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INVARIANT CLASSIFICATION OF MOLECULAR VIEWS IN ELECTRON-MICROGRAPHS

ULTRAMICROSCOPY 1990年第3期32卷 255-264页

作者： SCHATZ, M VANHEEL, M Fritz Haber Institute Max Planck Society Berlin Germany.

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 levels 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.

关键词： Fourier Analysis Fourier Analysis Frozen Sections Frozen Sections image processing, computer-assisted Macromolecular Substances Microcomputers Microscopy, Electron/methods

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computer-assisted, quantitative cytokine enzyme-linked immunospot analysis of human immune effector cell function

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BIOTECHNIQUES 1997年第6期22卷 1146-1149页

作者： Cui, Y Chang, LJ UNIV ALBERTA DEPT MED MICROBIOL & IMMUNOLHERITAGE MED RES CTREDMONTONAB T6G 2S2CANADA

Originally developed for detecting antibody production from B lymphocytes, the enzyme-linked immunospot (ELISPOT) assay was later modified to assess cytokine production from various immune effector cells. Although the ELISPOT assay carl detect antibody or cytokine production at the single-cell level, the visual counting of spots in a 96-well plate under a microscope makes this method unsuitable for handling large sample sizes. Here, we introduce a computer-assisted image analysis system to overcome this problem. This system makes the data analysis step of the ELISPOT assay convenient, objective, sensitive and suitable for handling large sample pools. Studies requiring lymphocyte proliferation assay, cytotoxic lymphocyte assay and precursor frequency assay can be conducted through the ELISPOT assay. This is demonstrated here using examples such as mixed lymphocyte allogeneic reactions and human immunodeficiency virus antigen-specific, cell-mediated immune responses.

关键词： Antigens, CD/analysis Cell Line Enzyme-Linked Immunosorbent Assay/methods Enzyme-Linked Immunosorbent Assay/methods HIV Antigens/immunology Humans image processing, computer-assisted Interferon-gamma/biosynthesis Interferon-gamma/immunology Lymphocyte Culture Test, Mixed Lymphocyte Culture Test, Mixed Sensitivity and Specificity T-Lymphocyte Subsets/immunology

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Applications of combined spectral lifetime microscopy for biology

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BIOTECHNIQUES 2006年第3期41卷 249-+页

作者： Yan, Long Rueden, Curtis T. White, John G. Eliceiri, Kevin W. Univ Wisconsin Lab Opt & Computat Instrumentat Madison WI USA

Live cell imaging has been greatly advanced by the recent development of new fluorescence microscopy-based methods such as multiphoton laser scanning microscopy, which can noninvasively image deep into live specimens and generate images of extrinsic and intrinsic signals. Of recent interest has been the development of techniques that can harness properties of fluorescence, other than intensity, such as the emission spectrum and excited state lifetime of a fluorophore. Spectra can be used to discriminate between fluorphores, and lifetime can be used to report on the microenvironment of flurophores. We describe a novel technique-combined spectral and lifetime imaging-which combines the benifits of multiphoton microscopy, spectral discrimination, and lifetime analysis and allows for the simultaneous collection of all three dimensions of data along with spatial and temporal information.

关键词： Animals Equipment Design Fluorescent Dyes/pharmacology Humans image processing, computer-assisted Microscopy, Confocal Microscopy, Fluorescence/methods Microscopy, Fluorescence, Multiphoton/methods Photons Photons

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The ATPase activity of the ChlI subunit of magnesium chelatase and formation of a heptameric AAA⁺ ring

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BIOCHEMISTRY 2003年第22期42卷 6912-6920页

作者： Reid, JD Siebert, CA Bullough, PA Hunter, CN Univ Sheffield Krebs Inst Biomolec Res Dept Mol Biol & Biotechnol Sheffield S10 2TN S Yorkshire England

The AAA(+) ATPase component of magnesium chelatase (ChlI) drives the insertion of Mg2+ into protoporphyrin IX;this is the first step in chlorophyll biosynthesis. We describe the ATPase activity, nucleotide binding kinetics, and structural organization of the ChII protein. A consistent reaction scheme arises from our detailed steady state description of the ATPase activity of the ChlI subunit and from transient kinetic analysis of nucleotide binding. We provide the first demonstration of metal ion binding to a specific subunit of any of the multimeric chelatases and characterize binding of Mg2+ to the free and MgATp(2-) bound forms of ChlI. Transient kinetic studies with the fluorescent substrate analogue TNP-ATP show that there are two forms of monomeric enzyme, which have distinct magnesium binding properties. Additionally, we describe the self-association properties of the subunit and provide a structural analysis of the multimeric ring formed by this enzyme in the presence of nucleotide. This single particle analysis demonstrates that this species has a 7-fold rotational symmetry, which is in marked contrast to most members of the AAA(+) family that tend to form hexamers.

关键词： Adenosine Triphosphatases/metabolism Adenosine Triphosphate/analogs & derivatives Adenosine Triphosphate/metabolism Chromatography, Gel Cryoelectron Microscopy/methods Hydrolysis image processing, computer-assisted Isomerism Kinetics Lyases/chemistry Lyases/metabolism Magnesium/chemistry Magnesium/metabolism Protein Structure, Tertiary Protein Subunits/metabolism Recombinant Proteins/chemistry Recombinant Proteins/metabolism

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A robust algorithm for the reconstruction of helical filaments using single-particle methods

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ULTRAMICROSCOPY 2000年第4期85卷 225-234页

作者： Egelman, EH Univ Virginia Hlth Sci Ctr Dept Biochem & Mol Genet Charlottesville VA 22908 USA

Some of the earliest methods for three-dimensional reconstruction from electron microscopic images were developed for helical objects. Single-particle methods have been used with great success for the three-dimensional reconstruction of macromolecular assemblies that have no internal symmetry or closed point group symmetries. An approach is presented for the application of single-particle methods to helical filaments that surmounts many of the difficulties of helical image analysis, including indexing, unbending and the need to find long helically symmetric filament segments. It is shown using both human Rad51 and E. coli RecA nucleoprotein filaments that this approach converges without user intervention to a stable solution, and that it has the potential to overcome many of the problems associated with image analysis of disordered helical polymers. The method can be applied transparently to structures where Bessel overlap would greatly complicate helical analysis. In addition, the procedure allows for the ab initio determination of helical symmetry, when no prior knowledge exists. (C) 2000 Elsevier Science B.V. All rights reserved.

关键词： Algorithms DNA, Single-Stranded/metabolism DNA, Single-Stranded/metabolism DNA-Binding Proteins/chemistry DNA-Binding Proteins/metabolism DNA-Binding Proteins/ultrastructure Escherichia coli/metabolism Humans image processing, computer-assisted Microscopy, Electron/methods Protein Conformation Rad51 Recombinase Rec A Recombinases/chemistry Rec A Recombinases/metabolism Rec A Recombinases/ultrastructure

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Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet

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SCIENCE 2002年第5585期297卷 1349-1352页

作者： Takahashi, N Kishimoto, T Nemoto, T Kadowaki, T Kasai, H Natl Inst Physiol Sci Dept Cell Physiol Okazaki Aichi 4448585 Japan Grad Univ Adv Studies Okazaki Aichi 4448585 Japan Univ Tokyo Grad Sch Med Dept Metab Dis Bunkyo Ku Tokyo 1138655 Japan

Insulin secretion from intact mouse pancreatic islets was investigated with two-photon excitation imaging. Insulin granule exocytosis occurred mainly toward the interstitial space, away from blood vessels. The fusion pore was unusually stable with a lifetime of 1.8 seconds. Opening of the 1.4-nanometer-diameter pore was preceded by unrestricted lateral diffusion of lipids along the inner wall of the pore, supporting the idea that this structure is composed of membrane lipids. When the pore dilated to 12 nanometers, the granules rapidly flattened and discharged their contents. Thus, our methodology reveals fusion pore dynamics in intact tissues at nanometer resolution.

关键词： Animals Cell Membrane/physiology Cell Membrane/ultrastructure Cell Polarity Cell Polarity Diffusion Exocytosis Extracellular Space Fluorescence Fluorescence Forskolin/pharmacology Glucose/pharmacology Guinea Pigs image processing, computer-assisted Insulin/secretion Insulin/secretion Intracellular Membranes/physiology Intracellular Membranes/ultrastructure Islets of Langerhans/blood supply Islets of Langerhans/physiology Islets of Langerhans/secretion Islets of Langerhans/ultrastructure Kinetics Membrane Fusion Membrane Lipids/physiology Mice Mice, Inbred ICR Permeability Pyridinium Compounds Quaternary Ammonium Compounds Quaternary Ammonium Compounds Rhodamines Secretory Vesicles/physiology Secretory Vesicles/ultrastructure

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Practical issues in imaging hydraulic conductivity through hydraulic tomography

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GROUND WATER 2008年第1期46卷 120-132页

作者： Illman, Walter A. Craig, Andrew J. Liu, Xiaoyi Univ Waterloo Dept Earth & Environm Sci Waterloo ON N2L 3G1 Canada Univ Iowa Iowa Inst Hydraul Res Hydrosci & Res Iowa City IA 52242 USA Univ Iowa Dept Civil & Environm Engn Dept Geosci Iowa City IA 52242 USA

Hydraulic tomography has been developed as an alternative to traditional geostatistical methods to delineate heterogeneity patterns in parameters such as hydraulic conductivity (K) and specific storage (S-s). During hydraulic tomography surveys, a large number of hydraulic head data are collected from a series of cross-hole tests in the subsurface. These head data are then used to interpret the spatial distribution of K and S-s using inverse modeling. Here, we use the Sequential Successive Linear Estimator (SSLE) of Yeh and Liu (2000) to interpret synthetic pumping test data created through numerical simulations and real data generated in a laboratory sandbox aquifer to obtain the K tomograms. Here, we define "K tomogram" as an image of K distribution of the subsurface (or the inverse results) obtained via hydraulic tomography. We examine the influence of signal-to-noise ratio and biases on results using inverse modeling of synthetic and real cross-hole pumping test data. To accomplish this, we first show that the pumping rate, which affects the signal-to-noise ratio, and the order of data included into the SSLE algorithm both have large impacts on the quality of the K tomograms. We then examine the role of conditioning on the K tomogram and find that conditioning can improve the quality of the K tomogram, but can also impair it, if the data are of poor quality and conditioning data have a larger support volume than the numerical grid used to conduct the inversion. Overall, these results show that the quality of the K tomogram depends on the design of pumping tests, their conduct, the order in which they are included in the inverse code, and the quality as well as the support volume of additional data that are used in its computation.

关键词： Algorithms image processing, computer-assisted Models, Theoretical Tomography Water Movements Water Movements Water Supply Water Supply

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PREPARATION OF FIGURES FOR PUBLICATION USING A DIGITAL COLOR PRINTER

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BIOTECHNIQUES 1993年第6期14卷 1006-1008页

作者： KNECHT, DA Department of Molecular and Cell Biology University of Connecticut Storrs 06269.

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Dynamic imaging of T cell-dendritic cell interactions in lymph nodes

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SCIENCE 2002年第5574期296卷 1873-1876页

作者： Stoll, S Delon, J Brotz, TM Germain, RN NIAID Lymphocyte Biol Sect Immunol Lab NIH Bethesda MD 20892 USA NCI Expt Immunol Branch NIH Bethesda MD 20892 USA

T cell immune responses begin within organized lymphoid tissues. The pace, topology, and outcomes of the cellular interactions that underlie these responses have, so far, been inferred from static imaging of sectioned tissue or from studies of cultured cells. Here we report dynamic visualization of antigen-specific T cells interacting with dendritic cells within intact explanted lymph nodes. We observed immunological synapse formation and prolonged interactions between these two coil types, followed by the activation, dissociation, and rapid migration of T cells away from the antigenic stimulus. This high-resolution spatiotemporal analysis provides insight into the nature of cell interactions critical to early immune responses within lymphoid structures.

关键词： Animals Antigen Presentation Antigens/immunology Antigens, CD Antigens, CD43 Cell Communication Cell Division Cell Movement Dendritic Cells/immunology Dendritic Cells/physiology Dendritic Cells/ultrastructure Fluorescent Dyes image processing, computer-assisted Intercellular Junctions/physiology Intercellular Junctions/ultrastructure Lymph Nodes/cytology Lymph Nodes/immunology Lymphocyte Activation Lymphocyte Activation Mice Mice, Transgenic Mice, Transgenic Microscopy, Confocal Recombinant Fusion Proteins/metabolism Sialoglycoproteins/analysis Sialoglycoproteins/genetics Sialoglycoproteins/metabolism T-Lymphocytes/immunology T-Lymphocytes/physiology T-Lymphocytes/ultrastructure

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Molecule-specific imaging with mass spectrometry and a buckminsterfullerene probe: Application to characterizing solid-phase synthesized combinatorial libraries

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2004年第12期126卷 3902-3909页

作者： Xu, JY Szakal, CW Martin, SE Peterson, BR Wucher, A Winograd, N Penn State Univ Dept Chem University Pk PA 16802 USA Univ Duisburg Gesamthsch Dept Phys D-45117 Essen Germany

We employ a newly developed buckminsterfullerene (C-60) primary ion beam with time-of-flight secondary ion mass spectrometry to create molecule-specific images of resin particles employed in the solid-phase synthesis of peptide combinatorial libraries. This new cluster ion source, when operated at an incident energy of 20 keV, is remarkably effective at desorbing small peptides directly from a polymer surface and opens new possibilities for characterizing large arrays of diverse sets of molecules. In addition, the C-60 ion beam may be focused to a spot of 1.5,mum in diameter, enabling molecule-specific images of single 100 mum resin particles to be acquired. We report three significant aspects associated with utilizing the C-60 projectile that show how this technology can be taken to a more advanced level, especially when compared to results obtained with more conventional atomic primary ions. First, the useful yield of molecular ions is generally observed to be enhanced by at least 3 orders of magnitude over those previously possible. Second, the energy dissipation process associated with the C-60 impact is most efficient at desorbing molecules on soft substrates such as polymer surfaces rather than harder substrates such as metals or semiconductors. Third, there is a greatly reduced tendency for insulating surfaces to build up excess charge, obviating the need for charge compensation. Using a small five-member peptide library as a model, we show that by utilizing the focusing properties of the C-60 beam, it is possible to assay the surface composition of 100-mum polymer beads at a rate of up to 10 particles/s. Moreover, even at the picomole level, there are enough sequence ions in the mass spectrum to determine a unique composition. The results illustrate the ability to quickly assay large libraries without the use of tags and suggest the strategy may be applicable to a range of high-throughput experiments.

关键词： Combinatorial Chemistry Techniques Combinatorial Chemistry Techniques image processing, computer-assisted Peptide Library Peptides/chemical synthesis Resins, Synthetic/chemistry Spectrometry, Mass, Fast Atom Bombardment Spectrometry, Mass, Secondary Ion

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