Multiphoton microscope upgrade

多光子显微镜升级

• 批准号: 8247216
• 负责人: David W Piston
• 金额: $ 43.59万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8247216
• 关键词: Animal Model; Area; Biomedical Research; Cells; Charge; Communities; Computer software; Confocal Microscopy; Contract Services; Data; Diabetes Mellitus; Financial compensation; Fluorescence Microscopy; Funding; Image; Label; Laboratories; Lasers; Life; Malignant Neoplasms; Microscope; Microscopy; Minor; Mus; Neurosciences; Penetration; Physiologic pulse; Productivity; Proteins; Qualifying; Research; Research Infrastructure; Resolution; Resource Sharing; Resources; Sapphire; Scanning; Spectrum Analysis; System; Techniques; Thick; Tissues; Training; Training and Education; United States National Institutes of Health; Universities; Vision; Width; Work; cellular imaging; instrument; research study; two-photon

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.

描述（由申请人提供）：我们申请资金将共享的双光子/共焦激光扫描显微镜（Carl Zeiss LSM 710 NLO）升级为具有相干变色龙视觉II-S Ti：Sapphire激光器的高效LSM 780 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符合条件的NIH资助的项目，其中特别包括使用双光子激发，并且系统的拟议改进对于目前资助的工作至关重要。现有的显微镜安装于2008年，并已非常好地服务于许多活细胞和组织实验。然而，我们已经发现，光谱成像对于利用新可用的多荧光蛋白标记的细胞和组织至关重要，并且所提出的科学问题也需要双光子激发进行深层组织成像。为了确保实验的连续性，我们建议升级LSM和钛：蓝宝石激光器，以允许主要用户的资助项目所需的深层组织光谱成像。此外，我们正在请求软件升级，以便根据几个主要和次要用户的需要，通过相关光谱从数据中提取最大量的信息。 与当前仪器一样，申报仪器将成为细胞成像共享资源（CISR）的一部分，Piston博士是该资源的科学总监，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和接受培训。在过去的12年里，使用费支持了双光子激发显微镜的服务合同（1999年至2008年为LSM 510，2008年为LSM 710），我们预计继续为LSM 780/Chameleon Vison II-S系统提供服务不会有任何困难。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的300多个实验室小组中拥有广泛的教育、培训和生产力记录。在过去的15年里，我们已经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，荧光相关光谱和去卷积显微镜的共享访问。这些技术最初都是由生物物理实验室使用的，但现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的动物模型（如小鼠）的成像，双光子激发是远远优于其他方法上级，并允许在6至10倍的水平比共聚焦显微镜深的高分辨率成像。拟议的仪器将继续是唯一的一般可用的双光子激发成像系统在范德比尔特大学。 公共卫生关系：细胞成分的精确三维排列是非常重要的，就像在细胞的生命过程中以及与外部刺激相互作用时这种排列的时间依赖性变化一样。了解这些组件的时间和空间组织需要我们同时监测多个信号。显微镜的最新进展，如高效率的光谱检测器结合双光子激发显微镜的要求，使我们能够观察这些安排和运动在活组织和整个动物细胞活力的影响最小。