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

获取用于关联光学和电子显微镜的共焦冷冻光学显微镜

• 批准号: 10796557
• 负责人: Lanying Zeng
• 金额: $ 25万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10796557
• 关键词: Acinetobacter; Administrative Supplement; Adsorption; Bacteria; Bacterial Infections; Bacteriophages; Biochemistry; Biophysics; Cells; Collaborations; Communities; Complement; Complex; Cryoelectron Microscopy; Electron Microscope; Electron Microscopy; Event; Fluorescence Microscopy; Goals; Imaging Device; In Situ; Infection; Light; Light Microscope; Maintenance; Microbial Biofilms; Molecular; Multi-Drug Resistance; Pathway interactions; Penetration; Pilum; Process; Pseudomonas; Pseudomonas aeruginosa; RNA; RNA Phages; Research; Research Personnel; Research Project Grants; Resolution; Structure; Testing; Texas; Three-Dimensional Imaging; Time; Virulence; Visualization; combat; experience; high resolution imaging; imaging capabilities; instrument; light microscopy; novel strategies; operation; pathogen; receptor; tomography

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.

项目概要： 主要研究者 (PI) 正在寻求行政补充以获得共聚焦 他们的研究项目“RNA 渗透到 细菌”（R01GM141659）。该研究旨在了解相互作用背后的分子机制 单链 RNA 噬菌体（ssRNA 噬菌体）与其宿主细菌之间的研究，重点关注 PP7 和 AP205 噬菌体，分别感染铜绿假单胞菌和不动杆菌属。 PI 希望 利用这些信息开发新方法来对抗多重耐药细菌感染 减轻这些病原体的毒力。 该团队计划研究噬菌体原位包装过程、噬菌体吸附途径以及 使用多尺度成像工具（包括荧光显微镜和 冷冻电子显微镜/断层扫描 (cryo-EM/ET)。他们已经获得了高分辨率冷冻电镜 噬菌体 PP7 和 AP205 单独或与宿主受体 IV 型菌毛复合的结构，并且具有 表征了与噬菌体感染后菌毛脱离相关的分子事件。 PI 需要共焦冷冻 CLEM，这将增强他们的成像能力并带来好处 他们的项目主要有两种方式。首先，cryo-CLEM 可以提供高分辨率成像和实时 观察能力，弥补了传统光学显微镜和冷冻电镜/电子断层扫描之间的差距。其次， 研究小组的目标是在假单胞菌生物膜和共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噬菌体的空间组织至关重要。 博士。 PI 张和曾与 Nikon 和 Linkam 合作创建了冷冻 CLEM 设置 根据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，紧邻 曾博士的实验室位于生物化学与生物物理系大楼。曾博士拥有超过15年的从业经验 具有荧光光学显微镜经验，将监督共焦的操作和维护 与张博士合作进行的冷冻 CLEM。该工具不仅支持 PI 当前的研究 项目，而且对德克萨斯 A&M 社区的未来研究也具有长期价值。