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.

项目摘要： 主要调查人员（PIS）正在寻求行政补充以获得共识 其研究项目的冷冻功能光/电子显微镜（冷冻-CLEM）“ RNA渗透到 细菌”（R01GM141659）。该研究旨在了解相互作用的分子机制 在单链RNA细菌（SSRNA噬菌体）及其宿主细菌之间，重点是PP7 和AP205噬菌体，分别感染了铜绿假单胞菌和杆菌属。 PIS希望 使用此信息来开发新方法来抵抗多药耐药细菌感染 减轻这些病原体的病毒。 该团队计划调查原位噬菌体包装过程，噬菌体吸附途径和 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的冷冻EM 噬菌体PP7和AP205的结构单独或与宿主接收器（IV型pili）的结构，并且具有 表征了与噬菌体感染后pili脱离有关的分子事件。 PIS请求共核冷冻-CLEM，这将增强其成像功能并受益 他们的项目以两种主要方式。首先，低分辨率可以提供高分辨率成像和实时 观察能力，弥合了传统光学显微镜与冷冻EM/ET之间的差距。其次， 团队的目标是在假单胞菌生物膜和共聚焦荧光显微镜下测试他们的发现 对于理解细菌细胞和感染过程中噬菌体的空间组织至关重要。 博士。 PIS Zhang和Zeng与Nikon和Linkam合作创建了一个Cryo-Clem设置 为他们的项目量身定制。该乐器将位于张博士的新装修的实验室中，该实验室就在 Zeng博士在生物化学和生物物理学大楼的实验室。 Zeng博士，拥有超过15年的时间 具有荧光光学显微镜的经验，将监督共焦的操作和维护 与张博士协调的冷冻-Clem。该工具不仅将支持PI的当前研究 项目还具有长期价值，可在得克萨斯州A＆M社区的未来研究中具有长期价值。