Aquisition of a confocal cryo-light microscope for correlative light and electron microscopy

项目摘要： 主要研究者（PI）正在寻求行政补充，以获得共聚焦 冷冻相关光/电子显微镜（cryo-CLEM），用于他们的研究项目，“RNA渗透到 细菌”（R 01 GM 141659）。该研究旨在了解相互作用的分子机制 单链RNA噬菌体（ssRNA噬菌体）与其宿主细菌之间的关系，重点是PP 7 和感染铜绿假单胞菌和不动杆菌属的AP 205菌株，分别私家侦探希望 利用这些信息开发新的方法来对抗多重耐药细菌感染， 减轻这些病原体的毒性。 该团队计划研究原位噬菌体包装过程，噬菌体吸附途径， 使用多尺度成像工具，包括荧光显微镜， 冷冻电子显微镜/断层摄影术（cryo-EM/ET）。他们已经获得了高分辨率的冷冻电镜 噬菌体PP 7和AP 205单独或与宿主受体IV型皮利复合的结构， 表征了与噬菌体感染后的皮利脱离相关的分子事件。 PI要求共焦冷冻CLEM，这将增强其成像能力并受益 他们的项目主要有两个方面。首先，低温CLEM可以提供高分辨率成像和实时成像。 观察能力，弥合了传统光学显微镜和冷冻EM/ET之间的差距。二是 研究小组的目标是在假单胞菌生物膜和共聚焦荧光显微镜的背景下测试他们的发现 对于理解感染过程中细菌细胞和细菌的空间组织至关重要。 张博士和曾博士，PI，与尼康和Linkam合作，创建了一个冷冻CLEM设置 为他们的项目量身定制。该仪器将位于张博士新装修的实验室，就在 博士生物化学与生物物理系大楼内，曾老师的实验室。曾博士，谁拥有超过15年的 具有荧光显微镜经验，将监督共聚焦显微镜的操作和维护 与张博士合作进行冷冻CLEM。该工具不仅将支持PI当前的研究， 项目，但也有长期的价值，为未来的研究在得克萨斯州A&M社区。

Project Summary: The Principle Investigators (PIs) are seeking an administrative supplement to obtain a confocal cryo-correlative light/electron microscope (cryo-CLEM) for their research project, "RNA Penetrations into Bacteria" (R01GM141659). The study aims to understand the molecular mechanisms underlying the interaction between single-stranded RNA bacteriophages (ssRNA phages) and their host bacteria, with a focus on PP7 and AP205 phages, which infect Pseudomonas aeruginosa and Acinetobacter spp., respectively. The PIs hope to use this information to develop new approaches to combat multi-drug-resistant bacterial infections by mitigating the virulence of these pathogens. The team plans to investigate the in situ phage packaging process, the phage adsorption pathway, and the RNA entry/pili detachment events using multi-scale imaging tools, including fluorescence microscopy and cryo-electron microscopy/tomography (cryo-EM/ET). They have already obtained high-resolution cryo-EM structures of both phage PP7 and AP205 alone or in complex with the host receptors, the type IV pili, and have characterized molecular events associated with pili detachment upon phage infection. The PIs request a confocal cryo-CLEM, which would enhance their imaging capabilities and benefit their project in two main ways. Firstly, a cryo-CLEM can provide both high-resolution imaging and real-time observation capabilities, bridging the gap between traditional light microscopy and cryo-EM/ET. Secondly, the team aims to test their findings in the context of Pseudomonas biofilms, and confocal fluorescence microscopy is essential for understanding the spatial organization of bacterial cells and phages during infection. Drs. Zhang and Zeng, the PIs, have collaborated with Nikon and Linkam to create a cryo-CLEM setup tailored to their project. The instrument will be located in Dr. Zhang's newly renovated lab, which is right next to Dr. Zeng's lab in the Department of Biochemistry and Biophysics building. Dr. Zeng, who has over 15 years of experience with fluorescence light microscopy, will supervise the operation and maintenance of the confocal cryo-CLEM in coordination with Dr. Zhang. This instrument will not only support the PIs’ current research project but also have long-term value for future research in the Texas A&M community.