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

细菌病原体在世界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发展诊断学和治疗学非常重要，特别是在后抗生素时代。尤其是细胞内的细菌病原体是一个巨大的挑战，因为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微生物药物。宿主-病原体的相互作用长期以来一直在静态培养(通过将细胞放置在塑料或玻璃培养皿上)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。或者，动物模型很少概括人类的感染过程，通常也不会以动态的方式解析亚细胞细节。最近，有机化合物和芯片上器官微流控技术作为实验系统出现，允许在更接近于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体内发病机制的理解。然而，鉴于这种样本固有的三维(3D)结构以及与感染相关的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，使其能够在时间和空间上解析生物和感染过程。提到的以这样或那样的形式研究感染的挑战也适用于研究宿主细胞对炎症等无菌侮辱的反应。在此，我们要求购买旋转圆盘共聚焦显微镜(SDCM)，用于在短时间尺度和长时间尺度上对活细胞(感染或无菌)3D样本进行快速可视化和量化。用于这一特定目的的SDCM的以下独特功能包括：(I)高速采集，这对于成像芯片上的器官设备、活细胞有机物或3D基质中的感染样本以及随着时间的推移尤其关键。激光扫描共聚焦显微镜(LSCM)与点扫描仪一起操作，对于多通道成像和厚样品可能需要几分钟的时间，而使用SDCM则需要几秒钟。在较厚的样品(例如，细胞单层的典型厚度为10-15μm)中，广场荧光成像与反卷积相结合会导致像差随样品厚度的增加而呈非线性增加。SDCM能够以类似于LSCM的方式从样品中获取薄的光学切片，但速度要快得多。(Iii)与经典的LSCM相比，SDCM表现出较低的光毒性和光漂白，这一功能对于精细分辨单个、快速移动的细菌、运动敏感的免疫细胞和其他类型表达活细胞生物传感器的细胞的成像至关重要。因此，一个致力于S2安全级别环境的最先进的SDCM将帮助我们将宿主与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.