HVEM OPERATION AND IMPROVEMENTS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 摘要： 奥尔巴尼HVEM是美洲最后一个，也是世界上仅有的两个HVEM之一，能够进行1000 keV的操作，并致力于生物研究（另一个在日本冈崎）。 HVEM主要用于协作和服务（CS）项目，涉及作为全细胞支架或厚塑料切片制备的生物标本的3-D结构研究。 当采用选择性染色时，可以使用厚达5 mm的标本。 厚标本可以通过立体对图像分析或电子断层扫描进行研究。 HVEM的穿透能力非常适合这项工作，因为在层析倾斜系列期间，在60 o倾斜时，有效截面厚度是0 o厚度的两倍，在70 o时是3倍。 高压电子显微镜的仪器分辨率约为0.5nm。 HVEM配备了许多适用于断层扫描的专用样品台以及能量色散X射线微量分析系统。 内部设计的增强视频速率CCD电视系统可用于观察具有非常低的电子辐射的图像，用于在断层扫描数据收集期间聚焦和跟踪图像。 HVEM用于厚切片的电子断层扫描有两个重要的优点，但通常不被重视。 首先，HVEM具有大的焦距，因此具有大的焦深。 因此，5000 nm厚的染色塑料切片可以倾斜60度，而图像边缘不会出现明显的模糊，而在倾斜的250 nm厚染色塑料切片的IVEM图像中可以看到一些模糊。 其次，HVEM的厚染色塑料切片的电子剂量通常比IVEM低三倍，因为IVEM需要非常小的物镜孔径来实现倾斜厚切片的足够焦深。 上一报告期： 去年，灯丝电池被更换，这是15年前的最后一次操作。 这需要拆除加速箱的圆顶，并建造一个新型电池架。 HVEM继续运作良好。 + Michael Marko于2008年11月10日在日本东京举行的高压电子显微镜合作研究国际研讨会上发表了题为“纽约奥尔巴尼生物HVEM：简史”的特邀演讲。 特殊HVEM应用 在厚的、选择性染色的切片中追踪神经回路一直是HVEM的经典用途，多年后，我们又有一位备受尊敬的神经科学家热情地使用我们的HVEM。 圣胡安的波多黎各-里奥皮德拉斯大学的Eduardo Rosa-Molinar博士正在研究将成人出生的运动神经元整合到现有神经回路中的替代机制（即间隙连接）（见科学子项目服务项目S-11）。 HVEM断层扫描选择性逆行标记的运动神经元细胞体，纤维，和终端，以及interneuron细胞体，纤维，和终端正在被用来可视化，重建三维，和模型树突状细胞间隙连接以及丝状接触。 Rosa-Molinar博士访问RVBC并亲自收集数据，并正在学习如何进行重建和可视化数据。 HVEM测量将与系统采样和树突样品的连续电子显微镜重建相结合，以获得准确的表面积估计值。

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.