Computer-Assisted Ontology Construction System: Focus on Bootstrapping Capabilities

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

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Computer Assisted Image Reconstruction of Oligomer Structure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100092323 ◽

1976 ◽

Vol 34 ◽

pp. 472-473

Author(s):

A.M. Jones ◽

A. Max Fiskin

Keyword(s):

Three Dimensional ◽

Depth Of Field ◽

Computer Assisted ◽

Projection Data ◽

Two Dimensional ◽

Single Particles ◽

Dimensional Reconstruction ◽

Computer Assisted Image ◽

The Fourier Transform ◽

Space Approach

If the tilt of a specimen can be varied either by the strategy of observing identical particles orientated randomly or by use of a eucentric goniometer stage, three dimensional reconstruction procedures are available (l). If the specimens, such as small protein aggregates, lack periodicity, direct space methods compete favorably in ease of implementation with reconstruction by the Fourier (transform) space approach (2). Regardless of method, reconstruction is possible because useful specimen thicknesses are always much less than the depth of field in an electron microscope. Thus electron images record the amount of stain in columns of the object normal to the recording plates. For single particles, practical considerations dictate that the specimen be tilted precisely about a single axis. In so doing a reconstructed image is achieved serially from two-dimensional sections which in turn are generated by a series of back-to-front lines of projection data.

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Biodegradable polyester neuroprostheses promote the reconnection of separated peripheral nerve stubs

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100130109 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1094-1095

Author(s):

Beverly L. Giammara ◽

Jennifer S. Stevenson ◽

Peggy E. Yates ◽

Robert H. Gunderson ◽

Jacob S. Hanker

Keyword(s):

Image Analysis ◽

Basement Membrane ◽

Periodic Acid ◽

Light And Electron Microscopy ◽

Computer Assisted ◽

Type Iii Collagen ◽

Image Analysis System ◽

Biodegradable Polyester ◽

Adult Rats ◽

Type Iv

An 11mm length of sciatic nerve was removed from 10 anesthetized adult rats and replaced by a biodegradable polyester Vicryl™ mesh sleeve which was then injected with the basement membrane gel, Matrigel™. It was noted that leg sensation and movement were much improved after 30 to 45 days and upon sacrifice nerve reconnection was noted in all animals. Epoxy sections of the repaired nerves were compared with those of the excised segments by the use of a variation of the PAS reaction, the PATS reaction, developed in our laboratories for light and electron microscopy. This microwave-accelerated technique employs periodic acid, thiocarbohydrazide and silver methenamine. It stains basement membrane or Type IV collagen brown and type III collagen (reticulin), axons, Schwann cells, endoneurium and perineurium black. Epoxy sections of repaired and excised nerves were also compared by toluidine blue (tb) staining. Comparison of the sections of control and repaired nerves was done by computer-assisted microscopic image analysis using an Olympus CUE-2 Image Analysis System.

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High-resolution measurement of birefringent fine structure in living cells using a new polarized light microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136647 ◽

1995 ◽

Vol 53 ◽

pp. 54-55

Author(s):

Rudolf Oldenbourg

Keyword(s):

Light Microscope ◽

Fluorescent Dyes ◽

Polarized Light ◽

Processing System ◽

Video Camera ◽

Molecular Organization ◽

Computer Assisted ◽

Image Recording ◽

Birefringent Crystal ◽

Technological Advances

The recent renaissance of the light microsope is fueled in part by technological advances in components on the periphery of the microscope, such as the laser as illumination source, electronic image recording (video), computer assisted image analysis and the biochemistry of fluorescent dyes for labeling specimens. After great progress in these peripheral parts, it seems timely to examine the optics itself and ask how progress in the periphery facilitates the use of new optical components and of new optical designs inside the microscope. Some results of this fruitful reflection are presented in this symposium.We have considered the polarized light microscope, and developed a design that replaces the traditional compensator, typically a birefringent crystal plate, with a precision universal compensator made of two liquid crystal variable retarders. A video camera and digital image processing system provide fast measurements of specimen anisotropy (retardance magnitude and azimuth) at ALL POINTS of the image forming the field of view. The images document fine structural and molecular organization within a thin optical section of the specimen.

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Computer-Assisted Analysis of Multi-Color Confocal Microscopic Datasets: Automated Light Microscopic Discrimination of Synaptic Relationships

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163885 ◽

1996 ◽

Vol 54 ◽

pp. 284-285

Author(s):

M Wessendorf ◽

A Beuning ◽

D Cameron ◽

J Williams ◽

C Knox

Keyword(s):

Nerve Fibers ◽

Confocal Scanning Laser Microscopy ◽

Computer Assisted ◽

Nerve Terminals ◽

Neuronal Somata ◽

Scanning Laser Microscopy ◽

Confocal Scanning ◽

Entire Cell ◽

Confocal Scanning Laser ◽

Assisted Analysis

Multi-color confocal scanning-laser microscopy (CSLM) allows examination of the relationships between neuronal somata and the nerve fibers surrounding them at sub-micron resolution in x,y, and z. Given these properties, it should be possible to use multi-color CSLM to identify relationships that might be synapses and eliminate those that are clearly too distant to be synapses. In previous studies of this type, pairs of images (e.g., red and green images for tissue stained with rhodamine and fluorescein) have been merged and examined for nerve terminals that appose a stained cell (see, for instance, Mason et al.). The above method suffers from two disadvantages, though. First, although it is possible to recognize appositions in which the varicosity abuts the cell in the x or y axes, it is more difficult to recognize them if the apposition is oriented at all in the z-axis—e.g., if the varicosity lies above or below the neuron rather than next to it. Second, using this method to identify potential appositions over an entire cell is time-consuming and tedious.

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Serial thin sectioning for pre- and post-embedding immunohistochemistry: Achieving consistent and reliable results for a 3-D reconstruction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014748x ◽

1993 ◽

Vol 51 ◽

pp. 328-329

Author(s):

Antonia M. Milroy

Keyword(s):

Nervous System ◽

Computer Assisted ◽

Serial Sectioning ◽

Electron Microscopic ◽

High Quality ◽

New Techniques ◽

3 Dimensional ◽

Multiple User ◽

Thin Sectioning ◽

Subcellular Level

In recent years many new techniques and instruments for 3-Dimensional visualization of electron microscopic images have become available. Higher accelerating voltage through thicker sections, photographed at a tilt for stereo viewing, or the use of confocal microscopy, help to analyze biological material without the necessity of serial sectioning. However, when determining the presence of neurotransmitter receptors or biochemical substances present within the nervous system, the need for good serial sectioning (Fig. 1+2) remains. The advent of computer assisted reconstruction and the possibility of feeding information from the specimen viewing chamber directly into a computer via a camera mounted on the electron microscope column, facilitates serial analysis. Detailed information observed at the subcellular level is more precise and extensive and the complexities of interactions within the nervous system can be further elucidated.We emphasize that serial ultra thin sectioning can be performed routinely and consistently in multiple user electron microscopy laboratories. Initial tissue fixation and embedding must be of high quality.

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New polarized-light microscope for fast and orientation independent measurement of birefringent fine structure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146254 ◽

1993 ◽

Vol 51 ◽

pp. 82-83

Author(s):

Rudolf Oldenbourg

Keyword(s):

Light Microscope ◽

Polarized Light ◽

Video Camera ◽

Limited Range ◽

Image Point ◽

Computer Assisted ◽

Optical Modulators ◽

Anisotropic Structures ◽

Long Time ◽

Computer Assisted Image

The polarized light microscope has the unique potential to measure submicroscopic molecular arrangements dynamically and non-destructively in living cells and other specimens. With the traditional pol-scope, however, single images display only those anisotropic structures that have a limited range of orientations with respect to the polarization axes of the microscope. Furthermore, rapid measurements are restricted to a single image point or single area that exhibits uniform birefringence or other form of optical anisotropy, while measurements comparing several image points take an inordinately long time.We are developing a new kind of polarized light microscope which combines speed and high resolution in its measurement of the specimen anisotropy, irrespective of its orientation. The design of the new pol-scope is based on the traditional polarized light microscope with two essential modifications: circular polarizers replace linear polarizers and two electro-optical modulators replace the traditional compensator. A video camera and computer assisted image analysis provide measurements of specimen anisotropy in rapid succession for all points of the image comprising the field of view.

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