2-photon microscope (Imaging / Electrophysiology)

双光子显微镜已经成为显示活组织内细胞过程的一种不可或缺的方法。这项现代技术依赖于红外光，它可以以比可见光更少的散射穿透组织，允许在比传统荧光显微镜更深的组织层中激发荧光团。这使得在马格德堡奥托-冯-盖里克大学的许多工作组中，急性切片准备中的细胞可以在大约150微米的深度内进行成像，这些切片通常被用作神经科学或免疫学研究的模型系统。尽管人们一直对急性切片制备工作感兴趣，但马格德堡医学院校区唯一可用的双光子显微镜是专门为动物体内实验定制的。为切片工作临时重新装备这些设置将太费力和太耗时，因此不可行。因此，所请求的双光子显微镜旨在作为用于组织切片工作的专用装置，特别是为用户提供将双光子成像与电生理记录相结合的可能性。新的显微镜将取代生理学研究所陈旧的无法操作的双光子原型(Till Photonics，最初购买于1999年)，其图像质量已不能满足当前标准。为了降低成本，现有的电生理实验仪器(微操作器、电动工作台等)而原始设置中的其他组件将在现代双光子显微镜中重复使用。除了在申请者的项目中使用显微镜(见表格“研究”)，它还将成为核心设施“多维显微镜和细胞诊断”的一部分，使其可供马格德堡州立大学所有感兴趣的研究人员使用。选定的显微镜配置将允许进行复杂的活细胞成像实验，其中红色和绿色荧光团可以同时以高空间和时间分辨率可视化。因此，显微镜需要包含两条独立的激光线路(一条可调谐飞秒激光和一条固定波长激光)，以允许两个荧光团在其最佳吸收截面附近独立激发。此外，显微镜扫描头的配置需要支持对切片制备内的线、区域和体积元素的快速和精确扫描。最后，探测器应具有快速选通机制(“门控光电倍增管”)，以避免在外部激光进行光遗传刺激期间大量光暴露可能导致探测器损坏的风险。总而言之，采购具有这种特性的最先进的双光子显微镜应该会大大促进马格德堡校区由DFG资助的几个研究项目的进展。

Two-Photon microscopy has become an indispensable method to visualize cellular processes within living tissue. Relying on infrared light that can penetrate tissue with less scattering than visual light, this modern technique permits excitation of fluorophores in significantly deeper tissue layers than conventional fluorescence microscopy. This allows for imaging of cells in a depth of approximately 150 µm in acute slice preparations, which are routinely used as model systems for neuroscience or immunological research in many work groups at the Otto-von-Guericke-University Magdeburg. Although there is an ongoing strong interest in work with acute slice preparations, the only available 2-photon microscopes at the Medical Faculty Magdeburg campus have been specifically customized for in vivo experiments with animals. A temporary retooling of these setups for slice work would be too laborious and too time consuming to be feasible. The requested 2-photon microscope is therefore intended as a dedicated setup for work with tissue slices, offering users in particular the possibility to combine 2-photon imaging with electrophysiological recordings. The new microscope will replace an inoperable outdated 2-photon prototype (Till Photonics, originally purchased in 1999) in the Institute of Physiology, whose image quality can no longer meet current standards. To reduce costs, existing instrumentation for electrophysiological experiments (micromanipulators, motorized stage etc.) and additional components from the original setup will be re-used with the modern 2-photon microscope. Apart from the use of the microscope in the applicants’ projects (see form sheets "Research"), it will also become a part of the core facility "Multidimensional microscopy and cellular diagnostics", making it available for all interested researchers at the OVGU Magdeburg. The chosen configuration of the microscope will allow for sophisticated live-cell imaging experiments, in which red and green fluorophors can be simultaneously visualized with high spatial and temporal resolution. Therefore, the microscope needs to contain two separate laser lines (one tuneable femtosecond laser and one fixed wavelength laser) that permit independent excitation of two fluorophores near their optimal absorption cross sections. Moreover, the configuration of the microscope scan head needs to support fast and precise scanning of lines, areas, and volume elements within the slice preparation. Lastly, the detectors should possess a rapid gating mechanism ("gated photomultipliers") to exclude the risk that massive light exposure during optogenetic stimulation with an external laser can cause any detector damage. Taken together, the procurement of a state–of-the-art 2-photon microscope with such properties should significantly facilitate the progress in several DFG-funded research projects at the campus Magdeburg.