Spot-scan Images Research Articles

Images of paraffin monolayer crystals with perfect contrast: Minimization of beam-induced specimen motion

Quantitative analysis of electron microscope images of organic and biological two-dimensional crystals has previously shown that the absolute contrast reached only a fraction of that expected theoretically from the electron diffraction amplitudes. The accepted explanation for this is that irradiation of the specimen causes beam-induced charging or movement, which in turn causes blurring of the image due to image or specimen movement. In this paper, we used three different approaches to try to overcome this image-blurring problem in monolayer crystals of paraffin. Our first approach was to use an extreme form of spotscan imaging, in which a single image was assembled on film by the successive illumination of up to 50,000 spots, each of a diameter of around 7 nm. The second approach was to use the Medipix II detector with its zero-noise readout to assemble a time-sliced series of images of the same area in which each frame from a movie with up to 400 frames had an exposure of only 500 electrons. In the third approach, we simply used a much thicker carbon support film to increase the physical strength and conductivity of the support. Surprisingly, the first two methods involving dose fractionation in space or time produced only partial improvements in contrast whereas the third approach produced many virtually perfect images, where the absolute contrast predicted from the electron diffraction amplitudes was observed in the images. We conclude that it is possible to obtain consistently almost perfect images of beam-sensitive specimens if they are attached to an appropriately strong and conductive support; however great care is needed in practice and the problem remains of how to best image ice-embedded biological structures in the absence of a strong, conductive support film.

Solution X-Ray Scattering-Based Estimation of Electron Cryomicroscopy Imaging Parameters for Reconstruction of Virus Particles

Structure factor amplitudes and phases can be computed directly from electron cryomicroscopy images. Inherent aberrations of the electromagnetic lenses and other instrumental factors affect the structure factors, however, resulting in decreased accuracy in the determined three-dimensional reconstruction. In contrast, solution x-ray scattering provides absolute and accurate measurement of spherically averaged structure factor amplitudes of particles in solution but does not provide information on the phases. In the present study, we explore the merits of using solution x-ray scattering data to estimate the imaging parameters necessary to make corrections to the structure factor amplitudes derived from electron cryomicroscopic images of icosahedral virus particles. Using 400-kV spot-scan images of the bacteriophage P22 procapsid, we have calculated an amplitude contrast of 8.0 ± 5.2%. The amplitude decay parameter has been estimated to be 523 ± 188 Å 2 with image noise compensation and 44 ± 66 Å 2 without it. These results can also be used to estimate the minimum number of virus particles needed for reconstruction at different resolutions.

Multivariate analysis of single unit cells in electron crystallography

High-resolution electron cryomicroscopy of two-dimensional protein crystals is associated with extremely noisy raw data in which even the crystal lattice often cannot be discerned. Correlation averaging procedures, aimed at calculating the total average of all unit cells of crystals in order to reduce noise, are now used routinely in electron crystallography. Multivariate statistical analysis (MSA) may be used for finding not only the average structure but also for quantifying the systematic departures from that average within the population of individual unit cells. We show that the MSA approach is applicable to single unit-cell images in the low-dose (<10 electrons/Å 2), high-resolution (<5 Å) realm using 400 keV electron spot-scan images of ice-embedded gp32∗I protein crystals. Our feasibility study opens a pathway toward exploiting these naturally occurring variations on the unit-cell theme in order to achieve higher-resolution three-dimensional reconstruction results, or to better understand the dynamic behaviour of molecules within two-dimensional crystals. We explain how single unit-cell images can be processed and classified into homogeneous groups, and we review how the results of such discriminate averaging may subsequently be exploited within the context of conventional “ h , k ”-space electron crystallographic approaches. Variations among the individual unit cells may thus be one of the most significant resolution-limiting factors currently experienced in electron crystallography. The quantitative assessment and exploitation of such variations may lead to an increased performance of electron crystallographic procedures.

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 x 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 degrees different from the reference data of Henderson et al. (1986), Ultramicroscopy, 19, 147-178, up to 8.8 A resolution, which corresponds to 0.8 of the Nyquist frequency of the camera.

Methods to Monitor and Improve the Performance of Specimen Holders for Transmission Electron Cryomicroscopy

One goal in electron microscopy of biological specimens is to improve the quality of data to equal the resolution capabilities of modem transmission electron microscopes. Radiation damage and beam- induced movement caused by charging of the sample, low image contrast at high resolution, and sensitivity to external vibration and drift in side entry specimen holders limit the effective resolution one can achieve. Several methods have been developed to address these limitations: cryomethods are widely employed to preserve and stabilize specimens against some of the adverse effects of the vacuum and electron beam irradiation, spot-scan imaging reduces charging and associated beam-induced movement, and energy-filtered imaging removes the “fog” caused by inelastic scattering of electrons which is particularly pronounced in thick specimens.Although most cryoholders can easily achieve a 3.4Å resolution specification, information perpendicular to the goniometer axis may be degraded due to vibration. Absolute drift after mechanical and thermal equilibration as well as drift after movement of a holder may cause loss of resolution in any direction.

The CM200 Feg-Twin Optimized for Cryo-Electron Microscopy

The requirements for cryo-electron microscopy are: good vacuum, high resolution under low-dose conditions at low magnification, low drift with cooled cryo-transfer holders and, in some cases, fast spotscan imaging. For our cryo microscopes we have introduced special factory tests in which these specifications will be met.In order to obtain high spatial frequencies at low magnification an appropriate specimen with a statistical distribution of small spacings and a fine-grained recording film is needed. We used amorphous tungsten on carbon as specimen and overexposed and underdeveloped Agfa Scienta film (Fig. 1). The laser diffractogram in Fig. 2 presents Thon rings out to 0.19 nm in a one-second exposure flood-beam image. The result of a similar experiment done with a cooled cryo-transfer holder is shown in Fig. 3. Flood-beam images of gold islands and carbon black on a thin carbon film were taken at 66 OOOx at liquid-nitrogen temperature with one second exposure under low-dose conditions. The laser diffractogram clearly shows Thon rings out to 0. 2 nm.

Performance of a slow-scan CCD camera for macromolecular imaging in a 400 kV electron cryomicroscope

The feasibility and limitations of a 1024 × 1024 slow-scan charge-coupled device (CCD) camera were evaluated for imaging in a 400kV electron cryomicroscope. Catalase crystals and amorphous carbon film were used as test specimens. Using catalase crystals, it was found that the finite (24 μm) pixel size of the slow-scan CCD camera governs the ultimate resolution in the acquired images. For instance, spot-scan images of ice-embedded catalase crystals showed resolutions of 8Å and 4Å at effective magnifications of 67,000 × and 132,000 ×, respectively. Using an amorphous carbon film, the damping effect of the modulation transfer function (MTF) of the slow-scan CCD camera on the specimen's Fourier spectrum relative to that of the photographic film was evaluated. The MTF of the slow-scan CCD camera fell off more rapidly compared to that of the photographic film and reached the value of 0.2 at the Nyquist frequency. Despite this attenuation, the signal-to-noise ratio of the CCD data, as determined from reflections of negatively-stained catalase crystals, was found to decrease to ~50% of that of photographic film data. The phases computed from images of the same negatively-stained catalase crystals recorded consecutively on both the slow-scan CCD camera and photographic film were found to be comparable to each other within 12 °. Ways of minimizing the effect of the MTF of the slow-scan CCD camera on the acquired images are also presented.

PTOOL: A Software Package for the Selection of Particles from Electron Cryomicroscopy Spot-Scan Images

PTOOL, a suite of tools packaged in an X Window System-based graphical user interface has been developed to allow consistent and accurate selection of single particles from low-contrast spot-scan and flood beam electron microscopy images. These user friendly and computationally efficient tools run on Unix-based workstations. Particles are selected using an automatic reference based selection method which incorporates extensive image preprocessing. An image division and subsequent image sewing mechanism allows for efficient particle selection independent of micrograph size. A particle viewing tool is provided which displays galleries of images and has options for performing image processing operations such as Fourier filtering, histogram equalization, and image masking. In addition, a tool is included to perform semiautomatic focal pair particle matching to identify the same particles on two micrographs.

Cryoelectron microscopy and image reconstruction of spherical viruses with spot scan and FEG technologies

The field emission gun electron microscope (FEG) is a tool that has the potential to achieve near atomic resolution information of biological macromolecules. The FEG provides a beam with higher spatial and temporal coherence and a better phase contrast transfer function than do microscopes with either tungsten or LaB6 filaments. The FEG is also ideal for spot scan imaging applications because it can produce a small, coherent and very bright spot. In spot scan mode the specimen is exposed to an array of nonoverlapping spots rather man a flood beam. This significantly reduces beam-induced specimen drift.Frozen-hydrated samples of cowpea chlorotic mottle (CCMV, Fig. 1A) and cowpea severe mosaic virus (CPSMV, Fig. IB) were examined on a Philips CM12 transmission electron microscope equipped with a standard LaB6 gun and on a Philips CM200 equipped with a field emission gun, respectively. The CM12 was operated at 120kV and was externally controlled by means of a spot scan imaging program which produced a series of 250 nm diameter spots on Kodak SO-163 sheet film.

Comparison of 13Å helical and 7Å crystallographic projection maps of frozen, hydrated acrosomal bundle from limulus sperm

Actin associates with crosslinking proteins to form bundles and networks. These assemblies can provide a scaffold to anchor organelles, a support for the cell membrane or can attach the cell to the extracellular matrix or to other cells. Each crosslinker must have at least two actin binding sites, one for each actin filament. Scruin, in the Limulus acrosomal bundle, shows no homology with other actin cross-linking proteins. However, it has two homologous domains also found in the sequence encoded by kelch, a gene in Drosophila that is important in nutrient transport into the oocyte, and in MIPP, a mouse placental protein.We have determined a 13Å helical 3-dimensional structure of the actin-scruin complex, which is the basic repeat unit of the acrosomal bundle in Limulus sperm and have fitted the F-actin filament of Holmes. Each scruin binds to two adjacent actin molecules along a single F-actin filament helix. In the present investigation, we used our high resolution (7Å) spot scan electron images of ice-embedded acrosomal bundles for further analysis of the actin atomic map and scruin-scruin contacts. Spot-scan images were obtained from the frozen, hydrated acrosomal bundles in a 400 kV electron cryomicroscope.

Correlation averaging and structural changes in GP32*I crystal unit cells

We compared correlation and Fourier averaging by analyzing 400 kV spot-scan electron images of iceembedded gp32*I crystals from a previous study using correlation averaging and multivariate statistical analysis (MSA). The set consisted of 9 spot-scan electron images (Fig. 1) from different micrographs. Unit cells of gp32*I crystal in each spot-scan image were located using cross-correlation map ofthe whole image with its central portion (an area of up to 10 unit cells). Each correlation peak in the map served as a center for picking up a patch of the same size as the reference used for mapcalculation. After averaging of the patches a new reference with improved signal-to-noise ratio was obtained and procedure was iterated several times using progressively smaller reference sizes. Final reference size was about 1-2 unit cells. This permitted us to follow local lattice variations. The technique does not use any assumption of crystallinity of the specimen and can be applied for highly distorted crystal lattices. Each spot-scan image was processed independently of the others. After the iterativeprocedure a final set of averaged images was obtained and structure factor amplitudes and phases were calculated from them using standard crystallographic procedures followed by merging all the spectra. Using the set of structure factors a projection map was generated which looked very similar to the 4 Å resolution map obtained using Fourier averaging technique (Fig. 2).

Automated electron tomography and CCD spot-scan imaging of biological macromolecules

Computer control of a transmission electron microscope (TEM) and digital recording of images have shown to be useful tools in recording 2D data sets of macromolecules and larger cellular structures with accurately defined imaging conditions.3D structure of individual molecules can be obtained by electron tomography. The method requires that projection data are collected over a large range of specimen tilts. With automated electron tomography images are recorded in digital format, and lateral specimen displacement together with focus changes are automatically compensated for. Typically, with our experimental set-up, we collect 75 projections of negatively stained preparations over ±75° and 30 projections of ice-embedded molecules over ±60° with a total dose of 20-50 e/Å2.By using the spot-scan imaging mode beam-induced movements of ice-embedded specimens are greatly reduced, thus largely avoiding the loss of resolution associated with specimen instability. With CCD spot-scan imaging the size of the spot is matched to the CCD chip (Fig. 1).

Charging phenomena observed on biological specimens in a 400-kV electron cryo-microscope

There is an ever present need in electron microscopy of biological specimens to improve the quality of the recorded data to approach the atomic resolution capability of the modem instruments. The method adopted which has yielded the only significant advance in this direction is to directly phase theelectron diffraction amplitudes of 2-dimensional crystals using the Fourier transforms of high resolution images. Cryo methods have improved the preservation and stability of the specimen to the relatively high electron dose irradiation which is required for imaging compared to electron diffraction. In the cases, however, where the actual power of the structure factors was compared with those that were transferred by the microscope’s imaging system, a large attenuation was measured. Spot-scan imaging has been shown to improve the image quality by reducing the beam-induced movement. Charging of the specimen appears to cause similar deleterious effects on image resolution, but it requires different methods aimed at resolution improvement. Proteins are insulators and because of secondary electron loss they will become positively charged during irradiation. Defocused diffraction brightfield imagescan display the charged nature of an irradiated specimen as an apparent increase in mass. A comparison wasmade of the degree of charging and discharging between using 100 and 400 keV electrons on streptavidin crystals in glucose and catalase crystals in amorphous ice. We show that when the region of the irradiated sample includes part of the supporting carbon film, the charge build-up is prevented.

Spot-scan imaging of ice-embedded catalase crystal on a slow-scan CCD camera in a 400-kV electron cryomicroscope

In protein electron crystallography, both low dose electron diffraction patterns and images are needed to provide accurate amplitudes and phases respectively for a 3-dimensional reconstruction. We have demonstrated that the Gatan 1024x1024 model 679 slow-scan CCD camera is useful to record electron diffraction intensities of glucose-embedded crotoxin complex crystal to 3 Å resolution. The quality of the electron diffraction intensities is high on the basis of the measured intensity equivalence ofthe Friedel-related reflections. Moreover, the number of patterns recorded from a single crystal can be as high as 120 under the constraints of radiation damage and electron statistics for the reflections in each pattern.A limitation of the slow-scan CCD camera for recording electron images of protein crystal arises from the relatively large pixel size, i.e. 24 μm (provided by Gatan). The modulation transfer function of our camera with a P43 scintillator has been determined for 400 keV electrons and shows an amplitude fall-off to 0.25 at 1/60 μm−1.

4-Å Projection Map of Bacteriophage T4 DNA Helix-Destabilizing Protein (gp32*I) Crystal by 400-kV Electron Cryomicroscopy

Ice-embedded crystals of bacteriophage T4 DNA helix-destabilizing protein gp32*I were imaged by computer-controlled spot-scanning on a 400-kV electron cryomicroscope. gp32*I crystals generally have different steps of thickness within a crystal; each step can have different symmetry. Multivariate statistical analysis enabled us to unambiguously select spot-scan images that consist entirely of one motif which were processed subsequently by crystallographic Fourier-averaging techniques. The computed phases of the resulting reflections were evaluated for symmetry in projection, and some of those images were correlated with independent thickness measurements of freeze-dried samples of the same crystals. The structure factors with pgg symmetry from nine spot-scan images were merged, and the mean figure of merit of merged phases was better than 0.9 for data at resolution up to 4 Å. A projection map was generated and showed multiple density peaks that corresponded to the high-resolution features of gp32*I.

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.

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 Å resolution. The intensity strength of the structure factors of this protein crystal are different at low ( > 10 Å) resolution but similar at high resolution ( < 10 Å) 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.

Defocus ramp correction in spot-scan imaging

Three-dimensional structures of a number of samples have been determined by electron crystallography. The procedures used in this work include recording images of fairly large areas of a specimen at high tilt angles. There is then a large defocus ramp across the image, and parts of the image are far out of focus. In the regions where the defocus is large, the contrast transfer function (CTF) varies rapidly across the image, especially at high resolution. Not only is the CTF then difficult to determine with sufficient accuracy to correct properly, but the image contrast is reduced by envelope functions which tend toward a low value at high defocus.We have combined computer control of the electron microscope with spot-scan imaging in order to eliminate most of the defocus ramp and its effects in the images of tilted specimens. In recording the spot-scan image, the beam is scanned along rows that are parallel to the tilt axis, so that along each row of spots the focus is constant. Between scan rows, the objective lens current is changed to correct for the difference in specimen height from one scan to the next.

Calculated dynamical scattering in a fibrous protein structure

Despite many technical, experimental and in principle difficulties, particularly in regard to radiation damage and stability of the structure at the atomic level, considerable progress has been made in the application of the combined electron microscopy/electron diffraction (EM/ED) technique to the solution of protein structures. The technique now represents a possible alternative to x-ray diffraction for those cases where the protein is difficult or impossible to grow as macrocrystals, provided that the molecule can be incorporated in microcrystals or two-dimensional ordered arrays. Encouraged by this we have recently recorded images and diffraction intensities from microcrystals of an alpha-helical coiledcoil protein extracted from the ootheca of the praying mantis. The ED data shows reflections out to beyond 2Å, though not uniformly in all directions. Fourier transforms of the corresponding digitised images show resolution out to nearly 3Å and this data is being used to phase the ED data. All data was recorded from frozen-hydrated specimens at 400kV in a super-fluid helium specimen stage of a JEM 4000EX electron microscope using spot-scan and low-dose imaging techniques. Further work aimed at collecting a 3-d data set to a resolution of 3Å using tilted crystals is continuing.

High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.