Wohlwend High Pressure Freezer for the BIDMC Electron Microscopy Core

 描述（由申请人提供）：本申请的主要和次要用户要求资金购买高压冷冻（HPF）机，以进行NIH资助项目的超微结构研究。该共享仪器的广泛、长期目标是提高电子显微镜（EM）的组织处理质量，特别是改善棕色脂肪组织（BAT）、骨骼肌、肠、肝和胃中线粒体、ER和其他细胞成分的超微结构。所要求的仪器是Wohlwend HPF，选择它是因为它的灵活性，易用性，出色的维修和服务记录，以及出色的性能。该机器将被放置在现有的EM设施中，该设施部分由Beth Israel Deaconess医疗中心提供支持，部分由NIH P30消化系统疾病中心资助。主要用户组包括5名获得NIH赠款者，1名拥有活跃生物医学研究计划的次要用户，以及专门用于外部用户的时间。每个用户的个人研究目标是不同的，但每个人都需要改善组织超微结构。Arany博士研究了不同代谢病理生理学模型中骨骼肌线粒体的变化。Cohen、固特异和Tseng博士研究了BAT和/或肝脏中线粒体变化的病理意义，这些变化与肥胖、运动或糖尿病中的能量代谢有关。Blumberg博士研究了肠道Paneth细胞在炎症性肠病发病机制中的作用，特别是炎症诱导的ER应激激活和未折叠蛋白反应。最后，Ha-gen博士研究了紧密连接异常在胃癌发病机制中的致病作用.在该领域的专家的帮助下收集的初步数据清楚地表明，与传统的处理技术相比，HPF之后的超快速冷冻替代方法导致组织形态的显着改善。每个项目的健康相关目标的总体益处将是HPF/SQFS限制细胞收缩、线粒体肿胀和破坏，以及线粒体基质、ER腔、细胞核和细胞质中重要蛋白质的提取。由于所要求的HPF机器能够冷冻更厚和更大的组织块，用户组能够制备许多厚塑料切片用于分析，然后制备许多薄切片用于EM分析，这些切片处于正确的方向并且没有冷冻损伤。与传统处理相比，使用HPF/SQFS的组织超微结构差异如此显著，以至于用户组不愿意继续研究，因为如果没有该机器的好处，就无法修复显著的伪影。在行政上，所要求的HPF将由EM核心总监和研究助理管理，他们将对仪器进行培训，维护和监督。将有一个当地咨询委员会来处理冲突和新用户的申请。最后，这一工具得到了强有力的机构支持，并有适当的预算、空间和财政承诺，以确保成功。

 DESCRIPTION (provided by applicant): The Major and Minor Users for this application request funds to purchase a High Pressure Freezing (HPF) machine to carry-out ultrastructural studies for NIH-funded projects. The broad, long-term objective of this shared instrument is to improve the quality of tissue processing for electron microscopy (EM), in particular to improve the ultrastructure of mitochondria, ER, and other cellular components in brown adipose tissue (BAT), skeletal muscle, intestine, liver, and stomach. The requested instrument is the Wohlwend HPF, which was chosen for its flexibility, ease of use, excellent repair and service record, and outstanding performance. The machine will be placed into an existing EM facility, which is partially supported by the Beth Israel Deaconess Medical Center and partially supported by an active NIH P30 Center Grant for digestive diseases. The Major User group consists of 5 investigators with NIH-funded grants, one Minor User, who has an active biomedical research pro- gram, and dedicated time for outside users. The individual research goal for each user is different, but each requires improved tissue ultrastructure. Dr. Arany studies changes in skeletal muscle mitochondria in different models of metabolic pathophysiology. Drs. Cohen, Goodyear, and Tseng study the pathological significance of mitochondrial changes in BAT and/or liver, which relate to energy metabolism in obesity, exercise, or diabetes. Dr. Blumberg studies the role of intestinal Paneth cells in the pathogenesis of Inflammatory Bowel Disease, in particular the inflammation-induced activation of ER stress and the unfolded protein response. Lastly, Dr. Ha- gen studies the pathogenic role of tight junction abnormalities in stomach cancer pathogenesis. Preliminary data, collected with the assistance of an expert in the field who was also recruited as a consultant for this application, clearly demonstrate that HPF followed by super-quick freeze substitution methods resulted in significantly improved tissue morphology compared to conventional processing techniques. The overall benefit to the health-related goals of each project will be that HPF/SQFS limited cell shrinkage, mitochondrial swelling and disruption, and the extraction of important proteins in the mitochondrial matrix, lumen of the ER, nuclei, and cytoplasm. With the capability of the requested HPF machine to freeze thicker and larger tissue pieces, the user groups were able to prepare numerous thick plastic sections for analysis followed by numerous thin sections for EM analysis that were in the proper orientation and free of freezing damage. The differences in tissue ultrastructure using HPF/SQFS compared to conventional processing were so striking that the User group is reluctant to continue their studies with significant artifacts that cannot be fixed without the benefit of this machine. Administratively, the requested HPF will be managed by the EM Core Director and Research Associate, who will do the training, upkeep, and oversight of the instrument. There will be a local advisory committee to handle conflicts and new user applications. Lastly, there is strong institutional support for this instrumentation, with an appropriate budget, space, and financial commitment to ensure success.