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Description: Relatively large values of objective-lens defocus must normally be used to produce detectable levels of image contrast for unstained biological specimens, which are generally weak phase objects. As a result, a subsequent restoration operation must be used to correct for oscillations in the contrast transfer function (CTF) at higher resolution. Currently used methods of CTF-correction assume the ideal case in which Friedel mates in the scattered wave have contributed pairs of Fourier components that overlap with one another in the image plane. This"ideal" situation may be only poorly satisfied, or not satisfied at all, as the particle size gets smaller, the defocus value gets larger, and the resolution gets higher. We have therefore investigated whether currently used methods of CTF correction are also effective in restoring the single-sideband image information that becomes displaced (delocalized) by half (or more) the diameter of a particle of finite size. Computer simulations are used to show that restoration either by"phase flipping" or by multiplying by the CTF recovers only about half of the delocalized information. The other half of the delocalized information goes into a doubly defocused"twin" image of the type produced during optical reconstruction of an in-line hologram. Restoration with a Wiener filter is effective in recovering the delocalized information only when the signal-to-noise ratio (S/N) is orders of magnitude higher than that which exists in low-dose images of biological specimens, in which case the Wiener filter approaches division by the CTF (i.e. the formal inverse). For realistic values of the S/N, however, the"twin image" problem seenwith a Wiener filter is very similar to that seen when either phase flipping or multiplying by the CTF are used for restoration. The results of these simulations suggest that CTF correction is a poor alternative to using a Zernike-type phase plate when imaging biological specimens, in ...
Date: March 28, 2008
Creator: Downing, Kenneth H. & Glaeser, Robert M.
Partner: UNT Libraries Government Documents Department

Historical background: Why is it important to improve automated particle selection methods?

Description: A current trend in single-particle electron microscopy is to compute three-dimensional reconstructions with ever-increasing numbers of particles in the data sets. Since manual--or even semi-automated--selection of particles represents a major bottleneck when the data set exceeds several thousand particles, there is growing interest in developing automatic methods for selecting images of individual particles. Except in special cases, however, it has proven difficult to achieve the degree of efficiency and reliability that would make fully automated particle selection a useful tool. The simplest methods such as cross correlation (i.e., matched filtering) do not perform well enough to be used for fully automated particle selection. Geometric properties (area, perimeter-to-area ratio, etc.) and the integrated ''mass'' of candidate particles are additional factors that could improve automated particle selection if suitable methods of contouring particles could be developed. Another suggestion is that data be always collected as pairs of images, the first taken at low defocus (to capture information at the highest possible resolution) and the second at very high defocus (to improve the visibility of the particle). Finally, it is emphasized that well-annotated, open-access data sets need to be established in order to encourage the further development and validation of methods for automated particle selection.
Date: August 14, 2003
Creator: Glaeser, Robert M.
Partner: UNT Libraries Government Documents Department

Water structure as a function of temperature from X-ray scatteringexperiments and ab initio molecular dynamics

Description: We present high-quality X-ray scattering experiments on pure water taken over a temperature range of 2 to 77 C using a synchrotron beam line at the advanced light source (ALS) at Lawrence Berkeley National Laboratory. The ALS X-ray scattering intensities are qualitatively different in trend of maximum intensity over this temperature range compared to older X-ray experiments. While the common procedure is to report both the intensity curve and radial distribution function(s), the proper extraction of the real-space pair correlation functions from the experimental scattering is very difficult due to uncertainty introduced in the experimental corrections, the proper weighting of OO, OH, and HH contributions, and numerical problems of Fourier transforming truncated data in Q-space. Instead, we consider the direct calculation of X-ray scattering spectra using electron densities derived from density functional theory based on real-space configurations generated with classical water models. The simulation of the experimental intensity is therefore definitive for determining radial distribution functions over a smaller Q-range. We find that the TIP4P, TIP5P and polarizable TIP4P-Pol2 water models, with DFT-LDA densities, show very good agreement with the experimental intensities, and TIP4P-Pol2 in particular shows quantitative agreement over the full temperature range. The resulting radial distribution functions from TIP4P-Pol2 provide the current best benchmarks for real-space water structure over the biologically relevant temperature range studied here.
Date: March 1, 2003
Creator: Hura, Greg; Russo, Daniela; Glaeser, Robert M.; Head-Gordon,Teresa; Krack, Matthias & Parrinello, Michele
Partner: UNT Libraries Government Documents Department

Extraction of macro-molecule images in cryo-EM micrographs

Description: Advances in Electron Microscopy and single-particle reconstruction have led to results at increasingly high resolutions. This has opened up the possibility of complete automation of single particle reconstruction. Main bottleneck in automation of single particle reconstruction is manual selection of particles in the micrograph. This paper describes a simple but efficient approach for segmentation of particle projections in the micrographs obtained using cryo-electron microscope. Changing the shape of objects to facilitate segmentation from the cluster and reconstructing its actual shape after isolation is successfully attempted. Both low-level and high-level processing techniques are used and the whole process is made automatic. Over 90 percent success in automatic particle picking is achieved. Several areas for improvement and future research directions are discussed.
Date: March 20, 2003
Creator: Adiga, Umesha P.S.; Malladi, Ravi & Glaeser, Robert M.
Partner: UNT Libraries Government Documents Department

Electron microscopy of biological macromolecules: Bridging the gapbetween what physics allows and what we currently can get

Description: The resolution achieved in low-dose electron microscopy of biological macromolecules is significantly worse than what can be obtained on the same microscopes with more robust specimens. When two-dimensional crystals are used, it is also apparent that the high-resolution image contrast is much less than what it could be if the images were perfect. Since specimen charging is one factor that might limit the contrast and resolution achieved with biological specimens, we have investigated the use of holey support films that have been coated with a metallic film before depositing specimens onto a thin carbon film that is suspended over the holes. Monolayer crystals of paraffin (C44H90) are used as a test specimen for this work because of the relative ease in imaging Bragg spacings at {approx}0.4 nm resolution, the relative ease of measuring the contrast in these images, and the similar degree of radiation sensitivity of these crystals when compared to biological macromolecules. A metallic coating on the surrounding support film does, indeed, produce a significant improvement in the high-resolution contrast for a small fraction of the images. The majority of images show little obvious improvement, however, and even the coated area of the support film continues to show a significant amount of beam-induced movement under low-dose conditions. The fact that the contrast in the best images can be as much as 25 percent-35 percent of what it would be in a perfect image is nevertheless encouraging, demonstrating that it should be possible, in principle, to achieve the same performance for every image. Routine data collection of this quality would make it possible to determine the structure of large, macromolecular complexes without the need to grow crystals of these difficult specimen materials.
Date: April 30, 2003
Creator: Typke, Dieter; Downing, Kenneth H. & Glaeser, Robert M.
Partner: UNT Libraries Government Documents Department


Description: A miniature electrostatic element has been designed to selectively apply a ninety-degree phase shift to the unscattered beam in the back focal plane of the objective lens, in order to realize Zernike-type, in-focus phase contrast in an electron microscope. The design involves a cylindrically shaped, biased-voltage electrode, which is surrounded by a concentric grounded electrode. Electrostatic calculations have been used to determine that the fringing fields in the region of the scattered electron beams will cause a negligible phase shift as long as the ratio of electrode length to the transverse feature-size is greater than 5:1. Unlike the planar, three-electrode einzel lens originally proposed by Boersch for the same purpose, this new design does not require insulating layers to separate the biased and grounded electrodes, and it can thus be produced by a very simple microfabrication process. Scanning electron microscope images confirm that mechanically robust devices with feature sizes of {approx}1 {micro}m can be easily fabricated. Preliminary experimental images demonstrate that these devices do apply a 90-degree phase shift between the scattered and unscattered electrons, as expected.
Date: September 20, 2006
Creator: Cambie, Rossana; Downing, Kenneth H.; Typke, Dieter; Glaeser,Robert M. & Jin, Jian
Partner: UNT Libraries Government Documents Department

Specimen charging on thin films with one conducting layer:Discussion of physical principles

Description: While the most familiar consequences of specimen charging in transmission electron microscopy can be eliminated by evaporating a thin conducting film (such as a carbon film) onto an insulating specimen, or by preparing samples directly on such a conducting film to begin with, a more subtle charging effect still remains. We argue here that specimen charging is in this case likely to produce a dipole sheet rather than a layer of positive charge at the surface of the specimen. A simple model of the factors that control the kinetics of specimen charging, and its neutralization, is discussed as a guide for experiments that attempt to minimize the amount of specimen charging. Believable estimates of the electrostatic forces and the electron optical disturbances that are likely to occur suggest that specimen bending and warping may have the biggest impact on degrading the image quality at high resolution. Electron optical effects are likely to be negligible except in the case of a specimen that is tilted to high angle. A model is proposed to explain how both the mechanical and electron-optical effects of forming a dipole layer would have much greater impact on the image resolution in a direction perpendicular to the tilt axis, a well-known effect in electron microscopy of two-dimensional crystals.
Date: April 15, 2003
Creator: Glaeser, Robert M. & Downing, Kenneth H.
Partner: UNT Libraries Government Documents Department

Specificity of anion-binding in the substrate-pocket ofbacteriorhodopsin

Description: The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff-base binding site, and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side-chain of S85. On the basis of these x-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K{sub d}, for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.
Date: August 30, 2003
Creator: Facciotti, Marc T.; Cheung, Vincent S.; Lunde, Christopher S.; Rouhani, Shahab; Baliga, Nitin S. & Glaeser, Robert M.
Partner: UNT Libraries Government Documents Department


Description: Beam-induced specimen movement may be the major factor that limits the quality of high-resolution images of organic specimens. One of the possible measures to improve the situation that was proposed by Henderson and Glaeser (Henderson and Glaeser, 1985), which we refer to here as 'stroboscopic image capture', is to divide the normal exposure into many successive frames, thus reducing the amount of electron exposure--and possibly the amount of beam-induced movement--per frame. The frames would then be aligned and summed. We have performed preliminary experiments on stroboscopic imaging using a 200-kV electron microscope that was equipped with a high dynamic range CCD camera for image recording and a liquid N{sub 2}-cooled cryoholder. Single-layer paraffin crystals on carbon film were used as a test specimen. The ratio F(g)/F(0) of paraffin reflections, calculated from the images, serves as our criterion for the image quality. In the series that were evaluated, no significant improvement of the F{sub image}(g)/F{sub image}(0) ratio was found, even though the electron exposure per frame was reduced by a factor of 30. A frame-to-frame analysis of image distortions showed that considerable beam-induced movement had still occurred during each frame. In addition, the paraffin crystal lattice was observed to move relative to the supporting carbon film, a fact that cannot be explained as being an electron-optical effect caused by specimen charging. We conclude that a significant further reduction of the dose per frame (than was possible with this CCD detector) will be needed in order to test whether the frame-to-frame changes ultimately become small enough for stroboscopic image capture to show its potential.
Date: August 1, 2006
Creator: Typke, Dieter; Gilpin, Christopher J.; Downing, Kenneth H. & Glaeser, Robert M.
Partner: UNT Libraries Government Documents Department

Experimental characterization and mitigation of specimen charging on thin films with one conducting layer

Description: Specimen charging may be one of the most significant factors that contribute to the high variability generally low quality of images in cryo-electron microscopy. Understanding the nature of specimen charging can help in devising methods to reduce or even avoid its effects and thus improve the rate of data collection as well as the quality of the data. We describe a series of experiments that help to characterize the charging phenomenon which has been termed the Berriman effect. The pattern of buildup and disappearance of the charge pattern have led to several suggestions for how to alleviate the effect. Experiments are described that demonstrate the feasible of such charge mitigation.
Date: April 4, 2003
Creator: Downing, Kenneth H.; McCartney, M.R. & Glaeser, Robert M.
Partner: UNT Libraries Government Documents Department

A Binary Segmentation Approach for Boxing Ribosome Particles in Cryo EM Micrographs

Description: Three-dimensional reconstruction of ribosome particles from electron micrographs requires selection of many single-particle images. Roughly 100,000 particles are required to achieve approximately 10 angstrom resolution. Manual selection of particles, by visual observation of the micrographs on a computer screen, is recognized as a bottleneck in automated single particle reconstruction. This paper describes an efficient approach for automated boxing of ribosome particles in micrographs. Use of a fast, anisotropic non-linear reaction-diffusion method to pre-process micrographs and rank-leveling to enhance the contrast between particles and the background, followed by binary and morphological segmentation constitute the core of this technique. Modifying the shape of the particles to facilitate segmentation of individual particles within clusters and boxing the isolated particles is successfully attempted. Tests on a limited number of micrographs have shown that over 80 percent success is achieved in automatic particle picking.
Date: June 24, 2003
Creator: Adiga, Umesh P. S.; Malladi, Ravi; Baxter, William & Glaeser, Robert M.
Partner: UNT Libraries Government Documents Department

High-throughput film-densitometry: An efficient approach to generate large data sets

Description: A film-handling machine (robot) has been built which can, in conjunction with a commercially available film densitometer, exchange and digitize over 300 electron micrographs per day. Implementation of robotic film handling effectively eliminates the delay and tedium associated with digitizing images when data are initially recorded on photographic film. The modulation transfer function (MTF) of the commercially available densitometer is significantly worse than that of a high-end, scientific microdensitometer. Nevertheless, its signal-to-noise ratio (S/N) is quite excellent, allowing substantial restoration of the output to ''near-to-perfect'' performance. Due to the large area of the standard electron microscope film that can be digitized by the commercial densitometer (up to 10,000 x 13,680 pixels with an appropriately coded holder), automated film digitization offers a fast and inexpensive alternative to high-end CCD cameras as a means of acquiring large amounts of image data in electron microscopy.
Date: July 14, 2004
Creator: Typke, Dieter; Nordmeyer, Robert A.; Jones, Arthur; Lee, Juyoung; Avila-Sakar, Agustin; Downing, Kenneth H. et al.
Partner: UNT Libraries Government Documents Department