Multiphoton microscope for fast intravital microscopy applications

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The proposed instrument is a high performance confocal microscope for applications on living cells, organs and small organisms. The instrument will be used across faculty boundaries, with preference given to SFB 1350 projects. SFB 1350 focuses on the pathophysiology and crosstalk between different cell types in the kidney. The study of this topic is challenging because it requires "insights" into the living tissue, since functions such as protein resorption and cell-cell interactions depend on the integrity of the complex tissue architecture. In addition to "high-resolution functional morphology" and finding nanoparticles in living animals, the focus of several projects will be on analyzing organelle function, e.g., mitochondria and lysosomes in specific cells of the kidney. Furthermore, the system will allow long-term experiments on cultured cells, organoids and small animals, e.g. Drosophila larvae. The planned experiments make high demands that cannot be met with the existing equipment: Sensitivity, spatial and temporal resolution: the study of small moving subcellular organelles requires very good spatial resolution (120-150 nm in XY) combined with fast image acquisition (40 Hz at 512x512). In this context, fast image acquisition requires particularly high scanner sensitivity to keep phototoxicity, bleaching and image noise low. Penetration depth and tissue protection: For measurements on organoids, isolated perfused organs and living animals, the penetration depth (80-120 µm) is of particular importance. High penetration depth and simultaneous use of different dyes requires excitation by a dual multiphoton laser and additional dedicated detection techniques. Spectral flexibility: The system should be spectrally flexible. On the excitation side, this is to be ensured by the integration of a sufficient number of laser lines or by tunable lasers. On the emission side, this should be implemented by the possibility of parallel recording of several channels and by spectrally flexible detection of the emitted light and its spectral analysis.The device must be able to meet these specifications simultaneously, as this is the only way to realize the analysis of subcellular structures and signals as well as cell-cell interactions in the depth of living tissue. This will, for example, provide new insights into the interaction of cells in the intact tissues of kidney and adrenal gland and provide impulses for the further development of the methods of intravital microscopy of the kidney and ex-vivo perfusion of kidney and adrenal gland already established in Regensburg. The system will thus enable us to contribute to the strengthening of biomedical research in Regensburg and to implement innovative research concepts.