Advanced confocal laser-scanning-microscope with live-imaging setup

所提出的设备应能够实现高通量/高分辨率共聚焦显微镜，这是必要的亚细胞（病理）的生理过程在固定组织的先进分析。该设备的配置应能够以380-900 nm的光谱灵活性对8-10种蛋白质进行多重标记，以便可以解析亚细胞过程（例如囊泡分类、蛋白酶体亚型鉴定、亚细胞区室化、免疫细胞鉴定）。另一方面，光谱的灵活性应提高信噪比，使复杂器官中低丰度蛋白质的可视化（肾脏自体荧光）是可行的。分辨率高达约。去卷积后的80-100 nm应该能够使亚细胞过程可视化。该设备必须配备活细胞显微镜，以可视化体内亚细胞过程具有高的时间和空间分辨率。该设备将用于推进研究计划“蛋白酶体-溶酶体蛋白质降解-炎症的信号中枢和驱动程序”的开发，该计划旨在了解1）导致缺陷蛋白质降解的病理机制过程，以及2）这种缺陷蛋白质降解作为炎症过程驱动程序的后果。使用活细胞显微镜对器官典型的小鼠和猪肾切片培养物进行亚细胞过程的体内分析也代表了生理学研究所的3R项目。调查这些主题是具有挑战性的，因为除了高通量高分辨率图像和超分辨率图像，“现场分析”，如细胞器功能，膜动力学，如内吞作用和外泌体形成，溶酶体定位和运动，分析蛋白酶体定位和活性的亚细胞微域内的焦点。计划中的实验对时间提出了很高的要求，现有的设备和显微镜时间无法满足这一要求。该设备必须能够同时满足这些规格，因为这是进行结构和信号的亚细胞分析以及细胞-细胞相互作用的唯一方法。因此，将获得对蛋白质降解系统的相互作用的新见解，并且可以确定它们对细胞稳态、细胞间通讯和免疫应答的影响。该系统将有助于1）加强UKE的生物医学研究，2）使生理学研究所的基础生理学研究的方法和方法面向未来，3）实施创新原则和3R研究概念。

The proposed device should enable high-throughput/high-resolution confocal microscopy, which is necessary for the advanced analysis of subcellular (patho)physiological processes in fixed tissues. The configuration of the device should enable multiple labeling of 8-10 proteins with a spectral flexibility of 380-900 nm, so that subcellular processes (e.g. vesicle classification, proteasome subtype identification, subcellular compartmentalization, immune cell identification) can be resolved. On the other hand, the spectral flexibility should improve the signal-to-noise ratio, so that the visualization of low-abundance proteins in complex organs (kidney autofluorescence) is feasible. A resolution up to approx. 80-100 nm after deconvolution should enable the visualization of subcellular processes. The device must be equipped for live-cell microscopy to visualize subcellular processes in vivo with a high temporal and spatial resolution. The device will be used to advance the development of the research initiative "Proteasome-lysosome protein degradation - signaling hub and driver of inflammation", which aims to understand the pathomechanistic processes that 1) lead to defective protein degradation, and 2) the consequences of this defective protein degradation as a driver of inflammatory processes. The in vivo analysis of subcellular processes using live-cell microscopy on organo-typical mouse and pig kidney slice cultures also represents the 3R project of the Institute of Physiology. Investigating these topics is challenging because, in addition to high-throughput high-resolution images and super-resolution images, "live analyses" such as organelle function, membrane dynamics such as endocytosis and ectosome formation, lysosome positioning and motility, analysis of proteasome localization and activity within subcellular microdomains are the focus. The planned experiments place high demands on time, which cannot be met with the available equipment and microscopy time. The device must be able to meet these specifications simultaneously, as this is the only way the subcellular analysis of structures and signals as well as cell-cell interactions can be performed. As a result, new insights into the interaction of the protein-degrading systems will be gained and their effects on cell homeostasis, cell-cell communication and on the immune response can be determined. The system will contribute to 1) strengthening biomedical research at the UKE, 2) making the methods and approaches of basic physiological research at the Institute of Physiology future-proof and 3) implementing the innovative principles and 3R research concepts.