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

具有实时成像设置的先进共焦激光扫描显微镜

• 批准号: 527193870
• 金额: --
• 依托单位: Universität Hamburg
• 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/527193870?language=en
• 关键词: Advanced; confocal; laser; scanning; microscope

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.

所提出的设备应能够实现高通量/高分辨率共焦显微镜，这对于固定组织中的亚细胞（病理）生理过程的高级分析是必要的。该设备的配置应能够对 8-10 种蛋白质进行多重标记，并具有 380-900 nm 的光谱灵活性，以便能够解决亚细胞过程（例如囊泡分类、蛋白酶体亚型识别、亚细胞区室化、免疫细胞识别）。另一方面，光谱灵活性应提高信噪比，从而使复杂器官（肾脏自发荧光）中低丰度蛋白质的可视化成为可能。分辨率高达约。去卷积后 80-100 nm 应能够实现亚细胞过程的可视化。该设备必须配备活细胞显微镜，以高时间和空间分辨率可视化体内亚细胞过程。该设备将用于推进“蛋白酶体-溶酶体蛋白质降解 - 信号中枢和炎症驱动因素”研究计划的开发，该计划旨在了解 1）导致蛋白质降解缺陷的病理机制过程，以及 2）这种有缺陷的蛋白质降解是炎症过程的驱动因素。使用活细胞显微镜对典型的小鼠和猪肾脏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。研究这些主题具有挑战性，因为除了高通量高分辨率图像和超分辨率图像之外，还需要“实时分析”，例如细胞器功能、膜动力学（例如内吞作用和胞外体形成）、溶酶体定位和运动、蛋白酶体定位分析亚细胞微域内的活性是焦点。计划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以及细胞间相互作用的亚细胞分析的唯一方法。因此，我们将获得对蛋白质降解系统相互作用的新见解，并确定它们对细胞稳态、细胞间通讯和免疫反应的影响。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研究方法和方法面向未来，3）实施创新原则和3R研究理念。