Computer-aided profiling of a unique broad-specific antibody and its application to an ultrasensitive fluoroimmunoassay for five N-methyl carbamate pesticides

Structurally-oriented biologists examine cells, tissues, organelles and macromolecules in order to gain insight into cellular and molecular physiology by relating structure to function. The understanding of these structures can be greatly enhanced by the use of techniques for the visualization and quantitative analysis of three-dimensional structure. Three projects from current research activities will be presented in order to illustrate both the present capabilities of computer aided techniques as well as their limitations and future possibilities.The first project concerns the three-dimensional reconstruction of the neuritic plaques found in the brains of patients with Alzheimer's disease. We have developed a software package “Synu” for investigation of 3D data sets which has been used in conjunction with laser confocal light microscopy to study the structure of the neuritic plaque. Tissue sections of autopsy samples from patients with Alzheimer's disease were double-labeled for tau, a cytoskeletal marker for abnormal neurites, and synaptophysin, a marker of presynaptic terminals.

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Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163903 ◽

1996 ◽

Vol 54 ◽

pp. 288-289

Author(s):

Greg V. Martin ◽

Ann L. Hubbard

Keyword(s):

Three Dimensional ◽

Integral Membrane Proteins ◽

Polarized Secretion ◽

Microscope Images ◽

Computer Aided ◽

Intracellular Organelles ◽

Cytoskeletal Elements ◽

Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

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A Sensitive On-Grid Immunoelectron Microscopic Procedure for Diagnosis of Rotavirus Infection

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s143192760000221x ◽

1980 ◽

Vol 38 ◽

pp. 506-507

Author(s):

M. F. Miller ◽

A. R. Rubenstein

Keyword(s):

Electron Microscopy ◽

Specific Antibody ◽

Immune Complexes ◽

Acute Gastroenteritis ◽

Plant Viruses ◽

Aggregation Process ◽

Microscopic Technique ◽

Fecal Material ◽

Present Communication ◽

Number Of Particles

Studies of rotavirus particles in humans, monkeys and various non-primates with acute gastroenteritis have involved detection of virus in fecal material by electron microscopy. The EM techniques most commonly employed have been the conventional negative staining (Fig. 1) and immune aggregation (Fig. 2) procedures. Both methods are somewhat insensitive and can most reliably be applied to samples containing large quantities of virus either naturaLly or as a result of concentration by ultracentrifugation. The formation of immune complexes by specific antibody in the immune aggregation procedures confirms the rotavirus diagnosis, but the number of particles per given microscope field is effectively reduced by the aggregation process. In the present communication, we describe use of an on-grid immunoelectron microscopic technique in which rotavirus particles are mounted onto microscope grids that were pre-coated with specific antibody. The technique is a modification of a method originalLy introduced by Derrick (1) for studies of plant viruses.

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