Upright two-photon microscope for intravital fluorescence imaging and photostimulation

双光子显微镜已经成为研究生命科学中丰富多样过程的一种既定方法。由于该方法在生物组织中的穿透深度高，操作方式相对简单，因此特别适合于中枢神经系统的实时成像。然而，近年来，双光子显微镜也越来越多地应用于生物研究的其他领域。双光子显微镜的应用包括快速亚细胞过程的荧光成像，如神经元过程或突触传递中的钙动力学，大细胞群的钙成像，或细胞形态和运动的慢性成像。随着大量信号分子和代谢物的遗传编码荧光指示剂的不断完善和发展，双光子显微镜在生物过程的研究中不断获得重要意义。除了成像，双光子激发也可以用来刺激光敏蛋白。此外，在这种情况下，该技术已经达到成熟的状态，可以用于在各种时空尺度上可靠地控制细胞过程的功能。鉴于这项技术在生物医学研究中的重要性，我们的目标是在曼海姆生命成像设施建立一个直立双光子显微镜，并将其提供给曼海姆医学院的研究界。应用将包括长期监测对脑肿瘤的免疫反应，脑癌细胞与神经元的相互作用，病理状态下的脑细胞动力学，如神经炎症，伤害性和体感周围神经元的光遗传学研究，心血管（病理）生理学和使用光遗传学操作的细胞网络的全光学研究，以及大脑中细胞和亚细胞功能的功能读出。该系统将允许双光子成像神经元动力学在体内和切片结合全息双光子刺激。我们申请的双光子系统具有以下特性和能力：1)与小动物（主要是啮齿动物）的活体成像兼容的直立显微镜，2)可移动的组织样本成像平台，如类器官或脑切片，3)具有大视场的多功能扫描系统，可以快速扫描大面积，并精确配置扫描路径，4)快速3D聚焦系统，用于厚组织的快速体积成像，5)两条飞秒脉冲红外激光线同时进行双色成像，6)利用合适的高能脉冲红外激光实现三维全息双光子刺激的能力。

Two-photon microscopy has become an established method for the investigation of a rich variety of processes in the life sciences. Due to the high penetration depth in biological tissue and the relatively simple mode of operation, this method is particularly suited for live imaging in the central nervous system. However, in recent years, two-photon microscopy has also been increasingly used in other fields of biological research. Applications for two-photon microscopy include fluorescence imaging of fast, subcellular processes, such as calcium dynamics in neuronal processes or synaptic transmission, calcium imaging of large cellular populations, or chronic imaging of cell morphology and motility. With the continuous improvement and development of genetically encoded fluorescent indicators for a large number of signaling molecules and metabolites, two-photon microscopy continues to gain significance for the investigation of biological processes. In addition to imaging, two-photon excitation can also be used to stimulate light-sensitive proteins. Also, in this case, the technology has reached a state of maturity that it can be used to reliably control the function of cellular processes at various spatiotemporal scales. Given the importance of this technology in biomedical research, we aim to establish an upright two-photon microscope in the Life Imaging Facility Mannheim and make it available to the research community at the Medical Faculty Mannheim. Applications will include long-term monitoring of immune responses to brain tumors, interaction of brain cancer cells with neurons, brain cell dynamics during pathological states, such as neuroinflammation, optogenetic investigation of nociceptive and somatosensory periphery neurons, cardiovascular (patho)physiology and all-optical investigations of cellular networks using optogenetic manipulation and functional read-out of cellular and subcellular function in the brain. The system will allow two-photon imaging of neuronal dynamics in vivo and in slices in conjunction with holographic two-photon stimulation. The two-photon system we apply for has the following properties and capabilities: 1) Upright microscope compatible with intravital imaging in small animals (mostly rodents), 2) a removable stage for imaging of tissue samples such as organoids or brain slices, 3) a versatile scanning system with a large field-of-view enabling fast scanning of large areas as well as precise configuration of the scan path, 4) a fast, 3D focusing system for fast volumetric imaging in thick tissue, 5) two femtosecond-pulsed infrared laser lines for simultaneous dual-color imaging, 6) capability of holographic, two-photon stimulation in 3D with an appropriate high-energy pulsed infrared laser.