1a. Objectives (from AD-416):
The long-term objectives of this project involve the application of electron and confocal microscopy for the observation of a wide range of sample materials impacting agricultural productivity, the goals of which include the protection of plants, animals and humans from various pathogens and parasites. The Beltsville Agricultural Research Center (BARC) Electron and Confocal Microscopy Unit (ECMU) serves the research projects at BARC that require electron and confocal microscopy data necessary to achieve their specific research objectives. The ECMU will use standard protocols as well as develop new technologies and methodologies as needed to meet the needs of its clientele. Over the next 5 years we will focus on the following objectives: 1) Develop and apply new techniques and methodologies in electron and confocal microscopy that facilitate the identification, characterization, and systematics of plant pathogens and other pests, and their interaction with their hosts; and 2) Provide technical support and expertise specific to individual research projects with BARC scientists and their collaborators for achieving previously unobtainable data and improvement of the quality of imaging results.
1b. Approach (from AD-416):
The Electron and Confocal Microscopy Unit (ECMU) is a core facility which provides collaborative assistance for the Beltsville Agricultural Research Center scientists in need of high resolution imaging in their research programs. The facility is equipped with state-of-the-art electron microscopes [transmission (TEM) and scanning (SEM)] and a confocal laser scanning microscope (CLSM). A scanning electron microscope equipped with a cryostage allows samples to be studied in the frozen state for the observation of ultra-delicate external structures of organisms and the study of behavioral interactions on their hosts. Variable pressure SEM requires minimal specimen preparation for rapid observation of specimens. The ECMU also possesses a high-resolution transmission electron microscope that is used to obtain detailed internal ultra-structural information from very thin sections of plastic embedded material. The CLSM permits the study of fluorescent organelles, tissue, proteins, cells with high color contrast and in 3 dimensions. Cellular and subcellular structures are easily observed and photographed using these approaches to provide compositional information as well. Techniques including critical point drying, freeze drying, freeze substitution, freeze etching, rapid freezing, negative staining, thin sectioning, sputter coating, high vacuum evaporation, immunogold labeling, single and multiplex fluorescent labeling are performed on a wide variety of sample types that arise from research projects at BARC. If existing methods are inadequate for appropriately viewing a particular specimen, new techniques, preparative methodologies, fluorochromes, specimen holders and equipment are designed and developed. Training will be provided to staff of collaborating scientists to assist in creating and enhancing images. The final result is dramatic, high-resolution, digitally-achievable images of many of the most important organisms affecting agriculture.
3. Progress Report:
Scientists at the Beltsville Agricultural Research Center and their collaborators are working on numerous and varied research projects and many of them have an occasional or frequent need to visualize samples using microscopy techniques provided by the Electron and Confocal Microscopy Unit (ECMU). There were ten different projects for which the ECMU provided support which included: characterization of viruses attacking bees; studies of animal parasites (Giardia and yeast tachyozoites); investigative studies of insects (mosquitoes and Hemlock Woolly Adelgid); detailed morphological examination of three genera of mites affecting citrus, oranges, lemons, and grapefruits; localization of gene insertions in tomato and soybeans; identification of plant tissues and organelles which store heavy metals for decontamination of soils; and food safety issues including: bacterial contamination of spinach and cabbage and cyptosporidium on the surface of peach, tomato, and apples.
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