure. The effectiveness of the solution is demonstrated and practical recommendations are made regarding its use.     

Direct three-dimensional reconstruction for macromolecular complexes from electron micrographs

An algorithm is presented for the direct reconstruction of a three-dimensional image from two-dimensional projections with directions randomly distributed over the unit sphere. An iterative algorithm for improving the rotational alignment of projections is described. The performance of these algorithms is illustrated using the reconstruction of a coil model from its projections with increasing random errors in their relative angular orientations.     

Bright field and hollow cone dark field electron microscopy of palladium catalysts

The usefulness of hollow cone dark field imaging for the study of Pd catalyst particles on γ-alumina substrates is investigated. By varying the depth of the specimen in the objective lens field, different Bragg reflections can be conveniently selected. Typically, about 70% of the particles appear in either the 111 or 200 Bragg reflections. Rather more particles can be visible in bright field because of other Bragg reflections as well as absorption effects. Size distributions in bright field and dark field are compared.     

Identification of the three-dimensional gel microstructure from transmission electron micrographs

Mass transport in gels depends crucially on local properties of the gel network. We propose a method for identifying the three‐dimensional (3D) gel microstructure from statistical information in transmission electron micrographs. The gel strand network is modelled as a random graph with nodes and edges (branches). The distribution of edge length, the number of edges at nodes and the angles between edges at a node are estimated from transmission electron micrographs by image analysis methods. The 3D network is simulated by Markov chain Monte Carlo, with a probability function based on the statistical information found from the micrographs. The micrographs are projections of stained gel strands in slices, and we derive a formula for estimating the thickness of the stained gel slice based on the total projected gel strand length and the number of times that gel strands enter or exit the slice.     

Low cost internegatives from electron micrographs of metal shadowed objects

Some observers prefer a contrast reversal of electron micrographs of metal shadowed objects, so that the metal-free shadows appear dark on the print. There are several ways of obtaining contrast reversal, and the reasons for using 35 mm internegatives are given and a setup for re-photography of electron microscopy films is described together with a modified enlarger. Both are equipped with nearpoint illumination, which can be diffused in a simple manner with minimal loss of intensity. Other features are a particularly rigid camera stand combined with a numbering device, and a glass- and scratch-free negative holder for the enlarger. The choice of lens for the re-photography is discussed.     

On dark field techniques in transmission electron microscopy.

Different dark field techniques in transmission electron microscopy are investigated. In particular, a displaced aperture technique is developed, which produces high quality off-axis dark field images. The essential feature of the method is that the chromatic aberration of the objective and intermediate lenses is compensated for with the aid of a one-stage beam deflector in the object plane of the intermediate lens. In addition, the image distortion and the effect of the curvature of the image field are minimized by reducing the coefficient of sperical aberration of the intermediate lens. Diffracted beams, forming angles alpha up to 2.5 degrees with the optical axis, can be used for dark field imaging. Resolutions better than 30 A for alpha = 2 degrees, and 17 A for alpha = 1 degree, are obtained.     

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.     

Improved high resolution image processing of bright field electron micrographs: II. Experiment

The new methods of nonlinear image processing are applied to high resolution experimental micrographs of chlorinated copper phthalocyanine taken on the Kyoto 500 kV electron microscope. With these new methods of image processing the phase and amplitude of the specimen transmission function are reconstructed from a defocus series of conventional transmission electron micrographs (bright field). Strong scattering, partial coherence and statistical noise have been included. Both of these new methods are based on the MAP (maximum a posteriori) criterion generalized to include reconstruction from multiple input images. In a companion paper (the first part of this two-part report) the theory behind these methods was presented and in this paper it is tested on actual experimental micrographs. A significant increase in resolution has been obtained with computer image processing. The point-to-point resolution obtained here with computer image processing of 500 kV electron micrographs is of the order of 1.2–1.4 Å which represents a 30–50% increase in resolution.     

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.