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复制和传播来避开宿主免疫系统和抗微生物药物，因此提出了一个重大挑战。宿主-病原体相互作用长期以来一直在静态培养中研究（通过将细胞置于塑料或玻璃皿上），这些静态培养不能有效地再现人类体内（病理）生理学。或者，动物模型很少重演人类的感染过程，并且通常不能以动态方式解析亚细胞细节。最近，类器官和器官芯片微流体技术已经成为实验系统，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，考虑到这些样本固有的三维（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，以便在时间和空间上解决生物和感染过程。感染研究中提到的挑战，以一种或另一种形式也适用于宿主细胞对无菌损伤如炎症的反应的研究。在此，我们请求购买一台旋转圆盘共聚焦显微镜（SDCM），用于在短时间内快速可视化和定量活细胞（感染或无菌）3D样品，但也可以是长时间尺度。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3D矩阵中的器官芯片设备、活细胞类器官或感染样本的成像以及随着时间的推移尤其重要。激光扫描共聚焦显微镜（LSCM）与点扫描仪一起工作，可以花费几分钟进行多通道成像和厚样品，而SDCM则需要几秒钟。(ii)SDCM减少了失焦光和像差。在厚样品中（即，>10-15 μm的典型细胞单层厚度），宽场落射荧光成像结合去卷积会导致像差，该像差倾向于以非线性方式作为样本厚度的函数增加。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LSCM相比，SDCM表现出较低的光毒性和光漂白，并且该特征对于个体、快速移动的细菌、运动敏感的免疫细胞和表达活细胞生物传感器的其他细胞类型的精细分辨活细胞成像变得至关重要。因此，一个致力于S2安全水平环境的最先进的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。