Development of a novel workflow for the efficient volume imaging of thick tissue samples with correlative light and electron microscopy

开发一种新颖的工作流程，通过相关光学和电子显微镜对厚组织样本进行有效的体积成像

• 批准号: ST/Y000595/1
• 负责人: Chang Guo
• 金额: $ 4.6万
• 依托单位: UK Health Security Agency
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=ST%2FY000595%2F1
• 关键词: Development; novel; workflow; efficient; volume

ConnectomX proposes to join an existing relationship between the UK Health Security Agency (UKHSA) and the Central Laser Facility (CLF) to collaboratively develop a novel correlative light and electron microscopy (CLEM) workflow. This workflow will be applied to a UKHSA project imaging brain tissue for the presence of environmental pollutant particles. Together, the workflow will be tested, and proof of concept data will be collected to prove the utility of the Katana ultramicrotome as a universal solution to volume CLEM of tissue samples. The ConnectomX Katana ultramicrotome will be installed onto an existing scanning electron microscope (SEM) at the CLF. This will allow versatile switching between serial block face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM). ConnectomX will produce software and hardware solutions as part of an ongoing and iterative research and development collaboration to improve and build on the CLEM workflow pioneered in this proposal.More specifically, the proposed workflow is to image entire brain tissue sections using multiphoton microscopy (MPM) at low resolution to identify smaller regions of interest (ROIs) impacted by nanoparticle infiltration. These ROIs will be identified related to blood brain barrier (BBB) permeability and will later be relocated in the SEM for high-resolution imaging. The ConnectomX Katana ultramicrotome can be mounted within the chamber of the SEM at the CLF to perform serial SBF-SEM on selected brain tissue sections containing ROIs identified in MPM. SBF-SEM utilises a diamond knife to cut away a thin layer of the sample, after which the surface is imaged. This process is repeated thousands of times to build up 3D visualisations of the structure of a sample. In this proposal, the Katana ultramicrotome will be used to acquire a low-resolution volume dataset within each brain tissue sample. Blood vessels or branding marks (made using MPM) in these low-resolution datasets will be used to relocate smaller ROIs with BBB permeability. Incorporation of the Katana into the CLEM workflow would ensure the imaging is performed in a more efficient and robust way by significantly reducing experiment time and increasing sample throughput. In addition, SBF-SEM data collected during the proposed study will be used to facilitate development of an automated ROI recognition software, which can be used to stop cutting once the ROI has been located. This can significantly reduce operator time at the SEM as well as reduce unnecessary data collection. For each sample, once the final ROI has been located using SBF-SEM, the Katana ultramicrotome will be unmounted from the SEM and FIB-SEM will be used to explore the nanoparticles on a subcellular level.Data collected from this project will contribute towards the development of modified hardware and workflows that allow for FIB-SEM data to be acquired with the Katana still on the sample stage in the microscope. In-situ SBF-SEM and FIB-SEM will lead the way to automating CLEM from the electron microscopy point of view. This will truly push forward the boundaries of bioimaging at the nanoscale which is vital to fully understand how organelles, cells, and tissues function.

ConnectomX提议加入英国卫生安全局（UKHSA）和中央激光设施（CLF）之间的现有关系，共同开发一种新的相关光学和电子显微镜（CLEM）工作流程。该工作流程将应用于UKHSA项目成像脑组织环境污染物颗粒的存在。同时，将测试工作流程，并收集概念验证数据，以证明Katana超微组作为组织样品体积CLEM的通用解决方案的实用性。ConnectomX Katana超显微组将安装在CLF现有的扫描电子显微镜（SEM）上。这将允许串行块面扫描电子显微镜（SBF-SEM）和聚焦离子束扫描电子显微镜（FIB-SEM）之间的多功能切换。ConnectomX将提供软件和硬件解决方案，作为持续不断的研发合作的一部分，以改进和构建该提案中首创的CLEM工作流程。更具体地说，提出的工作流程是使用低分辨率的多光子显微镜（MPM）对整个脑组织切片进行成像，以识别受纳米颗粒浸润影响的较小兴趣区域（roi）。这些roi将被识别为与血脑屏障（BBB）渗透率相关，随后将在扫描电镜中重新定位以进行高分辨率成像。ConnectomX Katana超微组可以安装在CLF的SEM腔室中，对含有MPM中识别的roi的选定脑组织切片执行串行SBF-SEM。SBF-SEM利用金刚石刀切割掉样品的薄层，然后对表面进行成像。这个过程重复数千次，以建立样品结构的3D可视化。在这项提议中，Katana超微组将用于获取每个脑组织样本内的低分辨率体积数据集。这些低分辨率数据集中的血管或标记（使用MPM制作）将用于重新定位具有血脑屏障渗透性的较小roi。将Katana整合到CLEM工作流程中，可以通过显着减少实验时间和提高样品吞吐量，确保以更有效和稳健的方式进行成像。此外，在拟议研究期间收集的SBF-SEM数据将用于促进自动化ROI识别软件的开发，该软件可用于一旦定位ROI就停止切割。这可以大大减少操作人员在扫描电镜上的时间，并减少不必要的数据收集。对于每个样品，一旦使用SBF-SEM确定了最终的ROI，将从SEM上卸下Katana超微组，然后使用FIB-SEM在亚细胞水平上探索纳米颗粒。从该项目中收集的数据将有助于改进硬件和工作流程的开发，以便在刀刀仍处于显微镜样品阶段时获取FIB-SEM数据。原位SBF-SEM和FIB-SEM将从电子显微镜的角度引领CLEM自动化的道路。这将真正推动纳米尺度生物成像的发展，这对于充分了解细胞器、细胞和组织的功能是至关重要的。