2-photon microscope (Imaging / Electrophysiology)

双光子显微镜已成为一种不可或缺的方法来可视化活组织内的细胞过程。依赖于红外光，可以穿透组织，散射比可见光少，这种现代技术允许激发荧光团在显着更深的组织层比传统的荧光显微镜。这允许在急性切片制备中对深度约为150 µm的细胞进行成像，这些切片制备通常用作马格德堡奥托-冯-格里克大学许多工作组的神经科学或免疫学研究的模型系统。虽然有一个持续的强烈兴趣与急性切片准备工作，唯一可用的2-光子显微镜在医学学院马格德堡校园已专门定制的动物体内实验。临时重新调整这些切片工作的设置将过于费力和耗时，是不可行的。因此，所要求的双光子显微镜预期作为组织切片工作的专用装置，特别为用户提供将联合收割机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研究所无法操作的过时的双光子原型（Till Photonics，最初于1999年购买），其图像质量不再符合当前标准。为了降低成本，现有的电生理实验仪器（显微操作器，电动平台等）。并且原始设置中的附加组件将与现代双光子显微镜一起重新使用。除了在申请人的项目中使用显微镜（见表格“研究”）外，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OVGU的所有感兴趣的研究人员使用。显微镜的选择配置将允许复杂的活细胞成像实验，其中红色和绿色荧光团可以同时可视化与高空间和时间分辨率。因此，显微镜需要包含两个单独的激光线（一个可调谐飞秒激光器和一个固定波长的激光器），允许独立激发两个荧光团附近的最佳吸收截面。此外，显微镜扫描头的配置需要支持切片制备内的线、区域和体积元素的快速和精确扫描。最后，检测器应具有快速门控机制（“门控光电倍增管”），以排除在使用外部激光进行光遗传学刺激期间大量曝光可能导致任何检测器损坏的风险。两者合计，采购一个国家的最先进的2-光子显微镜与这些属性应显着促进在校园马格德堡的几个DFG资助的研究项目的进展。

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 The new microscope will replace an inoperable outdated 2-photon prototype (Till Photonics, originally purchased in 1999) in the Institute of Physiology, whose image quality can no longer meet current standards.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.