The iBiOs platform for advanced live-cell imaging, ultrastructural analyses and correlative light and electron microscopy in SPP 2225

这项拟议的研究项目旨在开发先进的相关光学和电子显微镜(Clem)成像技术，用于分析宿主和病原体的相互作用。目前常规成像方法的局限性包括时空分辨率不足，难以解释结构复杂的宿主-病原体相互作用，以及缺乏理解微生物病原体退出策略所需的3D信息。最近的技术进步，如高压冷冻/冷冻替代(HPF-FS)和超分辨(SR)Clem，使宿主-病原体相互作用的高分辨率可视化成为可能。IBios平台提供了一系列先进的荧光和电子显微镜系统，即将晶格光片显微镜(LLSM)成像与2D/3D电子显微镜、切片Clem和近红外标记(Nirb)相结合，以在极化融合细胞和组织中生成标志物。该设施的独特之处在于，它能够在BL2/S2条件下将活细胞成像与随后的冷冻固定病原体样本的EM相结合，并将用于开发和优化成像工作流程，以分析SPP 2225财团内的病原体退出策略。在SPP 2225内研究病原体的大量项目负责人已经表达了对iBios成像平台的兴趣。由此产生的数据将有助于病原体退出策略的比较分析，满足对分析病原体从宿主隔间或宿主细胞逃逸的定制方法和创新工作流程的需要。

The proposed research project aims to develop advanced correlative light and electron microscopy (CLEM) imaging techniques for the analysis of host-pathogen interactions. Current limitations of conventional imaging methods include inadequate spatio-temporal resolution, difficulties in interpreting structurally complex host-pathogen interactions, and lack of 3D information needed to understand microbial pathogen exit strategies. Recent technological advances, such as high pressure freezing/freeze substitution (HPF-FS) and super-resolution (SR) CLEM, have enabled high-resolution visualisation of host-pathogen interactions. The iBiOs platform provides a range of advanced fluorescence and electron microscopy systems, i.e. combining lattice light-sheet microscopy (LLSM) imaging with 2D/3D electron microscopy, on-section CLEM and near-infrared branding (NIRB) to generate landmarks in polarised confluent cells and tissues. The facility is unique in its ability to combine live cell imaging with subsequent cryo-fixation for EM of pathogenic samples under BL2/S2 conditions and will be used to develop and optimise imaging workflows to analyse pathogen exit strategies within the SPP 2225 consortium. A large number of project leaders studying pathogens within SPP 2225 have already expressed interest in the iBiOs imaging platform. The resulting data will be useful for comparative analyses of pathogen exit strategies, addressing the need for tailored methods and innovative workflows to analyse pathogen escape from host compartments or host cells.