Electron tomography and immunogold labeling of plant cells

Studies of the biology of Pneumocystis carinii (PC) are of increasing importance because this extracellular pathogen is a frequent source of severe pneumonia in patients with acquired immunodeficiency syndrome (AIDS) and is a leading cause of mortality in these patients. Immunoelectron microscopic localization of antigenic sites on the surface of PC would improve the understanding of these sites and their role in pathenogenisis of the disease and response to chemotherapy. The purpose of this study was to develop a methodology for visualizing immunoreactive sites on PC with transmission electron microscopy (TEM) using immunogold labeled probes.Trophozoites of PC were added to spinner flask cultures and allowed to grow for 7 days, then aliquots of tissue culture fluid were centrifuged at 12,000 RPM for 30 sec. Pellets of organisims were fixed in either 1% glutaraldehyde, 0.1% glutaraldehyde-4% paraformaldehyde, or 4% paraformaldehyde for 4h. All fixatives were buffered with 0.1M Na cacodylate and the pH adjusted to 7.1. After fixation the pellets were rinsed in 0.1M Na cacodylate (3X), dehydrated with ethanol, and immersed in a 1:1 mixture of 95% ethanol and LR White resin.

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Automated electron tomography: from data collection to image processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136507 ◽

1995 ◽

Vol 53 ◽

pp. 26-27

Author(s):

Weiping Liu ◽

Jennifer Fung ◽

W.J. de Ruijter ◽

Hans Chen ◽

John W. Sedat ◽

...

Keyword(s):

Image Processing ◽

Data Collection ◽

Structural Information ◽

Imaging Technique ◽

Electron Tomography ◽

Ccd Camera ◽

Automated Data Collection ◽

Full Control ◽

Transmission Electron ◽

Amorphous Structures

Electron tomography is a technique where many projections of an object are collected from the transmission electron microscope (TEM), and are then used to reconstruct the object in its entirety, allowing internal structure to be viewed. As vital as is the 3-D structural information and with no other 3-D imaging technique to compete in its resolution range, electron tomography of amorphous structures has been exercised only sporadically over the last ten years. Its general lack of popularity can be attributed to the tediousness of the entire process starting from the data collection, image processing for reconstruction, and extending to the 3-D image analysis. We have been investing effort to automate all aspects of electron tomography. Our systems of data collection and tomographic image processing will be briefly described.To date, we have developed a second generation automated data collection system based on an SGI workstation (Fig. 1) (The previous version used a micro VAX). The computer takes full control of the microscope operations with its graphical menu driven environment. This is made possible by the direct digital recording of images using the CCD camera.

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SEM study of the morphological effects of kinetin and zeatin on the growth and development of the apical meristem in wheat

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141263 ◽

1995 ◽

Vol 53 ◽

pp. 978-979

Author(s):

G. M. Hutchins ◽

J. S. Gardner

Keyword(s):

Growth And Development ◽

Furfuryl Alcohol ◽

Apical Meristem ◽

Plant Cells ◽

Triticum Aestivum L ◽

Morphological Development ◽

Naturally Occurring ◽

Chinese Spring Wheat ◽

Sem Study ◽

Moist Environment

Cytokinins are plant hormones that play a large and incompletely understood role in the life-cycle of plants. The goal of this study was to determine what roles cytokinins play in the morphological development of wheat. To achieve any real success in altering the development and growth of wheat, the cytokinins must be applied directly to the apical meristem, or spike of the plant. It is in this region that the plant cells are actively undergoing mitosis. Kinetin and Zeatin were the two cytokinins chosen for this experiment. Kinetin is an artificial hormone that was originally extracted from old or heated DNA. Kinetin is easily made from the reaction of adenine and furfuryl alcohol. Zeatin is a naturally occurring hormone found in corn, wheat, and many other plants.Chinese Spring Wheat (Triticum aestivum L.) was used for this experiment. Prior to planting, the seeds were germinated in a moist environment for 72 hours.

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