Upright two-photon microscope for intravital fluorescence imaging and photostimulation

用于活体荧光成像和光刺激的正置双光子显微镜

• 批准号: 537547683
• 金额: --
• 依托单位: Ruprecht-Karls-Universität Heidelberg
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2024
• 资助国家: 德国
• 起止时间: 2023-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/537547683?language=en
• 关键词: Upright; two; photon; microscope; intravital

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.

双光子显微镜已成为研究生命科学中各种丰富过程的既定方法。由于生物组织的高穿透深度和相对简单的操作模式，该方法特别适合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，例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基因编码荧光指示剂的不断改进和发展，双光子显微镜在生物过程的研究中不断获得重要意义。除了成像之外，双光子激发还可用于刺激光敏蛋白。此外，在这种情况下，该技术已达到成熟状态，可用于可靠地控制各种时空尺度的细胞过程的功能。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中建立立式双光子显微镜，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，例如神经炎症、伤害性和体感周围神经元的光遗传学研究、心血管（病理）生理学和所有-使用光遗传学操作对细胞网络进行光学研究以及大脑中细胞和亚细胞功能的功能读出。该系统将允许与全息双光子刺激相结合，对体内和切片中的神经元动力学进行双光子成像。我们申请的双光子系统具有以下特性和功能：1) 与小动物（主要是啮齿类动物）活体成像兼容的正置显微镜，2) 用于组织样本（例如类器官或脑切片）成像的可拆卸载物台，3)具有大视场的多功能扫描系统，可快速扫描大面积以及精确配置扫描路径，4) 快速 3D 聚焦系统，用于厚组织中的快速体积成像，5) 两个用于同时双色成像的飞秒脉冲红外激光线，6) 使用适当的高能脉冲红外激光进行 3D 全息、双光子刺激的能力。