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

细菌性病原体在世界范围内构成巨大威胁，了解它们与人类细胞的相互作用对于开发诊断和治疗方法非常重要，特别是在后抗生素时代。特别是细胞内细菌病原体，由于它们可以通过隐藏在宿主细胞内进行复制和传播来避开宿主免疫系统和抗菌药物，因此提出了重大挑战。宿主-病原体相互作用长期以来一直在静态培养（通过将细胞置于塑料或玻璃培养皿中）中进行研究，这种培养不能有效地概括人体体内（病理）生理学。另外，动物模型很少概括人类的感染过程，通常不能以动态的方式解决亚细胞细节。最近，类器官和器官芯片微流控技术作为实验系统出现，允许在更接近模拟体内环境的条件下一次调节一个参数。这大大增加了我们对发病机制如何在体内展开的理解。然而，考虑到这些样品固有的三维（3D）结构和感染相关过程的不同时间尺度（从几秒到几天），需要特定的显微镜模式来解决时间和空间上的生物和感染过程。上述各种形式的感染研究面临的挑战也适用于宿主细胞对诸如炎症等无菌损伤的反应的研究。在此，我们要求购买旋转盘共聚焦显微镜（SDCM），以便在短时间和长时间尺度上快速可视化和定量活细胞（感染或无菌）3D样品。SDCM用于此特定目的的以下独特功能包括：(i)高速采集，这对于在3D矩阵和时间内成像器官芯片设备，活细胞类器官或感染样本至关重要。与点扫描仪操作的激光扫描共聚焦显微镜（LSCMs）可以在几分钟内完成多通道成像和厚样品，而与SDCM相比，只需几秒钟。（ii） SDCM减少焦外光和像差。在较厚的样品中（即，10-15 μm的细胞单层的典型厚度），宽视场荧光成像与反褶积相结合，导致像差倾向于以非线性的方式增加，作为样品厚度的函数。sdcm能够以类似于LSCMs的方式从标本中获取薄光学切片，但速度要快得多。（iii）与经典LSCMs相比，SDCM表现出更低的光毒性和光漂白，这一特征对于个体、快速移动的细菌、运动敏感的免疫细胞和其他表达活细胞生物传感器的细胞类型的精细分辨活细胞成像至关重要。因此，一个专门用于S2安全级别环境的最先进的SDCM将帮助我们将宿主-病原体相互作用的研究推进到我们校园前所未有的水平。

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.