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

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，以合作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项目成像脑组织的环境污染物颗粒的存在。同时，将对工作流程进行测试，并收集概念验证数据，以证明Katana超薄切片机作为组织样本体积CLEM的通用解决方案的实用性。ConnectomX Katana超薄切片机将安装在CLF现有的扫描电子显微镜（SEM）上。这将允许在连续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）之间进行多功能切换。ConnectomX将提供软件和硬件解决方案，作为持续和迭代研发合作的一部分，以改进和建立本提案中开创的CLEM工作流程。更具体地说，拟议的工作流程是使用多光子显微镜（MPM）以低分辨率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感兴趣区域（ROI）。这些ROI将被识别为与血脑屏障（BBB）渗透性相关，随后将在SEM中重新定位以进行高分辨率成像。ConnectomX Katana超薄切片机可以安装在CLF的SEM室内，对选定的含有MPM中识别出的ROI的脑组织切片进行连续SBF-SEM。SBF-SEM利用金刚石刀切掉样品的薄层，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。在该提案中，Katana超薄切片机将用于采集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品牌标记（使用MPM制作）将用于重新定位具有BBB渗透性的较小ROI。将Katana纳入CLEM工作流程将通过显着减少实验时间和增加样品吞吐量来确保以更有效和更稳健的方式进行成像。此外，在拟定研究期间收集的SBF-SEM数据将用于促进自动ROI识别软件的开发，一旦定位ROI，该软件可用于停止切割。这可以显著减少SEM的操作时间，并减少不必要的数据收集。对于每个样品，一旦使用SBF-SEM定位了最终的ROI，Katana超薄切片机将从SEM上卸下，FIB-SEM将用于在亚细胞水平上探索纳米颗粒。从该项目收集的数据将有助于改进硬件和工作流程的开发，允许在显微镜中的样品台上仍然使用Katana获取FIB-SEM数据。原位SBF-SEM和FIB-SEM将从电子显微镜的角度自动化CLEM的方式。这将真正推动纳米级生物成像的边界，这对于充分了解细胞器，细胞和组织的功能至关重要。

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.