Multiphoton microscope for fast intravital microscopy applications

该仪器是一种高性能共聚焦显微镜，适用于活细胞、器官和小生物。该仪器将跨学院边界使用，优先考虑SFB 1350项目。sfb1350主要关注肾脏不同类型细胞之间的病理生理和串扰。这一课题的研究具有挑战性，因为它需要对活组织的“洞察”，因为蛋白质吸收和细胞-细胞相互作用等功能依赖于复杂组织结构的完整性。除了“高分辨率功能形态学”和在活体动物中发现纳米颗粒外，几个项目的重点将放在分析细胞器功能上，例如肾脏特定细胞中的线粒体和溶酶体。此外，该系统将允许在培养细胞、类器官和小动物（如果蝇幼虫）上进行长期实验。计划中的实验对灵敏度、空间和时间分辨率提出了现有设备无法满足的高要求：小型运动亚细胞器的研究需要非常好的空间分辨率（XY为120- 150nm）和快速的图像采集（512x512为40hz）。在这种情况下，快速图像采集需要特别高的扫描仪灵敏度，以保持低光毒性，漂白和图像噪声。穿透深度和组织保护：对于类器官、离体灌注器官和活体动物的测量，穿透深度（80-120µm）尤为重要。高穿透深度和同时使用不同的染料需要激发双多光子激光器和额外的专用检测技术。光谱灵活性：系统应具有光谱灵活性。在激发方面，这是通过集成足够数量的激光线或可调谐激光器来保证的。在发射方面，这应该通过多个通道并行记录的可能性以及通过对发射光的光谱柔性检测及其光谱分析来实现。该设备必须能够同时满足这些规格，因为这是实现亚细胞结构和信号以及活组织深处细胞-细胞相互作用分析的唯一途径。例如，这将为完整肾脏和肾上腺组织中细胞的相互作用提供新的见解，并为进一步发展已经在雷根斯堡建立的肾脏活体显微镜和肾脏和肾上腺的离体灌注方法提供动力。因此，该系统将使我们能够为加强雷根斯堡的生物医学研究做出贡献，并实施创新的研究概念。

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.