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Wet electron microscopy with quantum dots

BIOTECHNIQUES 2006年第3期41卷 295-298页

作者： Timp, Winston Watson, Nicki Sabban, Alon Zik, Ory Matsudaira, Paul MIT Whitehead Inst Cambridge MA 02139 USA Quantomix Corp Ness Ziona Israel

Wet electron microscopy (EM) is a new imaging method with the potential to allow higher spatial resolution of samples. In contrast to most EM methods, it requires little time to perform and does not require complicated equipment or difficult steps. We used this method on a common murine macrophage cell line, IC-21, in combination with various stains and preparations, to collect high resolution images of the actin cytoskeleton. Most importantly, we demonstrated the use of quantum dots in conjunction with this technique to perform light/electron correlation microscopy. We found that wet EM is a useful tool that fits into a niche between the simplicity of light microscopy and the high spatial resolution of EM.

关键词： Actins/metabolism Animals Cell Line Cytoskeleton/metabolism Fluorescent Dyes/pharmacology image processing, computer-assisted Macrophages/metabolism Mice Microscopy, Electron/instrumentation Microscopy, Electron/methods Quantum Dots Quantum Dots Scattering, Radiation Time Factors

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Optimized filter set and viewing conditions for the S65T mutant of GFP in living cells

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BIOTECHNIQUES 1996年第2期21卷 220-&页

作者： Zylka, MJ Schnapp, BJ Harvard Medical School Boston MA USA.

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SIZE OF THE EXPORT CHANNEL IN THE FLAGELLAR FILAMENT OF SALMONELLA-TYPHIMURIUM

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ULTRAMICROSCOPY 1993年第1-4期49卷 417-425页

作者： RUIZ, T FRANCIS, NR MORGAN, DG DEROSIER, DJ Rosenstiel Basic Medical Sciences Research Center Brandeis University Waltham MA 02254 USA

The size of the putative export channel in the bacterial flagellar filament appears small (25 angstrom) in studies done by electron microscopy but large (60 angstrom) in studies done by X-ray diffraction. We have undertaken additional studies by electron microscopy to examine some of the possible causes of the difference. A comparison of three-dimensional image reconstructions of native and reconstituted filaments rules out the presence or absence of flagellin monomers in the export channel as the source of the variation in apparent channel size. The channel seen in reconstructions from both kinds of filaments is 25 angstrom in diameter. The difference in the previous studies is more probably a result of artifacts introduced in either the X-ray or the electron microscopical methodology, Comparisons of three-dimensional reconstructions from images of filaments embedded in various stains (anionic, cationic and neutral) and in ice, taken at a range of defocuses, rule out the two most likely sources of artifact in electron microscopy (i.e., staining artifacts and defocus phase contrast). Based on these studies we suggest that the channel seen in the image reconstructions is free of exported flagellin monomers, that its true diameter is about 25 angstrom, and, therefore, that the flagellin monomer must be unfolded to pass along it.

关键词： Flagella/ultrastructure image processing, computer-assisted Microscopy, Electron Salmonella typhimurium/ultrastructure Tissue Embedding/methods Tissue Embedding/methods

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MEASUREMENT OF LOW CALCIUM CONCENTRATIONS IN CRYOSECTIONED CELLS BY PARALLEL-EELS MAPPING

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ULTRAMICROSCOPY 1993年第1-4期49卷 225-234页

作者： LEAPMAN, RD HUNT, JA BUCHANAN, RA ANDREWS, SB NINCDS LAB BETHESDA MD 20892 USA NINCDS NEUROBIOL LAB BETHESDA MD 20892 USA LEHIGH UNIV DEPT MAT SCI & ENGN BETHLEHEM PA 18015 USA

Electron energy loss spectroscopy (EELS) in the scanning transmission electron microscope provides a high sensitivity for microanalysis of certain important biological elements such as calcium whose physiological concentrations in cells are rather low. Application of parallel-EELS mapping to the analysis of freeze-dried cryosections of rapidly frozen tissue provides a means of detecting small amounts of calcium in structures with diameter approximately 50 nm. Detector pattern noise due to channel gain variations can be reduced by acquiring difference spectra at each pixel. By segmenting nitrogen maps that reflect the structure through the protein distribution it is possible to sum spectra from specific compartments. These are then processed by fitting reference spectra for the Ca L23-edge and the carbon background. It has been found that useful data can be collected at 100 keV beam energy from freeze-dried cryosections of cerebellar cortex cut to nominal thickness of 100 nm. The analysis results in a sensitivity of +/- 0.4 mmol Ca/kg dry weight with a total acquisition time of 400 s, a significant improvement over that achievable with energy-dispersive X-ray spectroscopy.

关键词： Animals Calcium/analysis Cerebellar Cortex/chemistry Cerebellar Cortex/cytology Cryopreservation Cryopreservation image processing, computer-assisted Mice Microscopy, Electron, Scanning Transmission/methods Spectrum Analysis/methods

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A MARKER-FREE ALIGNMENT METHOD FOR ELECTRON TOMOGRAPHY

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ULTRAMICROSCOPY 1995年第3-4期58卷 393-402页

作者： LIU, Y PENCZEK, PA MCEWEN, BF FRANK, J NEW YORK STATE DEPT HLTH WADSWORTH CTR LABS & RESALBANYNY 12201 SUNY ALBANY DEPT PHYSALBANYNY 12222 SUNY ALBANY DEPT BIOMED SCIALBANYNY

In electron tomography of biological specimens, fiducial markers are normally used to achieve accurate alignment of the input projections. We address the problem of alignment of projections from objects that are freely supported and do not permit the use of markers. To this end we present a new alignment algorithm for single-axis tilt geometry based on the principle of Fourier-space common lines. An iterative scheme has been developed to overcome the noise-sensitivity of the common-line method. This algorithm was used to align a data set that was not amenable to alignment with fiducial markers.

关键词： Algorithms image processing, computer-assisted Microscopy, Electron/methods

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Nanografting versus Solution Self-Assembly of α,ω-Alkanedithiols on Au(111) Investigated by AFM

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LANGMUIR 2008年第20期24卷 11661-11668页

作者： Yu, Jing-Hang Ngunjiri, Johnpeter N. Kelley, Algemon T. Gano, Jayne C. Louisiana State Univ Dept Chem Baton Rouge LA 70803 USA Ctr Biomodular Multi Scale Syst Baton Rouge LA 70803 USA Agilent Technol Nanotechnol Measurements Div Chandler AZ 85226 USA

The solution self-assembly of alpha,omega-alkanedithiols onto Au(111) was investigated using atomic force microscopy (AFM). A heterogeneous surface morphology is apparent for 1,8-octanedithiol and for 1,9-nonanedithiol self-assembled monolayers (SAMs) prepared by solution immersion as compared to methyl-terminated n-alkanethiols. Local views from AFM images reveal a layer of mixed molecular orientations for alpha,omega-alkanedithiols, which evidence surface structures with heights corresponding to both lying-down and standing-up orientations. For dithiol SAMs prepared by solution self-assembly, the majority of alpha,omega-alkanedithiol molecules chemisorb with both thiol end groups bound to the Au(111) surface with the backbone of the alkane chain aligned parallel to the surface. However, AFM images disclose that there are also islands of standing molecules scattered throughout the surface. To measure the thickness of alpha,omega-alkanedithiol SAMs with angstrom sensitivity, methyl-terminated n-alkanethiols with known dimensions were used as molecular rulers. Under conditions of spatially constrained self-assembly, nanopattems of alpha,omega-alkanedithiols written by narografting formed monolayers with heights corresponding to an upright configuration.

关键词： Adsorption Chemistry, Physical/methods image processing, computer-assisted Materials Testing Metal Nanoparticles/chemistry Microscopy, Atomic Force/methods Nanoparticles/chemistry Nanotechnology/methods Solutions/chemistry Sulfhydryl Compounds/chemistry Surface Properties

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COVERING EVENTS IN EIGENimageS OF BIOMOLECULES

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ULTRAMICROSCOPY 1991年第3-4期38卷 305-317页

作者： HARAUZ, G CHIU, DKY UNIV GUELPH DEPT COMP & INFORMAT SCI GUELPH N1G 2W1 ONTARIO CANADA

Multivariate statistical analysis of a large set of micrographs of biological macromolecules involves the computation of eigenimages representing principal features, on the basis of which similar views of the complexes can be grouped. It is not generally clear what these eigenimages represent physically and which ones should be used in the classification process. In this paper, hierarchical maximum entropy discretisation and event covering are used to (1) detect statistically significant relationships in the eigenimages, (2) select the most relevant eigenimages for classifying biomolecular projections, and (3) build a prototype of the biomolecular complex under study.

关键词： Algorithms Cluster Analysis computer Simulation DNA, Superhelical/ultrastructure DNA, Superhelical/ultrastructure image processing, computer-assisted Models, Biological Multivariate Analysis Software

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Liquid crystal behavior of single-walled carbon nanotubes dispersed in biological hyaluronic acid solutions

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2007年第30期129卷 9452-9457页

作者： Moulton, Simon E. Maugey, Maryse Poulin, Philippe Wallace, Gordon G. Univ Wollongong ARC Ctr Excellence Electromat Sci Intelligent Polymer Res Inst Wollongong NSW 2500 Australia Univ Bordeaux 1 CNRS UPR Ctr Rech Paul Pascal F-8641 Bordeaux France

We report the spontaneous liquid crystal phase separation of nanotubes (single-walled carbon nanotubes, SWNTs) stabilized in aqueous biological (hyaluronic acid, HA) solutions. Sonication of SWNTs in solutions of HA produced well-dispersed single-phase isotropic dispersions which, over time, phase separated into dispersions containing birefringent nematic domains in equilibrium with an isotropic phase. The time required for phase separation to occur was shown to depend on the concentration of SWNT and HA, with the attractive interactions between the SWNT and HA shifting the onset of the phase separation toward lower concentration. This phase separation is accompanied by an increase in the dispersion viscosity with this increase qualitatively matching the degree of phase separation. The formation of ordered phases in biological media can offer wide opportunities for processing conducting biomaterials with aligned and oriented domains.

关键词： Hyaluronic Acid/chemistry image processing, computer-assisted Liquid Crystals/chemistry Microscopy Nanotechnology Nanotubes, Carbon/chemistry Phase Transition Solubility Solutions/chemistry Time Factors Viscosity Water/chemistry

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EELS ELEMENTAL MAPPING WITH UNCONVENTIONAL METHODS .2. APPLICATIONS TO BIOLOGICAL SPECIMENS

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ULTRAMICROSCOPY 1990年第3期34卷 179-203页

作者： TREBBIA, P MORY, C LAB PHYS SOLIDE F-91405 ORSAY FRANCE

This article presents two applications of image analysis and processing using the unconventional methods described in the companion paper (part I). Both the information analysis via relative entropy measurement and mapping and the factorial analysis of correspondence are demonstrated to be valuable tools for building an elemental map from a set of noisy energy-filtered images recorded in an analytical transmission electron microscope. Although the only technique considered here is electron energy loss spectroscopy, there is no doubt that such methods can be applied to a wide variety of similar problems;only a reduced number of underlying hypotheses are needed.

关键词： Algorithms Animals Freeze Drying Freeze Drying Growth Plate/chemistry Growth Plate/ultrastructure image processing, computer-assisted Mathematics Microscopy, Electron/methods Mitochondria/chemistry Mitochondria/ultrastructure Phosphorus/analysis Rats Uranium/analysis

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Atomic force microscopy for characterization of the biomaterial interface

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BIOMATERIALS 1998年第4-5期19卷 441-454页

作者： Siedlecki, CA Marchant, RE Case Western Reserve Univ Dept Biomed Engn Cleveland OH 44106 USA Case Western Reserve Univ Dept Macromol Sci Cleveland OH 44106 USA

The molecular processes that occur at the interface of an implanted biomaterial determines the host response, including phenomena such as protein adsorption, conformational changes and subsequent interactions with cellular components. Until recently, such processes could not be observed directly. Over the past decade, atomic force microscopy (AFM) has provided mechanistic insights into the molecular level interactions that occur at the biomaterial interface. Several unique operational modes have been developed which utilize intermittent contact with the sample and decrease applied shear forces. These dynamic modes also can be used to study the role of different structural components on biomaterial micromechanical properties. Force detection techniques allow molecular level studies of individual receptor-ligand binding events, and force mapping for determining structure/function relationships. Advancements in tip manufacturing, image processing techniques, the use of model surfaces and labeling all have contributed to the advancement of the AFM as a state-of-the-art research instrument. In this report, we examine the applicability of the AFM to the study of biomaterials and cell/molecular interactions. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.

关键词： Adsorption Biocompatible Materials/chemistry computer Simulation image processing, computer-assisted Microscopy, Atomic Force/instrumentation Microscopy, Atomic Force/methods Models, Chemical Protein Conformation Proteins/chemistry Surface Properties

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