Spinning disk confocal microscope for visualizing and quantifying host-bacteria interactions

用于可视化和量化宿主-细菌相互作用的转盘共聚焦显微镜

• 批准号: 527320650
• 金额: --
• 依托单位: Eberhard Karls Universität Tübingen
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2023
• 资助国家: 德国
• 起止时间: 2022-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/527320650?language=en
• 关键词: Spinning; disk; confocal; microscope; visualizing

Bacterial pathogens pose a great threat worldwide, and understanding their interaction with human cells is important for developing diagnostics and therapeutics, especially in a post-antibiotic era. Intracellular bacterial pathogens, in particular, present a significant challenge since they can avoid the host immune system and antimicrobial drugs by hiding within host cells to replicate and spread. Host-pathogen interactions have long been studied in static cultures (by placing cells on plastic or glass dishes) that do not effectively recapitulate human in vivo (patho)physiology. Alternatively, animal models rarely recapitulate the infection process in humans and typically do not resolve subcellular detail in a dynamic manner. Recently, organoids and organ-on-chip microfluidic technologies have emerged as experimental systems allowing to modulate one parameter at a time under conditions that more closely mimic in vivo settings. This has added significantly to our understanding of how pathogenesis might unfold in vivo. However, given the inherent three-dimensional (3D) architecture of such samples and the varying time scales of infection-related processes (ranging from seconds to days), specific microscopy modalities are required that enable to resolve biological and infection processes in time and space. The challenges mentioned for the study of infections, in one form or another also apply to the study of host cell responses to sterile insults such as inflammation. Herein, we request the purchase of a spinning disk confocal microscope (SDCM) for fast visualization and quantification of live-cell (infected or sterile) 3D samples over short but also long time scales. The following unique features of SDCM for this specific purpose include, (i) high speed of acquisition, which is critical especially for imaging organ-on-chip devices, live-cell organoids or infected samples in 3D matrices and over time. Laser scanning confocal microscopes (LSCMs) operating with point scanners can take minutes for multi-channel imaging and thick samples, as opposed to seconds with a SDCM. (ii) SDCM reduces out of focus light and aberrations.In thick samples (i.e., >10-15 μm typical thickness of a cell monolayer), wide-field epifluorescence imaging coupled with deconvolution causes aberrations that tend to increase in a non-linear fashion as a function of the specimen thickness. SDCMs are capable of acquiring thin optical sections from specimens in a manner similar to LSCMs, but much faster. (iii)SDCM exhibits lower phototoxicity and photobleaching compared to classical LSCMs and this feature becomes critical for finely resolved live-cell imaging of individual, fast-moving bacteria, motile sensitive immune cells and other cell types expressing live-cell biosensors. Thus, a state-of-the-art SDCM dedicated to an S2 safety level environment would help us advance the study of host-pathogen interactions to an unprecedented level at our campus.

细菌病原体在全球范围内构成了巨大的威胁，了解它们与人类细胞的相互作用对于开发诊断和治疗很重要，尤其是在抗抗生素后时代。特别是细胞内细菌病原体提出了一个重大挑战，因为它们可以通过隐藏在宿主细胞内复制和扩散来避免宿主免疫系统和抗菌药物。长期以来，长期以来一直在静态培养物（通过将细胞放在塑料或玻璃菜肴上）在体内（PATHO）生理学中不概括人类的人体（通过将细胞放置在塑料或玻璃菜肴上）。最近，器官和芯片上的微流体技术已经成为实验系统，允许在一个更紧密地模拟体内设置的条件下一次调节一个参数。这已经大大添加了我们对发病机理如何在体内展开的理解。但是，鉴于此类样品的继承三维（3D）结构以及与感染相关过程的不同时间尺度（从秒到几天范围内），需要特定的显微镜模态，使能够在时间和空间中解决生物学和感染过程。一种或另一种形式的研究研究所提到的挑战也适用于宿主细胞对无菌损伤（例如炎症）的反应。在此，我们要求购买旋转磁盘共聚焦显微镜（SDCM），以快速可视化和量化活细胞（感染或无菌）3D样品，这是在短时但长时间尺度上的。 SDCM出于此特定目的的以下独特功能包括，（i）高速获取，这对于在3D材料和随着时间的时间中的成像器官芯片设备，活细胞器官或受感染的样品至关重要。用点扫描仪运行的激光扫描共聚焦显微镜（LSCM）可以花费几分钟的多渠道成像和较厚的样品，而不是使用SDCM的秒数。 （ii）SDCM从聚焦的光和畸变中减少。在厚的样品（即>10-15μm细胞单层的典型厚度），宽场落荧光成像与反向逆溶性相连的近落荧光成像，并导致异常导致异常，这些异常倾向于以非线性方式增加，并且具有非线性的厚度，并且具有标本厚度的功能。 SDCM能够以类似于LSCM的方式从规格中获取薄光学部分，但更快。 （iii）与经典LSCM相比，SDCM表现出较低的光毒性和光漂白，并且此功能对于单个，快速移动细菌，运动敏感的免疫电池和表达活细胞生物传感器的其他细胞类型的单个，快速移动细菌的活细胞成像至关重要。这是一个专门针对S2安全水平环境的最先进的SDCM，将有助于我们将宿主 - 病原体相互作用的研究提高到我们校园前所未有的水平。