Multi-Function Laser Scanning Microscope

描述（由申请人提供）：我们申请资金购买共享双光子/共聚焦激光扫描显微镜（卡尔蔡司LSM710 NLO），作为老化的蔡司LSM510系统的替代。现有的显微镜最初是在1998年安装的，已经升级了几次（例如，在2002年增加了META探测器），但我们现在被告知，卡尔蔡司将在2009年停止对这款较旧的LSM510/Axiovert100系统的服务支持。为了确保实验的连续性，我们将在新系统上使用大部分现有的光学器件和附属硬件，包括最近购买的相干变色龙Ti：蓝宝石激光器。目前的系统有6个主要用户，集中在三个广泛的研究领域：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，其中特别包括使用双光子激发，因此在服务合同下保证系统的可靠性至关重要。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。过去八年，现有LSM510 NLO的服务合约的使用费一直维持不变，我们预计LSM710继续维持这一安排不会有任何困难。此外，CISR还将根据项目需要帮助确定和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，并且可以在比共聚焦显微镜深6倍的水平上进行高分辨率成像。所提出的仪器将是范德比尔特大学唯一普遍可用的双光子激发成像系统。利用双光子激发，我们开发了NAD(P)H， Ca2+指示染料，量子点和GFP突变体的体内定量激光扫描成像方案，以及光化学激活方案。在过去的几年中，我们使用这些新方法来阐明涉及肿瘤生长和转移，糖尿病病理生理和神经递质动力学的许多生物学机制。由于目前的显微镜即将缺乏服务支持，所要求的仪器对于继续这一研究势头至关重要。公共卫生相关性：在过去十年中，很明显，生物细胞不能被视为进行反应的“化学物质袋”。细胞成分的精确三维排列是非常重要的，在细胞生命周期和与外界刺激相互作用时，这种排列的时间依赖性变化也是非常重要的。显微镜技术的最新进展，如本文所要求的双光子激发显微镜，使我们能够在对细胞活力影响最小的情况下观察活细胞中的这些排列和运动。

DESCRIPTION (provided by applicant): We request funds to purchase a shared two-photon/confocal laser scanning microscope (Carl Zeiss LSM710 NLO), as a replacement for an aging Zeiss LSM510 system. The existing microscope was originally installed in 1998 and has been upgraded several times (for instance, adding the META detector in 2002), but we have now been informed that Carl Zeiss will discontinue service support for this older LSM510/Axiovert100 system in 2009. To assure experimental continuity, we will utilize much of our existing optics and accessory hardware on the new system, including the recently purchased Coherent Chameleon Ti:Sapphire laser. The current system has 6 major users focused in three broad 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, so the guaranteed reliability of a system under service contract is crucial. 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 contract for the existing LSM510 NLO over the last eight years, and we foresee no difficulty in continuing that arrangement for the LSM710. In addition, the CISR will also help identify and 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 fold deeper than with confocal microscopy. The proposed instrument will be the only generally available two-photon excitation imaging system available at Vanderbilt University. Using two-photon excitation, we have developed in vivo quantitative laser scanning imaging protocols for NAD(P)H, Ca2+ indicator dyes, quantum dots, and GFP mutants, as well as photochemical activation protocols. Over the last few years, we have used these new approaches to elucidate many biological mechanisms involved in tumor growth and metastasis, diabetic pathophysiology, and neurotransmitter dynamics. Because of the imminent lack of service support for the current microscope, the requested instrumentation is critical to continue this research momentum. PUBLIC HEALTH RELEVANCE: Over the last decade, it has become apparent that the biological cell cannot be regarded as a "bag of chemicals" that carries out reactions. 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. Recent advances in microscopy, such as the two-photon excitation microscope requested here, allow us to watch these arrangements and movements in living cells with minimal effects on cell viability.