Use of spot-scan procedure for recording low-dose micrographs of beam-sensitive specimens

Electron micrographs of monolayer crystals of paraffin have been recorded with a spot-scan mode of imaging which uses a small 50 nm diameter moving beam. In comparison with normal stationary beam images using 5 μm illuminating beams, the spot-scan micrographs show a higher and more consistent contrast from the 3.8–4.2 Å hydrocarbon chain spacings. On average the improvement in contrast is twofold, but this still leaves scope for further improvement: the best spot-scan images still do not quite reach the level of contrast calculated theoretically from electron diffraction. We believe that the cause of the low contrast in paraffin images must be specimen motion caused by radiation damage rather than a charging effect, for two reasons. First, it does not occur in control images of vermiculite, a non-beam-sensitive mineral, when treated identically. Secondly, although charging might still be a problem with paraffin, when images are taken with the objective aperture in the column, a procedure which is normally expected to reduce charging, no improvement in contrast is found. Thus we think that the use of the small beam minimizes the effect of specimen motion on image contrast by minimizing the specimen area exposed at each instant and therefore the resultant image blurring. Further improvements with even smaller illumination beam diameters might be expected.     

Structure of purple membrane from halobacterium halobium: recording,measurement and evaluation of electron micrographs at 3.5 Å resolution

Electron micrographs of the purple membrane have been recorded using liquid nitrogen and liquid helium cooling on three cryoelectron microscopes. The best micrographs show optical diffraction spots, arising from the two-dimensional crystal, out to resolutions of around 6 Å. Large areas of several of these micrographs have been analysed using a procedure which determines the strength of the very weak high resolution Fourier components of the image of the crystal. The procedure consists of reciprocal space filtering followed by real space correlation analysis to characterise image distortions, removal of the distortions by interpolation, and finally extraction of the amplitudes and phases of the Fourier components from the distortion-corrected image of the crystal. These raw image amplitudes and phases are then used, together with previously measured amplitude and phase information, to refine the beam tilt and crystal tilt, phase origin and amount of defocus and astigmatism of the image. The phases can then be corrected for the effects of the contrast transfer function, beam tilt and phase origin. The amplitudes of all the spots which are expected to be strong from their known electron diffraction intensity are observed to be significantly above the background noise level, and the independent phases from different images, and from symmetry-related directions in the same image, show excellent agreement out to a resolution of 3.5 Å. Although only images from untilted or slightly tilted ( < 5°) crystals have been analysed using the procedure described in this paper, a simple additional step enables analysis of images at any tilt angle, providing a complete practical method for high resolution analysis of images of two-dimensional crystalline arrays.     

Contrast analysis of cryo-images of n-paraffin recorded at 400***kV out to 2·1 Å resolution

N-Paraffin was used as a test specimen for evaluating the relative merits of 400-kV versus 100-kV electron microscopy in recording data for electron crystallographic analysis of beam-sensitive materials. The parameter used for comparison, the relative contrast R, is the ratio of amplitudes from the computed Fourier transform of images and amplitudes from an electron diffraction pattern from the same crystal. R will thus be a measure of the contrast from an experimental image relative to that of a perfect image. Electron diffraction patterns and bright-field images were recorded at 400 kV at a specimen temperature of ?167°C. Using the flood-beam imaging technique the best R-value is 0 08 for all reflections in the resolution zone from 4 to 3 Å. This value is equivalent to that found at 100 kV. In the resolution zone from 3 to 2 Å we have found R — 0 02. Using the spot-scan imaging technique, on the other hand, R was measured to be 0·42 for the reflections between 4- and 3-Å resolution. This amount of relative contrast is 1·7 times that observed at 100 kV. Reflections at 3–2 Å displayed an R-value of 0 05. Besides obtaining higher R-values when applying the spot-scan imaging technique at 400 kV, we observe a higher yield of images with isotropic diffraction and/or higher resolution reflections. Various contrast-attenuating factors, including the modulation transfer function of the photographic film and the cryo-holder, envelope functions for spatial and temporal coherence and lens and high-tension instabilities, the contrast transfer function and lastly the radiation damage effects, have been considered in interpreting the observed image contrast. Overall, use of 400 kV in combination with spot-scan does offer important improvements in contrast levels, which can be very useful in determining the three-dimensional structure from protein crystals.     

Reliability of phases retrieved from 400-kV spot-scan images of purple membranes acquired on a slow-scan CCD camera

Glucose-embedded purple membranes were used as a test specimen to evaluate the reliability of phases retrieved from 400-kV spot-scan images acquired on a 1024 × 1024 slow-scan CCD camera. This specimen was chosen because it represents a broad class of low-contrast radiation-sensitive biological objects and its structure is well established. The amplitudes of computed reflections from these images were strongly damped by the modulation transfer function of the camera. Nevertheless, their phases on average were < 12° different from the reference data of Henderson et al . (1986), Ultramicroscopy , 19 , 147–178, up to 8.8 Å resolution, which corresponds to 0.8 of the Nyquist frequency of the camera.     

Computer-controlled spot-scan imaging of crotoxin complex crystals with 400 keV electrons at near-atomic resolution.

400 keV electrons yield a better relative image contrast than 100 keV electrons for a beam-sensitive organic crystal when spot-scan imaging is used [J. Brink and W. Chiu, J. Microscopy 161 (1991) 279]. A FORTRAN 77 program has been written to operate the spot-scan imaging system on a computer workstation under the VMS operating system which is interfaced serially to the JEOL4000 electron microscope. We demonstrate the application of this implementation by imaging crotoxin complex crystals embedded in either vitreous ice or glucose to 2.5 A resolution. The intensity strength of the structure factors of this protein crystal are different at low (> 10 A) resolution but similar at high resolution (< 10 A) for the two embedding media as expected from their scattering contrast difference. Based on our experience as judged from the electron diffraction patterns of highly tilted crystals, flat crystals embedded in glucose can be readily obtained. Furthermore, our spot-scan imaging system also has the option of correcting the focus gradient that is present in images of tilted specimens.     

Image matching between experimental and simulated high‐resolution electron micrographs of sapphire on the orientation

The effects of imaging parameters have been studied on their roles of the severe mismatches between experimental and simulated high‐resolution transmission electron micrographs of sapphire along the direction. Image simulation and convergent‐beam electron diffraction techniques have been performed on misalignments of the electron beam and the crystal specimen. Based on this study, we have introduced an approach to achieve reliable simulation for experimental images of sapphire on the projection by the use of iterative digital image matching.     

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.     

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.     

A method for producing hollow cone illumination electronically in the conventional transmission microscope.

An electronic device manipulates the primary beam in the conventional transmission microscope to produce a hollow cone of illumination with its apex located at the specimen. The device uses the existing tilt coils of the microscope, and modulates the D.C. signals to both x and y tilt directions simultaneously with various waveforms to produce Lissajous figures in the back-focal plane of the objective lens. Electron diffraction patterns can be recorded which reflect the manner in which the direct beam is tilted during exposure of a micrograph. In the bright-field imaging mode the device provides a microscope transfer function without zeros in all spatial directions and has been used to obtain high resolution images which are also free from the effect of chromatic aberration. A standard second condenser aperture is employed and the width of the cone annulus is readily controlled by defocusing the second condenser lens. The cone azimuthal angle is also controlled electronically; hence the device can also be used in the dark-field imaging mode. This device has been applied to imaging both amorphous and crystalline materials including biomolecular specimens.     

On specimen tilt for electron holography of semiconductor devices

Electron holography can be successfully used for potential mapping on a nanometer scale. It relies on the fact that the phase of the electron wave is proportional to the electrostatic potential in the specimen. However, this proportionality is valid only in a kinematical condition, which is achieved by proper specimen orientation with respect to the electron beam. In this report, we examine experimentally in detail the specimen orientations of silicon devices that minimize dynamical diffraction. The tilt of the specimen from the edge-on position to a favorable orientation causes certain interfaces in the specimen to smear. We describe the smearing by a transfer function and compare it to wave transfer function of the microscope. The maximal specimen tilt alphamax that does not cause smearing greater than the resolution r of the microscope is alphamax = arctan(5.70r/t), where t is the specimen thickness.     

Determination of the illuminating angle and defocus spread in transmission electron microscopy

In high resolution electron microscopy the beam divergence and defocus spread are important factors determining the resolution limit of the microscope. In this paper a straightforward method is proposed to estimate the illuminating angle (beam divergence) and the parameter of defocus spread using optical diffraction patterns from a through focal series of electron micrographs of a thin amorphous film.     

Image reconstruction from sparse data in synchrotron-radiation-based microtomography

Synchrotron-radiation-based microcomputed-tomography (SR-μCT) is a powerful tool for yielding 3D structural information of high spatial and contrast resolution about a specimen preserved in its natural state. A large number of projection views are required currently for yielding SR-μCT images by use of existing algorithms without significant artifacts. When a wet biological specimen is imaged, synchrotron x-ray radiation from a large number of projection views can result in significant structural deformation within the specimen. A possible approach to reducing imaging time and specimen deformation is to decrease the number of projection views. In the work, using reconstruction algorithms developed recently for medical computed tomography (CT), we investigate and demonstrate image reconstruction from sparse-view data acquired in SR-μCT. Numerical results of our study suggest that images of practical value can be obtained from data acquired at a number of projection views significantly lower than those used currently in a typical SR-μCT imaging experiment.     

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.     

Three-dimensional view of the chromatin in freeze-fractured chicken erythrocyte nuclei.

The freeze-fracture thaw-fix (FfTF) technique described in earlier papers is applied in the present work to more detailed study of the chicken erythrocyte, by transmission replicas and high resolution scanning electron microscopy (3 nm scan beam size). The three-dimensional structure of the chromatin, and possibly the non-histone protein matrix, of fractured nuclei is to a large extent retained in this method of preparation and seen in stereomicrographs. In these micrographs the helical sub-structure of the 25 nm chromatin strands can be seen at about the same resolution as that of previously published micrographs in which extracted chromatin is viewed by negative contrast or after metal shadowing. The useful resolution of the secondary electron micrographs, for a suitably mounted specimen, is shown to be as good as that of transmission micrographs of platinum-carbon replicas of the same material.     

Effects of surface relaxation on convergent-beam electron diffraction analysis of stress in silicon

Convergent‐beam electron diffraction on cross‐sectional transmission electron microscopy specimens can map strains in the silicon substrate of microelectronics devices with high spatial resolution. However, at shallow depths below the interface, most of the diffraction lines within a convergent‐beam electron diffraction pattern are split, rendering pattern interpretation impossible in the classic way. The splitting effect was systematically analysed for a variety of materials, and the same qualitative behaviour that can be explained by stress relaxation at the surfaces of the thin transmission electron microscopy specimen was observed. The effects of surface relaxation are modelled by finite elements simulations. The results predict well the experimental magnitude of the splitting for a variety of diffraction lines at different positions below the interface, but fail to simulate the intensity of the secondary lines. Possible reasons for such discrepancies are discussed and assessed.     

Towards 1.0 Å resolution: taking advantage of dynamical scattering, and the benefits for structure retrieval

This paper is an exploration of the behaviour of high-resolution transmission electron microscope (HRTEM) images at up to 1 Å resolution. The ultimate limits to HRTEM (structure) resolution and the manner in which strong scattering may lead to weak diffraction in heavy fcc metals are discussed. A resolution of 1.0 Å is somewhat better than the ultimate resolution presently achievable in a 400-kV electron microscope. In heavy metals, such as platinum, it is found that the lattice fringe contrast is very low in the [011] projection, but that fringe contrast may be improved by imaging in the [111] projection. For atomic resolution imaging of the heavy metals in the [111] projection a resolution of 1.2 Å is required. For the study of oxygen position in high-temperature superconducting (HTS) oxides a resolution of between 1.2 and 1.4 Å is required. At better than 1.2 Å resolution the thick crystal images in HTS oxides remain simple and are easily interpreted. At such resolution all atomic columns are separated for the HTS [010] projection and the dynamical diffraction effects improve the contrast of oxygen atoms relative to the metal atoms.     

Determination of the principal distance and the location of the perspective centre in low magnification SEM photogrammetry.

The principal distance, D, from the centre of perspective in the SEM optical projection to the tilt axis of the specimen stage must be accurately determined before photogrammetric evaluation of stereoscopic pairs of micrographs can proceed. A precise procedure for measuring D is described in which the specimen stage X micrometer is used to measure the width of the field scanned for a particular width of the CRT, when the specimen stage is moved along the electron beam axis by amounts measured with the stage Z micrometer. The Z micrometer is calibrated with an external dial gauge. A plot of field width against Z extrapolated to zero gives the location of the perspective centre. In SEM photogrammetry, it is usual to leave the lens currents unchanged whilst recording the stereo-pairs. The values of D measured with a constant final lens current show that the perspective centre is located close to the final aperture in its conventional position. Previous determinations of D for Stereoscans have used a changing lens current to keep the specimen in focus at varying Z, and found a virtual centre several millimetres above the final aperture. The value of D so obtained should only be used if the micrographs were recorded with dynamic or automatic focusing systems.     

Crystallographic analysis of thin specimens

Electron backscattering diffraction (EBSD) is commonly used on bulk samples for crystallographic material characterization. In this work, the technique was applied on transmission electron microscopy (TEM)-type thin specimens, prepared with a focused ion beam. Orientation maps were successfully collected on specimens made of a Cu₃Au copper–gold alloy. As compared to EBSD analysis on a bulk specimen, an improved pattern quality and a high spatial resolution (well below 10 nm) were obtained. Furthermore, a clear improvement of the signal-to-noise ratio with decreasing sample thickness was observed.     

Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice.

It is shown that the contrast in high-resolution electron micrographs of biological macromolecules, illustrated by a study of TMV in ice, falls considerably below the level which should theoretically be attained. The factors which contribute to the low contrast include radiation damage, inelastic scattering, specimen movement and charging. Future progress depends on improved understanding of their contributions and relative importance. Contrast is defined as the amplitude of a particular Fourier component extracted from an image in comparison to that expected by extrapolation from separate electron or X-ray diffraction measurements. The fall in contrast gets worse with increased resolution and is particularly serious at 10 A and beyond for specimens embedded in vitreous ice, a method of specimen preparation which is otherwise particularly desirable because of the expectation that the embedded molecules should be well preserved in a near-native environment. This low contrast at high resolution is the principal limitation to atomic-resolution structure determination by electron microscopy. In spite of good progress in the direction of better images, it remains a major problem which prevents electron microscopy from becoming a simple and rapid method for biological atomic structure determination.     

Preparation of frozen-hydrated specimens for high resolution electron microscopy

A method is presented for preserving the high resolution structure of biological membranes in a frozen-hydrated environment for electron microscopy. The technique consists of sandwiching a specimen between two carbon films and then waiting while some of the solvent evaporates. When the solvent layer is judged to be of an appropriate thickness, the specimen is then frozen in liquid nitrogen. The specimen can then be inserted into the precooled stage of an electron microscope. Electron diffraction studies of the purple membrane of Halobacterium halobium recorded at -120 degrees C have shown that the structure can be preserved to a resolution of 3.5 A. The main advantage of this method over previous techniques is that the hydrating conditions can be accurately controlled.