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; 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.

所提出的设备应启用高通量/高分辨率共聚焦显微镜，这对于对固定组织中亚细胞（病情）生理过程进行高级分析是必不可少的。该设备的配置应启用具有光谱柔韧性为380-900 nm的8-10个蛋白质的多重标记，因此可以重新溶解亚细胞过程（例如囊泡分类，蛋白酶体亚型识别，亚细胞隔室化，免疫细胞识别）。另一方面，光谱柔韧性应提高信噪比，因此，复杂器官（肾脏自动荧光）中低丰度蛋白的可视化是可行的。最高约为。反卷积后的80-100 nm应能够可视化亚细胞过程。该设备必须配备用于活细胞显微镜的设备，以使体内的亚细胞过程具有高时空和空间分辨率。该设备将用于推进研究计划的开发“蛋白酶体 - 溶酶体蛋白降解 - 信号枢纽和炎症的驱动器”，该计划旨在了解病理学过程，即1）1）导致这种有缺陷的蛋白质降解的后果，而蛋白质降解的后果是炎症过程的驱动因素。在有机典型小鼠和猪肾切片培养物上使用活细胞显微镜对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之外，“实时分析”，例如细胞器功能，诸如膜动力学（例如内吞作用和内吞作用形成，溶酶体定位和运动率，分析）蛋白酶体的定位和亚细胞微生物组中的活性。计划的实验可以按时提出很高的需求，这无法与可用的设备和显微镜时间满足。该设备必须能够同时满足这些规格，因为这是可以执行结构和信号的亚细胞分析以及可以进行细胞细胞相互作用的唯一方法。结果，将获得对蛋白质降解系统相互作用的新见解，并且可以确定它们对细胞稳态，细胞 - 细胞通信和免疫反应的影响。该系统将有助于1）在UKE上加强生物医学研究，2）在生理学研究所未来和3）实施创新原理和3R研究概念的生理学基础研究的方法和方法。