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.

双光子显微镜已经成为研究生命科学中丰富多样的过程的一种成熟的方法。由于该方法在生物组织中的穿透深度较高，操作方式相对简单，特别适合于中枢神经系统的活体成像。然而，近年来，双光子显微镜在生物研究的其他领域也得到了越来越多的应用。双光子显微镜的应用包括快速亚细胞过程的荧光成像，如神经元突起或突触传递中的钙动力学，大细胞群体的钙成像，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完善和发展，双光子显微镜在生物过程研究中的意义不断增强。除了成像，双光子激发也可以用来刺激光敏蛋白质。此外，在这种情况下，该技术已经达到了成熟的状态，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中建立一个直立式双光子显微镜，并将其提供给曼海姆医学院的研究社区。应用将包括对脑肿瘤的免疫反应的长期监测，脑癌细胞与神经元的相互作用，病理状态(如神经炎症)期间的脑细胞动力学，伤害性和躯体感觉外周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对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。该系统将允许对活体和切片中的神经元动力学进行双光子成像，并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式显微镜；2)可拆卸的平台，用于对有机组织或脑切片等组织样品进行成像；3)多用途扫描系统，具有大视场，能够快速扫描大面积并精确配置扫描路径；4)快速三维聚焦系统，用于厚组织的快速体积成像；5)两条飞秒脉冲红外激光线路，用于同时进行双色成像；6)利用适当的高能脉冲红外激光进行全息、双光子三维刺激。