Purchase of a FEI Titan Krios for 3-D EM

描述（由申请人提供）：佛罗里达州立大学（FSU）的生物科学成像资源（BSIR）是佛罗里达州和东南部冷冻电子显微镜和3-D电子显微镜的焦点。没有其他大学的设施有类似的重点或设施。本申请申请资金用于更换和升级该设施的中心部件，即FEI CM 300-FEG电子显微镜，并配备Titan-Krios，这是一种具有增强的冷冻电镜和电子断层扫描功能的机器人电子显微镜。我们的CM 300-FEG显微镜已经有15年的历史了，在停机时间和我们在该领域实施其他人生产的仪器和软件开发的能力方面都显示出它的年龄。该仪器缺少一个成像滤光片，这对于现在涉及厚标本的工作至关重要，并且对于电子显微镜的新发展（如相位板和像差校正器）的改造将变得越来越成问题。请求的Titan-Krios将支持主要BSIR用户的研究。Kenneth Taylor， Kenneth Roux和Thomas Roberts，新教员Scott Stagg博士和来自长期合作伙伴Michael Chapman博士实验室的访客，他最近搬到了俄勒冈健康与科学大学。本文描述了三个将受益于现代电子显微镜的小项目。Titan-Krios还将能够支持远程用户，使其能够方便地向大学以外的用户提供广泛的功能，并成为东南地区真正的研究资源。利用电子断层扫描和单粒子方法研究薄玻璃体冷冻水化悬浮液是FSU一个庞大且日益增长的冷冻电镜研究重点，但获得足够的数据，无论是作为倾斜序列还是投影，目前都是许多项目的瓶颈。Titan-Krios的机器人能力以及运行最先进的自动化EM软件（如Leginon）的能力将缓解这一瓶颈。电子断层扫描是三个申请小组的主要重点，而其他两个小组正在开始探索其可能性。Titan-Krios的双轴断层扫描能力将提高冷冻水合、快速冷冻、冷冻替代、塑料嵌入生物标本重建的通量和质量。Titan-Krios将支持NIH资助的以下研究：(1)肌源性肌肉产生力的基本机制，这与心肌功能的健康相关；(2)平滑肌肌球蛋白调节，与高血压健康相关；(3)与癌症健康相关的细胞迁移相关结构；(4)艾滋病毒和SIV病毒的结构，这是了解艾滋病的基础；(5)腺相关病毒作为基因治疗载体的开发；(6)美国国立卫生研究院（NIH）对COPII囊泡结构的相关研究，这些囊泡与乳糜微粒滞留病和颅透镜缝发育不全等货物运输缺陷有关。

DESCRIPTION (provided by applicant): The Biological Science Imaging Resource (BSIR) at Florida State University (FSU) is the focal point for cryoelectron microscopy and 3-D electron microscopy in the state of Florida and the Southeast. No other University facility has a comparable emphasis or facilities. This application requests funds to replace and upgrade the center piece of this facility, an FEI CM 300-FEG electron microscope with a Titan-Krios, a robotic electron microscope with enhanced capabilities for cryoEM and electron tomography. Our CM 300-FEG microscope is 15 years old and is showing its age both in terms of down time and our ability to implement instrumentation and software developments produced by others in the field. The instrument lacks an imaging filter which is crucial for work that now involves thick specimens, and will become increasing problematic for retrofit of new developments in electron microscopy such as phase plates and aberration correctors. The requested Titan-Krios will support the research of the major BSIR users, Drs. Kenneth Taylor, Kenneth Roux and Thomas Roberts, new faculty member Dr. Scott Stagg and visitors from the lab of a long standing collaborator, Dr. Michael Chapman, who recently moved to Oregon Health & Science University. Three minor projects are described that will benefit from access to a modern electron microscope. The Titan-Krios will also be able to support remote users, making it possible to offer its extensive capabilities conveniently to users outside of the university and become a true research resource for the Southeast. The study of thin, vitreous, frozen hydrated suspensions, both using electron tomography and single particle methods is a large and growing cryoEM research emphasis at FSU, but obtaining sufficient data, either as tilt series or projections is currently a bottleneck in a number of projects. The robotic capabilities of the Titan-Krios as well as the ability to run state of the art automated EM software, such as Leginon, will alleviate this bottleneck. Electron tomography is a major emphasis of three applicant groups while the other two are beginning to explore the possibilities. The dual axis tomography capabilities of the Titan-Krios will improve both throughput and quality of reconstructions obtained from both frozen hydrated as well as rapidly frozen, freeze substituted, plastic embedded biological specimens. The Titan-Krios will support NIH funded research in (1) the fundamental mechanism of force generation in a myogenic muscle, which has health relevence to cardiac muscle function; (2) smooth muscle myosin regulation, which has health relevance in hypertension; (3) structures involved in cell migration, which has health relevence in cancer; (4) the structure of HIV and SIV viruses which are fundamental to understanding AIDS; (5) the development of Adeno Associated Virus as a gene therapy vector and (6) NIH related research into the structure of COPII vesicles, which are implicated in defects of cargo trafficking including chylomicron retention disease and cranio-lenticulo-sutural displasia.