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MAGNIFICATION MISMATCHES BETWEEN MICROGRAPHS - CORRECTIVE PROCEDURES AND IMPLICATIONS FOR STRUCTURAL-ANALYSIS

ULTRAMICROSCOPY 1992年第1-4期46卷 175-188页

作者： ALDROUBI, A TRUS, BL UNSER, M BOOY, FP STEVEN, AC NIAMSD DIV COMP RES & TECHNOLCOMP SYST LABBETHESDAMD 20892 NIAMSD STRUCT BIOL RES LABBETHESDAMD 20892

Quantitative structural analysis from electron micrographs of biological macromolecules inevitably requires the synthesis of data from many parts of the same micrograph and, ultimately, from multiple micrographs. Higher resolutions require the inclusion of progressively more data, and for the particles analyzed to be consistent to within ever more stringent limits. Disparities in magnification between micrographs or even within the field of one micrograph, arising from lens hysteresis or distortions, limit the resolution of such analyses. A quantitative assessment of this effect shows that its severity depends on the size of the particle under study: for particles that are 100 nm in diameter, for example, a 2% discrepancy in magnification restricts the resolution to approximately 5 nm. In this study, we derive and describe the properties of a family of algorithms designed for cross-calibrating the magnifications of particles from different micrographs, or from widely differing parts of the same micrograph. This approach is based on the assumption that all of the particles are of identical size: thus, it is applicable primarily to cryo-electron micrographs in which native dimensions are precisely preserved. As applied to icosahedral virus capsids, this procedure is accurate to within 0.1-0.2%, provided that at least five randomly oriented particles are included in the calculation. The algorithm is stable in the presence of noise levels typical of those encountered in practice, and is readily adaptable to non-isometric particles. It may also be used to discriminate subpopulations of subtly different sizes.

关键词： Algorithms Capsid/ultrastructure Cryopreservation Cryopreservation image Enhancement/methods image processing, computer-assisted/methods Microscopy, Electron/methods Models, Theoretical Simplexvirus/ultrastructure

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AUTOMATIC FOCUS CORRECTION FOR SPOT-SCAN IMAGING OF TILTED SPECIMENS

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ULTRAMICROSCOPY 1992年第1-4期46卷 199-206页

作者： DOWNING, KH Life Sciences Division Lawrence Berkeley Laboratory Berkeley CA 94720.

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.

关键词： Artifacts image Enhancement/methods image processing, computer-assisted/methods Microscopy, Electron, Scanning/instrumentation Microscopy, Electron, Scanning/methods Models, Theoretical

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FLOPPING POLYPEPTIDE-CHAINS AND SULEIKAS SUBTLE IMPERFECTIONS - ANALYSIS OF VARIATIONS IN THE ELECTRON MICROGRAPH OF A PURPLE MEMBRANE CRYSTAL

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ULTRAMICROSCOPY 1993年第1-4期49卷 387-396页

作者： FRANK, J CHIU, W HENDERSON, R SUNY ALBANY DEPT BIOMED SCIALBANYNY 12222 BAYLOR COLL MED VERNA & MARRS MCLEAN DEPT BIOCHEMHOUSTONTX 77030 MRC MOLEC BIOL LABCAMBRIDGEENGLAND

Patch averaging, a method of data analysis combining correlation averaging with multivariate statistical analysis, was applied to a high-resolution image of a purple membrane thin crystal. The observed variational patterns fall in two categories: non-crystallographic, related to small electron-optical distortions of the microscope, and crystallographic. conceivably related to variations in the locations of flexible segments of the polypeptide chain on the surface of the membrane.

关键词： Bacteriorhodopsins/chemistry Crystallography image processing, computer-assisted/methods image processing, computer-assisted/statistics & numerical data Microscopy, Electron Multivariate Analysis

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SPECTRA - A SYSTEM FOR processing ELECTRON imageS OF CRYSTALS

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ULTRAMICROSCOPY 1993年第3期48卷 251-264页

作者： SCHMID, MF DARGAHI, R TAM, MW BAYLOR COLL MED WM KECK CTR COMPUTAT BIOLHOUSTONTX 77030

SPECTRA is a program system that combines well tested and familiar algorithms and advanced graphics tools for (i) the display and indexing of the Fourier transform of images of crystalline specimens, (ii) refinement of the reciprocal lattice, (iii) output of amplitudes and phases of reflections and (iv) lattice distortion correction through cross-correlation map analysis. Because of the ease of identifying and indexing reflections, the user can include many reflections for reciprocal lattice refinement, leading to more accurate lattice determination. Automatic indexing is much faster than entering indices by hand, with less operator error. The user can run the procedure that corrects for lattice distortions with either standard parameters or storable parameters tailored to a particular specimen. SPECTRA's primary function is as a control program which acts as an interface between existing programs and the user. It was written in C with routines for the display, user interface and peak list search. Communication between the separate processes that make up SPECTRA is via the UNIX pipe mechanism and command files, the latter so that users may run, test or replace them or even run them as stand-alone programs outside this system.

关键词： Crystallography Fourier Analysis Fourier Analysis image processing, computer-assisted/methods Microscopy, Electron Software

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AUTOMATIC PARTICLE PICKING FROM ELECTRON-MICROGRAPHS

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ULTRAMICROSCOPY 1995年第3-4期58卷 381-391页

作者： LATA, KR PENCZEK, P FRANK, J SUNY ALBANY DEPT BIOMED SCIALBANYNY 12222

A computer program for automatic particle picking based on textural methods is proposed. The technique relies on the evaluation of certain textural parameters for data windows containing single particles, and those containing undesirable material. These parameters are manipulated by a discriminant analysis routine for determining the rules of classification between the different categories. The effectiveness of the method was demonstrated by application to electron micrographs of 70S Escherichia coli ribosomes.

关键词： Algorithms Discriminant Analysis Escherichia coli/ultrastructure image processing, computer-assisted/methods Microscopy, Electron/methods Particle Size Ribosomes/ultrastructure Software Design

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Channel surfing: Creating different color combinations from multiple-label images

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BIOTECHNIQUES 2001年第4期30卷 756-+页

作者： Paddock, S Univ Wisconsin Dept Mol Biol Howard Hughes Med Inst Madison WI 53706 USA

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CONCENTRATION EVALUATION OF CHROMATIN IN UNSTAINED RESIN-EMBEDDED SECTIONS BY MEANS OF LOW-DOSE RATIO-CONTRAST IMAGING IN STEM

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ULTRAMICROSCOPY 1993年第1-4期49卷 235-251页

作者： BOHRMANN, B HAIDER, M KELLENBERGER, E EUROPEAN MOLEC BIOL LAB W-6900 HEIDELBERGGERMANY

Quantitative STEM with the imaging mode of ratio-contrast was investigated in order to evaluate the local concentration of DNA in situ for different kinds of DNA plasms in terms of intracellular packing densities (p,d.). The ability of ratio imaging to suppress thickness variations provided the basis to use unstained sections from cryofixed and freeze-substituted material. The DNA p.d. within the nucleoid of E coli was determined to be about 100 mg ml-1. Quantitative data concerning the p.d. of DNA in condensed eukaryotic chromatin assuming equal amounts of DNA and protein were evaluated for the first time: approximately 400 mg ml-1 chromatin which corresponds to 200 mg ml-1 DNA. The p.d. of DNA in chromosomes from the dinoflagellate Amphidinium carterae, a eukaryote devoid of histones and with only small relative amounts of histone-like protein, was also found to be of the order of 200 mg ml-1. The highest p.d. of DNA was measured for the head of the bacteriophage T4 with more than 800 mg ml-1, in fair agreement with previous calculations. The results provide further support for a condensation mode of low protein chromatins that involves a liquid-crystalline organization of the DNA filaments.

关键词： Animals Chromatin/chemistry Dinoflagellida/chemistry Dinoflagellida/ultrastructure DNA/analysis Epoxy Resins Escherichia coli/chemistry Escherichia coli/ultrastructure Euglena/chemistry Euglena/ultrastructure image processing, computer-assisted/methods Microscopy, Electron, Scanning Transmission/methods Tissue Embedding/methods Tissue Embedding/methods

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methods and applications of three-color confocal imaging

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BIOTECHNIQUES 1997年第1期22卷 120-&页

作者： Paddock, SW Hazen, EJ DeVries, PJ Howard Hughes Medical Institute Laboratory of Molecular Biology University of Wisconsin Madison 53706 USA. paddock@facstaff.wisc.edu

A simple method for constructing two- and three-color merged images from grayscale confocal fluorescence images using Adobe Photoshop(TM) is outlined. Various computer methods for manipulating and displaying the image... 详细信息

关键词： Animals Drosophila/embryology Embryo, Nonmammalian/cytology Fluorescence Fluorescence image processing, computer-assisted/methods Microscopy, Confocal/methods Software Videotape Recording

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PHOSPHOR image-ANALYSIS OF HUMAN P53 PROTEIN ISOFORMS

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BIOTECHNIQUES 1995年第2期18卷 292-299页

作者： MERRICK, BA PENCE, PM HE, C PATTERSON, RM SELKIRK, JK National Institute of Environmental Health Sciences Research Triangle Park NC USA.

Phosphor imaging was evaluated for detection, quantitation and resolution of multiphosphorylated protein isoforms separated by two-dimensional gel electrophoresis. A nuclear phosphoprotein, p53, was isolated by immunoprecipitation after biosynthetic labeling with S-35, P-32 or P-33 in cultured human cells. Of the three radionuclides, S-35 was the most sensitive in detection after a 1-week exposure, although shorter exposure times were effective. In dividing cells, 11 S-35-labeled isoforms were found, of which 10 were phosphorylated by P-33 and P-32. Exposure of phosphonuclides for one half-life showed that P-33 radiolabeling produced better resolution among isoforms than P-32 but was less sensitive in detection. Volume integration showed phosphorylated isoforms comprised from 1% to 25% of total isoform signal. The relative phosphorylation of each p53 isoform wa estimated by normalizing P-33 or P-32 isoform volumes with the corresponding S-35 volume and showed progressive phosphorylation of acidic isoforms. Additionally, phosphor imaging capably detected quantitative changes among individual isoforms after experimental modulation of the isoform pattern by serum deprivation. The described electrophoretic isolation and quantitation procedures should find general application in discerning active and inactive phosphoisoforms for eventual identification.

关键词： Electrophoresis, Polyacrylamide Gel Humans image processing, computer-assisted/methods Isomerism Phosphorus Radioisotopes Precipitin Tests Precipitin Tests Sulfur Radioisotopes Tumor Suppressor Protein p53/classification Tumor Suppressor Protein p53/isolation & purification

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Multicolor imaging: The important question of co-localization

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BIOTECHNIQUES 2001年第6期30卷 1240-+页

作者： Smallcombe, A Biorad Labs Ltd Hemel Hempstead HP2 7TD Herts England

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