This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: The Albany HVEM is the last in the Americas, and one of only two in the world, that is capable of 1000 keV operation and is dedicated to biological studies (the other is in Okazaki, Japan). The HVEM is used primarily for collaborative and service (CS) projects involving the study of the 3-D structure of biological specimens prepared as whole cell mounts or thick plastic sections. Specimens as thick as 5mm can be used when selective stains are employed. Thick specimens can be studied by analysis of stereo-pair images or by electron tomography. The penetrating power of HVEM is ideal for this work because at 60o tilt during a tomographic tilt series, the effective section thickness is twice the 0o thickness, and it is three times thicker at 70o. The instrumental resolution of the HVEM is about 0.5 nm. The HVEM is equipped with a number of specialized specimen stages suitable for tomography, as well as an energy-dispersive x-ray microanalysis system. An in-house designed intensified video-rate CCD TV system can be used to observe the image with very low electron irradiation, for focusing and following the image during tomographic data collection. There are two important benefits of the HVEM for electron tomography of thick sections that are often not appreciated. First, the HVEM has a large focal length and so a large depth of focus. Thus, stained plastic sections as thick as 5000 nm can be tilted to 60 degrees without noticeable blurring at the edges of the image, while some blurring is seen in IVEM images of tilted, 250-nm-thick stained plastic sections. Second, the electron dose with thick stained plastic sections is typically three times lower with the HVEM than with the IVEM, since the latter needs a very small objective aperture to achieve adequate depth of focus for tilted thick sections. Previous reporting period: Last year, the filament batteries were replaced, an operation last done 15 years ago. This required removal of the dome of the accelerator tank and construction of a rack for new-style batteries. The HVEM continues to operate well. + Michael Marko gave an invited talk entitled """"""""Biological HVEM at Albany, NY: A brief history"""""""" at the International Workshop on Collaborative Research with High-Voltage Electron Microscopy, Tokyo, Japan, November 10, 2008. Special HVEM application The tracing of neural circuitry in thick, selectively stained sections has long been a classical use of HVEM, and after many years we again have a well-respected neuroscientist enthusiastically using our HVEM. Dr. Eduardo Rosa-Molinar of the University of Puerto Rico-Rio Piedras, San Juan is studying an alternative mechanism (i.e. gap junctions) of integrating adult-born motor neurons into existing neural circuits (see Scientific Subproject Service Project S-11). HVEM tomography of selectively retrogradely labeled motor neurons cell bodies, fibers, and terminals as well as interneuron cell bodies, fibers, and terminals is being used to visualize, reconstruct three-dimensionally, and model dendrodendritic gap junctions as well as filamentous contacts. Dr. Rosa-Molinar visits the RVBC and collects the data personally, and is learning how to make reconstructions and visualize the data. HVEM measurements will be combined with systematic sampling and serial electron microscopic reconstruction of samples from the dendrites, in order to yield accurate surface area estimates.
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