Fast scanning two photon microscope with confocal unit for intravital and reflectance imaging

目前的建议涉及到一种先进的双光子显微镜，能够同时记录多色功能信号，如钙事件，并在自由行为的动物的大视场上进行高分辨率的亚细胞形态学记录。所要求的设备是在Hertie临床脑研究所（HIH）成功建立Nicolas Snaidero博士研究小组的核心，此外，还将为HIH和综合神经科学中心（CIN）的许多建立良好的小组提供技术解决方案，作为这些研究所之间虚拟成像设施的一部分。事实上，目前HIH和CIN有几台双光子显微镜已经被大量使用，由于主要的技术限制，不适合上述项目。事实上，本申请中描述的创新项目使该工具对申请人和整个机构绝对必要。本提案中列出的研究小组的主要科学目标集中在脑和脊髓中胶质细胞、上皮细胞、免疫细胞和神经元细胞之间的功能和形态相互作用，以及发育中的类器官和器官，如肺和肝。先进的双光子成像将与复杂的模型、行为研究和转化方法相结合，研究炎症环境中轴突和神经胶质病理以及免疫细胞的迁移、神经退行性疾病模型中的斑块动力学和免疫调节、神经递质和多动症对连通性和大脑发育的影响。为了实现这些目标，需要配备两个Ti:Sapphire激光器的多光子显微镜，其中一个具有扩展到1300nm的红外范围，并结合快速谐振串联扫描仪和高性能非扫描外部探测器。所要求的系统具有可调节的激发/检测的通用性，允许各种荧光团的最佳成像，自适应阶段与大型行为设置兼容。此外，共聚焦组件的集成允许共聚焦显微镜和光谱共聚焦反射显微镜将髓鞘的无标记成像与活体成像结合起来。最后，系统升级的可能性，以执行2P活体荧光寿命成像提供了独特的机会，扩大动态应用，特别是在代谢状态研究和亚细胞分子变化。

The present proposal concerns a state of the art two photon microscope capable to record simultaneous multi-chromatic functional signals, such as calcium events, and perform high resolution subcellular morphological recordings on large fields of view in freely behaving animals.The requested equipment is central to the successful establishment of Dr. Nicolas Snaidero’s research group at the Hertie Institute for Clinical Brain research (HIH) and will in addition, provide technical solutions for many well establishes groups at the HIH and at the Center for Integrated Neuroscience (CIN) as part of the virtual imaging facility between these institutes. Indeed, currently the HIH and CIN have several two photon microscopes that are already heavily used and not suitable for the described projects due to major technical limitations. Indeed the innovative projects described in this application make this instrument absolutely necessary for the applicants and the entire institution.The Major scientific targets of the research groups listed in this proposal focus on the functional and morphological interaction between glial, epithelial, immune and neuronal cells in the brain and spinal cord but also in developing organoids and organs such as lung and liver. Advanced two photon imaging will be combined with complex models, behavioral studies and translational approaches to investigate axonal and glial pathologies as well as migration of immune cells in inflamed milieu, plaque dynamics and immune modulation in neurodegenerative disease models, neurotransmitter and hyperactivity on connectivity and brain development.To achieve these objectives a multi photon microscope equipped with two Ti:Sapphire lasers, one of which with an extended IR range up to 1300nm combined with fast resonant tandem scanner and high performance non descanned external detectors is required. The versatile nature of the requested system with adjustable excitation/detection allows optimal imaging of a wide variety of fluorophores and the adaptive stage is compatible with large behavioral setups. Furthermore, the integration of confocal components allows concomitant 2P and Spectral Confocal Reflectance Microscopy to combine label free imaging of myelin sheath with intravital imaging. Finally, the possibility for the system to be upgraded in order to perform 2P intravital Fluorescence Life time Imaging offers the unique opportunity to broaden dynamic applications especially towards metabolic state investigations and subcellular molecular changes.