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

• Subject Area: Medicine
• Term: Funded in 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 527193870

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.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Fortgeschrittenes Konfokales Laser-Scanning Mikroskop mit Live-Imaging Konfiguration
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Universität Hamburg
• Leader: Professorin Dr. Catherine Meyer-Schwesinger