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Quantitative estimation of F-actin in single growth cones

methods-A COMPANION TO methods IN ENZYMOLOGY 1999年第4期18卷 487-492页

作者： Schindelholz, B Reber, BFX Univ Bern Dept Pharmacol CH-3010 Bern Switzerland

Growth cones at distal ends of elongating neurites are characterized by a bunch of motile filopodia. Filamentous actin (F-actin) is the supporting cytoskeletal structure of growth cone filopodia. Normal growth cone motility requires balanced polymerization and depolymerization rates of F-actin. If this balance is disturbed, growth cone shape is altered and extension may fail. image acquisition by confocal scanning microscopy was used as a very efficient tool to optically isolate single growth cones from the rest of the cell to study morphological and physiological behavior. The relative F-actin content (r.a.c.) of a single growth cone area was defined as a parameter describing different growth cone states. To estimate r.a.c., a double-labeling technique was applied. F-actin was selectively labeled by fluorescent rhodamine-conjugated phalloidin and total protein was unspecifically labeled by 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF). The r.a.c. was calculated by rationing and averaging digitized rhodamine and DTAF fluorescence of single growth cone areas. Subsequently, r.a.c, was used as a numeric descriptor of the variable F-actin underlying morphological structures of growth cones. The method allowed an analysis of local changes in growth cone morphology measured as a change in F-actin due to signaling events. It can be used to quantify ligand-receptor effects at subcellular areas of intact cells, (C) 1999 Academic Press.

关键词： Actins/analysis Animals Bradykinin/pharmacology Cytochalasin D/pharmacology Cytoskeleton/chemistry Cytoskeleton/drug effects Fluorescent Antibody Technique Fluorescent Antibody Technique Growth Cones/chemistry Growth Cones/drug effects image processing, computer-assisted/methods Kinetics Microscopy, Confocal/methods Nucleic Acid Synthesis Inhibitors/pharmacology PC12 Cells Rats Spermine/pharmacology

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Three-dimensional imaging by deconvolution microscopy

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methods-A COMPANION TO methods IN ENZYMOLOGY 1999年第3期19卷 373-385页

作者： McNally, JG Karpova, T Cooper, J Conchello, JA NCI Lab Receptor Biol & Gene Express NIH Bethesda MD 20892 USA Washington Univ Inst Biomed Comp St Louis MO 63110 USA Washington Univ Dept Cell Biol St Louis MO 63110 USA

Deconvolution is a computational method used to reduce out-of-focus fluorescence in three-dimensional (3D) microscope images. It can be applied in principle to any type of microscope image but has most often been used to improve images from conventional fluorescence microscopes. Compared to other forms of 3D light microscopy, like confocal microscopy, the advantage of deconvolution microscopy is that it can be accomplished at very low light levels, thus enabling multiple focal-plane imaging of light-sensitive living specimens over long time periods. Here we discuss the principles of deconvolution microscopy, describe different computational approaches for deconvolution, and discuss interpretation of deconvolved images with a particular emphasis on:what artifacts may arise. (C) 1999 Academic Press.

关键词： Artifacts image processing, computer-assisted/methods Microscopy, Fluorescence/instrumentation Microscopy, Fluorescence/methods Microscopy, Fluorescence/standards

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Scanning microphotolysis: Three-dimensional diffusion measurement and optical single-transporter recording

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methods-A COMPANION TO methods IN ENZYMOLOGY 1999年第4期18卷 508-517页

作者： Peters, R Kubitscheck, U Univ Munster Inst Med Phys & Biophys D-48149 Munster Germany

Scanning microphotolysis (SCAMP) is a combination of fluorescence microphotolysis and confocal laser scanning microscopy. A laser scanning microscope is equipped with an optical switch able to modulate the power or/and wavelength of the laser beam in less than a microsecond while a dedicated computer program is employed to precisely coordinate scanning process and laser beam modulation. By these means it becomes possible to vary the power or/and wavelength of the laser beam during scanning at a precision of one resolution element. Patterns of almost arbitrary design can be written into the object by photolysis, e.g., photobleaching or photoactivation. The dissipation of the photolysis pattern by diffusion or other types of molecular transport can be followed at confocal resolution and used to characterize the transport process. SCAMP can be employed in conjunction with single-photon or multiphoton excitation. Furthermore, it can be easily installed on virtually any confocal laser scanning microscope. We summarize at first the conceptual and practical basis of SCAMP. Then, two novel applications are discussed: (i) measurements of translational diffusion coefficients in truly three-dimensional systems at diffraction-limited resolution, and (ii) optical recording of single transporters in membrane patches. (C) 1999 Academic Press.

关键词： Diffusion image processing, computer-assisted/instrumentation image processing, computer-assisted/methods Microscopy, Confocal/instrumentation Microscopy, Confocal/methods Microscopy, Fluorescence/instrumentation Microscopy, Fluorescence/methods Neurosciences/instrumentation Optics and Photonics Patch-Clamp Techniques/instrumentation Patch-Clamp Techniques/instrumentation Photolysis

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Stereo-4-D reconstruction and animation from living fluorescent specimens

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BIOTECHNIQUES 1998年第6期24卷 1006-+页

作者： Mohler, WA White, JG Univ Wisconsin Mol Biol Lab Madison WI 53706 USA

We present a novel approach to the viewing and analysis of 4-dimensional (4-D) data sets recorded from live fluorescent samples. With stereo-4-D reconstructions, the observer manipulates a rotatable projection of the full 3-dimensional (3-D) specimen while simultaneously controlling animation of the recording forward or backward in time. The result is a unique lifelike perspective on the development of an entire living subject. Here, we apply this technique to the observation of the cell membranes of developing Caenorhabditis elegans. Embryos labeled with the vital plasma membrane probe FM(TM) 4-64 were imaged by multiphoton laser scanning fluorescence microscopy, yielding 4-D data sets of entire embryos over several hours of development. Stereo 4-D and standard focal-plane 4-D viewing of these novel time-lapse recordings provide the observer with detail at both the subcellular and whole-animal level from a single data set and produce a unique record of the lineage, cell shape changes, cell contacts and morphogenetic dynamics that make up embryogenesis. The procedures by which stereo-4-D reconstructions are created and viewed rely on public domain software running on a personal computer and should therefore be accessible by a general audience. Data output utilizes the versatile and well-supported QuickTime(TM) animation format. Additional features allow for stereo-4-D reconstruction of isolated 3-D volumes of interest from within the larger specimen.

关键词： Animals Caenorhabditis elegans/embryology Database Management Systems Databases, Factual image processing, computer-assisted/instrumentation image processing, computer-assisted/methods Microscopy, Fluorescence/methods Microscopy, Video/instrumentation Microscopy, Video/methods Software Video Recording/methods

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Evaluation of intraocular foreign bodies by spiral computed tomography and multiplanar reconstruction

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OPHTHALMOLOGY 1998年第2期105卷 307-312页

作者： Lakits, A Steiner, E Scholda, C Kontrus, M Univ Vienna Dept Ophthalmol A-1090 Vienna Austria Univ Vienna Dept Radiol A-1090 Vienna Austria

Objective: This study aimed to evaluate the ability of spiral computed tomography (CT) and multiplanar reconstruction: in the assessment of intraocular foreign bodies. Design: The study design was experimental. Material: Three foreign bodies (metal, glass, and plastic) implanted into the globe of a cadaveric head were studied, Intervention: Spiral CT in the axial plane was used. Beam collimation and table increment were both 3 mm, whereby overlapping axial slices were reconstructed at 1-mm intervals, The data were transferred to a workstation, and reconstructions in the coronal and sagittal plane were performed. Main Outcome Measures: The ability to detect and evaluate localization, shape, and size of the foreign bodies in all three imaging planes was assessed. Results: All foreign bodies were detected in the axial and in both reconstructed planes, The metal foreign body caused imaging artifacts in the axial plane. In the reconstructed planes, imaging artifacts were less severe, and evaluation of localization and shape was improved. Glass and plastic foreign bodies were well seen in all planes, and shape and localization were well demonstrated, Appropriate window settings improved determination of size of the different foreign bodies. Conclusions: Spiral CT of the orbit and multiplanar reconstruction offers a promising tool for evaluation of intraocular foreign bodies. Advantages of spiral CT compared to conventional CT are data acquisition in only one axial plane;shortened examination time;reduced radiation exposure;reduced imaging artifacts from metallic foreign bodies in the reconstructed planes;and demonstration of localization, shape, and size of the foreign body in three imaging planes.

关键词： Eye Foreign Bodies/radiography Eye Foreign Bodies/radiography Glass Humans image processing, computer-assisted/methods Metals Models, Anatomic Orbit/radiography Plastics Tomography, X-Ray Computed/methods

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Digital image processing measurement of the upper eyelid contour in Graves disease and congenital blepharoptosis

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OPHTHALMOLOGY 1998年第5期105卷 913-918页

作者： Cruz, AAV Coelho, RP Baccega, A Lucchezi, MC Souza, ADA Ruiz, EES Univ Sao Paulo Sch Med Ribeirao Preto Dept Ophthalmol Sao Paulo Brazil Univ Sao Paulo Sch Med Ribeirao Preto Dept Phys & Math Sao Paulo Brazil

Objective: This study used image processing techniques to quantify the upper eyelid contour of patients with Graves upper eyelid retraction and congenital blepharoptosis. Design: The study design was a cross-sectional study. Participants: A total of 29 patients with Graves disease, 22 patients with congenital blepharoptosis, and 50 patients with no history of eye disease participated. Intervention: The images of the palpebral fissure of all participants were transferred to a personal computer and processed with NIH image 1.55 software. Main Outcome Measures: The following parameters were analyzed: the curvature of the upper eyelid contour, the position of the contour peak relative to the midline, and the ratio between the temporal and nasal upper quadrant areas of the palpebral fissure. Results: All upper eyelid contours could be fitted with second-degree polynomial functions. The mean temporal/ nasal area ratio was 1.33 mm in patients with Graves disease, 0.92 mm in patients with blepharoptosis, and 1.04 mm in control subjects. The peak of the upper eyelid contour was found to be lateral to the midline in control subjects (1.05 mm) and in patients with Graves disease (2.09 mm). In patients with blepharoptosis, the peak was 0.69 mm medial to the midline. Overall, the distance between the midpupil and the upper eyelid margin was correlated with several factors: the degree of curvature, the position of the peak of the eyelid contour, and the temporal/nasal area ratio. Conclusions: In Graves eyelid retraction, the curvature of the upper eyelid is enhanced, the peak of the contour is displaced laterally, and the temporal upper quadrant area is increased. Conversely, in congenital blepharoptosis, the eyelid is almost flat, the peak of the contour is displaced medially, and the upper quadrant area is diminished. The lateral segment of the upper eyelid is more involved than the nasal segment in both Graves upper eyelid retraction and congenital blepharoptosis.

关键词： Adolescent Adult Aged Blepharoptosis/complications Blepharoptosis/congenital Child Child, Preschool Cross-Sectional Studies Eyelids/pathology Female Graves Disease/complications Humans image processing, computer-assisted/methods Male Middle Aged Signal processing, computer-assisted

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Correcting data shifts in gel files created by model 377 DNA sequencers

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BIOTECHNIQUES 1998年第6期24卷 1002-1003页

作者： O'Brien, KM Ironside, MA Athanasiou, MC Basit, MA Evans, GA Garner, HR Univ Texas SW Med Ctr Eugene McDermott Ctr Human Growth & Dev Dallas TX 75235 USA

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Reconstructing fragmented gel files created by model 377 DNA sequencers

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BIOTECHNIQUES 1998年第6期24卷 1004-1005页

作者： O'Brien, KM Schageman, JJ Evans, GA Garner, HR Univ Texas SW Med Ctr Eugene McDermott Ctr Human Growth & Dev Dallas TX 75235 USA

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Emulation of an ELISA reader and quantitative image analysis using the computer program Digital-OD

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BIOTECHNIQUES 1998年第6期24卷 998-1001页

作者： Jansen, S Kriegesmann, B Brenig, B Univ Gottingen Tierarztl Inst Mol Biol Abt D-37073 Gottingen Germany

Here, we present a Microsoft(R) Windows(R) application that has been written to emulate a microplate reader. After scanning a standard 96-well microplate, color gradations in the slots can be analyzed quantitatively and be used for the estimation of enzyme kinetics, binding assays or concentration determination. In a test, series accuracy of the program was determined We show that the returned optical densities correlated highly with the true concentrations (r >0.97). Thus, the software is useful for a wide number of applications. Further the program is easy to handle and is available free of charge.

关键词： Enzyme-Linked Immunosorbent Assay/instrumentation Enzyme-Linked Immunosorbent Assay/instrumentation Enzyme-Linked Immunosorbent Assay/methods Enzyme-Linked Immunosorbent Assay/methods image processing, computer-assisted/instrumentation image processing, computer-assisted/methods Reproducibility of Results Signal processing, computer-assisted Software

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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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