Multiphoton microscope upgrade

描述（由申请人提供）：我们申请资金将共享双光子/共聚焦激光扫描显微镜（卡尔蔡司LSM710 NLO）升级到具有相干变色龙视觉II-S Ti：蓝宝石激光器的高效LSM780 NLO，该激光器将提供色散补偿和窄脉冲宽度，以实现最大深度穿透。目前的系统有6个主要用户，集中在三个研究领域：癌症、糖尿病和神经科学，这些用户将构成拟议仪器的主要用户群。所有这些主要用户都有合格的nih资助项目，具体包括双光子激发的使用，并且对系统提出的改进对当前资助的工作至关重要。现有的显微镜安装于2008年，在许多活细胞和组织实验中发挥了很好的作用。然而，我们发现光谱成像对于利用新获得的多荧光蛋白标记的细胞和组织至关重要，并且正在提出的科学问题也需要双光子激发进行深部组织成像。为了保证实验的连续性，我们建议对LSM和Ti:Sapphire激光器进行升级，以允许主要用户资助项目所需的深层组织光谱成像。此外，我们要求软件升级，以便根据几个主要和次要用户的需要，通过相关光谱学从数据中提取最大数量的信息。与目前的仪器一样，拟议的仪器将成为细胞成像共享资源（CISR）的一部分，其中活塞博士是科学主任，威尔斯博士是常务董事。所有主要用户都可以通过已建立的CISR基础设施使用该仪器并接受培训。在过去的12年里，双光子激发显微镜（LSM510从1999年到2008年，LSM710从2008年开始）的服务合同都是由使用费支撑的，我们预计LSM780/变色龙vision II-S系统继续保持这种安排不会有困难。此外，CISR还将根据项目需要培训双光子仪器的新用户。该资源在范德比尔特大学拥有超过300个实验室小组的教育、培训和生产力方面的广泛跟踪记录。在过去的15年里，我们引入了共聚焦显微镜、活细胞成像、双光子激发、全内反射（TIRF）显微镜、荧光相关光谱和反褶积显微镜的共享访问。这些技术都是从更多的生物物理实验室开始使用的，但已被一般生物医学研究界广泛使用。对于厚的完整组织或活体动物模型（如小鼠）的成像，双光子激发远优于其他方法，并且允许高分辨率成像，其深度是共聚焦显微镜的6至10倍。该仪器将继续是范德比尔特大学唯一普遍可用的双光子激发成像系统。

DESCRIPTION (provided by applicant): We request funds to upgrade a shared two-photon/confocal laser scanning microscope (Carl Zeiss LSM710 NLO) to the high-efficiency LSM780 NLO with the Coherent Chameleon Vision II-S Ti:Sapphire laser, which will provide dispersion compensation and narrow pulse widths to allow maximal depth penetration. The current system has 6 major users focused in three research areas: cancer, diabetes, and neuroscience, and these users will constitute the major user group for the proposed instrument. All of these major users have qualifying NIH-funded projects that specifically include the use of two-photon excitation, and the proposed improvements in the system are crucial for the currently-funded work. The existing microscope was installed in 2008, and has served very well for many live cell and tissue experiments. However, we have discovered that spectral imaging is critical to take advantage of newly-available multi-fluorescent protein labeled cells and tissues, and that the scientific questions being asked also need with two-photon excitation for deep tissue imaging. To assure experimental continuity, we propose to upgrade both the LSM and the Ti:Sapphire laser to permit the deep-tissue spectral imaging required for the funded projects of the major users. In addition, we are requesting software upgrades needed to extract the maximal amount of information from the data via Correlation Spectroscopy as needed by several major and minor users. As with the current instrument, the proposed instrument will be part of the Cell Imaging Shared Resource (CISR), of which Dr. Piston is the Scientific Director and Dr. Wells is the Managing Director. All major users will have access to the instrument and training through the established CISR infrastructure. Usage charges have supported the service contracts for two-photon excitation microscopes (LSM510 from 1999 to 2008, and LSM710 since 2008) over the last 12 years, and we foresee no difficulty in continuing that arrangement for the LSM780/Chameleon Vison II-S system. In addition, the CISR will also train new users of the two-photon instrument as their projects require. The Resource has an extensive track record of education, training, and productivity with over 300 lab groups at Vanderbilt University. Over the last 15 years, we have introduced shared access to confocal microscopy, live cell imaging, two-photon excitation, total internal reflection (TIRF) microscopy, fluorescence correlation spectroscopy, and deconvolution microscopy. These techniques all began with use by the more biophysical laboratories, but have become widely used by the general biomedical research community. For imaging of thick intact tissues or live animal models (such as the mouse), two-photon excitation is far superior to other approaches and permits high-resolution imaging at a level 6 to 10 fold deeper than with confocal microscopy. The proposed instrument will continue to be the only generally available two-photon excitation imaging system available at Vanderbilt University. PUBLIC HEALTH RELEVANCE: The exact three-dimensional arrangement of the cellular components is tremendously important, as are the time-dependent changes in this arrangement during the life of the cell and upon interaction with external stimuli. Understanding the temporal and spatial organization of these components requires us to monitor multiple signals simultaneously. Recent advances in microscopy, such as the high-efficiency spectral detectors combined with two-photon excitation microscopy as requested here, allow us to watch these arrangements and movements in living tissues and whole animals with minimal effects on cell viability.