Leica TCSSP2RS two-photon microscope for in vivo imaging

描述(申请人提供)：双光子显微镜的最新发展使人们能够看到深度接近0.5毫米的组织内部。这使得研究活体器官中细胞的运动和相互作用成为可能。这项技术显然在免疫学方面有着重要的未来。所需的活体双光子成像系统是徕卡微系统公司的TCS SP2 RS(共振扫描)共聚焦显微镜，带有红外激光。这是对活动细胞进行快速、深层组织成像的最先进技术，这是成像淋巴器官等组织中细胞与细胞相互作用所必需的。该用户群在成像技术方面有很强的背景。在免疫学的广泛定义下，他们对广泛的学科感兴趣。T细胞对病原体的反应、细胞与细胞间的相互作用、肿瘤的生长和分化是人们特别感兴趣的问题。这些过程发生在淋巴组织中的特殊隔间，因此不能用其他显微镜技术在体内进行分析。徕卡TCS SP2 RS仪器以直立显微镜为基础，可轻松安装大量组织或活体动物，用于活体显微镜。该仪器的迈泰激光器是软件可调的，允许相对较快的波长变化(在几秒钟内)。谐振式振镜扫描头是系统的关键部件。共振扫描是目前最快的方法，接近视频帧速率。该仪器具有带可调带通滤波器的内部探测器。软件控制的电动狭缝被用来通过棱镜分割的一系列波长。这种方法比使用干涉滤光片更好，因为它允许对给定荧光团的每个通道进行完全自由的优化，并具有更好的穿透效率。此外，双通道外部(非去扫描)探测器允许最大限度地利用散射荧光。总体而言，该仪器将允许用户组从根本上提高他们在理解发育和疾病中的免疫系统方面进行实验的能力。

DESCRIPTION (provided by applicant): The recent development of two-photon microscopy has made it possible to see within tissues to depths approaching 0.5mm. This makes it feasible to investigate the movements and interactions of cells in living organs. This technology clearly has an important future in immunology. The Intravital two-photon imaging system requested is the Leica Microsystems TCS SP2 RS (resonance scanning) confocal microscope, with infrared laser. This is the state-of-the-art for fast, deep-tissue imaging of motile cells, which is what is required to image cell-cell interactions in tissues such as the lymphoid organs. The user group have a strong background in imaging techniques. They are interested in a wide range of subjects under the broad definition of immunology. There is a particular interest in T cell activation in response to pathogens, cell-cell interactions, tumor growth, and in differentiation. These processes occur in specialized compartments in lymphoid tissues and are therefore not amenable to analysis in vivo by other microscopic techniques. The Leica TCS SP2 RS instrument is based on an upright microscope to allow easy mounting of bulk tissues or living animals, for intravital microscopy. The instrument's MaiTai laser is software tunable to allow relatively fast wavelength changes (within several seconds). The scanning head with resonant galvanometer is a critical component of the system. Resonant scanning is currently the fastest method, approaching video frame rates. The instrument has internal detectors with adjustable bandpass filters. Software controlled motorized slits are used to pass a range of wavelengths split by the prism. This approach is superior to using interference filters because it allows full freedom in optimization of each channel for given fluorophore and has better pass-through efficiency. In addition, 2-channel external (non-descanned) detectors allow maximum utilization of scattered fluorescence. Overall, this instrument will allow the user group to radically improve their ability to perform experiments on understanding the immune system in development and disease.