Cryo Upgrade of UMB EM Core Facility

描述(由申请人提供)：我们申请资金用于购买冷冻-EM样品制备设备，包括高压冷冻机(HPF)、自动冷冻替代系统、冷冻-超显微切割机、立式冷冻装置和现有的透射电子显微镜Tecnai T12(FEI)的低温升级。这些仪器将被集成到一个新重组的电子显微镜(EM)核心设施中，该核心设施是马里兰大学巴尔的摩大学(UMB)、马里兰大学生物技术研究所(UMBI)、马里兰大学巴尔的摩县大学(UMBC)和其他邻近研究机构研究人员的唯一核心设施。低温样品制备技术具有更好的抗原保存性和更高的超微结构空间分辨率。低温电子显微镜的升级，结合了低温样品架和低剂量成像技术，将使研究人员能够以高分辨率观察冷冻的水合生物样本，这是UMB和其他周边研究所的研究人员所没有的选项。本申请中描述的许多主要用户研究项目(Bavoil、Nataro、Donnenberg、Russell和Bloch博士)将使用继HPF、冷冻替代和低温树脂包埋之后的免疫金技术，以增强抗原检测和超微结构保存。主要用户Donnenberg博士和Bavoil博士将使用插入式冷冻设备在玻璃冰中制备纯化的蛋白质复合体或微生物，然后在低温电子显微镜中进行高分辨率的超微结构分析，而不使用重金属染色。此外，与HPF结合使用的快速转移系统允许通过光学和电子显微镜对同一标本进行相关研究。这一功能补充了新收购的蔡司Duo激光扫描共聚焦显微镜(2008年通过SIG 1S10RR02454801购买，Thomas Blanped，研究员，该应用的主要用户)的活细胞成像能力，以及越来越多的研究项目在UMB/Umbi园区研究活细胞。主要用户布兰皮德博士和马丁博士计划利用这一功能来加强他们的活细胞成像研究。这笔赠款基于资助的项目，在这些项目中，低温样品制备和低温EM技术比传统的EM方法在动物细胞、整个组织和微生物方面提供了实质性的改进。这一研究界正在进行的其他项目也将立即受益于新兴市场核心设施的扩展能力。

DESCRIPTION (provided by applicant): We request funds toward the purchase of cryo-EM sample preparation equipment, including a high pressure freezer (HPF), an automatic freeze substitution system, a cryo-ultramicrotome, a plunge freezing device and the cryo upgrade of an existing transmission electron microscope (TEM) Tecnai T12 (FEI). These instruments will be integrated into a newly re-organized electron microscopy (EM) core facility, the only core facility for researchers at the University of Maryland Baltimore (UMB), University of Maryland Biotechnology Institute (UMBI), University of Maryland Baltimore County (UMBC) and other neighboring research institutes. It is well established that cryo sample preparation techniques offers better antigen preservation and enhanced spatial resolution of ultrastructures. The cryo TEM upgrade, incorporating a cryo sample holder and low dose imaging technologies will allow researchers to observe frozen hydrated biological samples at high resolution, an option that has not been available to researchers at UMB and other surrounding institutes. Many of the major user research projects (Drs. Bavoil, Nataro, Donnenberg, Russell and Bloch) described in this application will utilize immunogold techniques following HPF, freeze substitution and resin embedding at low temperature to enhance antigen detection and ultrastructural preservation. Major users Drs. Donnenberg and Bavoil will use the plunge freeze device to prepare purified protein complexes or microorganisms in vitreous ice and subsequently perform high resolution ultrastructure analysis in the cryo TEM without the use of heavy metal staining. Furthermore, the rapid transfer system used in combination with the HPF allows correlative studies of the same specimen by both light and electron microscopy. This feature complements the capability of live cell imaging of the newly acquired Zeiss Duo laser scanning confocal microscope (purchased in 2008 via SIG 1S10RR02454801, Thomas Blanpied, investigator, major user on this application) and the growing number of research projects studying live cells on the UMB/UMBI campus. Major users Drs. Blanpied and Martin plan to use this feature to enhance their live cell-imaging research. This grant is based upon funded projects for which the cryo sample preparation and cryo EM techniques offer substantial improvements over conventional EM methods for animal cells, whole tissues and microorganisms. Other ongoing projects in this research community will also immediately benefit from the expanded capability of the EM core facility.