Fluorescence Lifetime Imaging Microscope

荧光寿命成像显微镜

• 批准号: 8448049
• 负责人: David J Hall
• 金额: $ 50.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-27 至 2014-06-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8448049
• 关键词: Adhesions; Biomedical Research; California; Cancer Center; Cell membrane; Computer software; Computers; Discrimination; Energy Transfer; Engineering; Environment; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; Hypersensitivity; Image; Immunology; Institutes; Institution; Kinetics; Lasers; Measurement; Measures; Medicine; Membrane Potentials; Microbubbles; Microscope; Microscopy; Monitor; Organism; Peptide Hydrolases; Photons; Physiologic pulse; Proteins; Request for Applications; Research; Research Infrastructure; Research Project Grants; Resource Sharing; Resources; Scanning; Sickle Cell Anemia; Spectrum Analysis; Superoxides; Techniques; Technology; Time; Training; United States National Institutes of Health; Universities; cancer imaging; caspase-8; data acquisition; design; detector; fluorescence imaging; fluorescence microscope; fluorophore; in vivo; instrument; molecular imaging; nanoparticle; neutrophil; protein protein interaction

DESCRIPTION (provided by applicant): This application requests funds for the purchase of a Fluorescence Lifetime Imaging Microscope (FLIMscope) as a Shared Instrument for the In Vivo Imaging Shared Resource (IVISR) at the University of California, San Diego (UCSD) Moores Cancer Center. There is no dedicated, shared FLIMscope at UCSD or local institutions and it is a clear unmet need. We have assembled a dozen outstanding users, including Dr. Roger Tsien (Nobel Laureate), that have all expressed the significant benefit a FLIMscope would bring to their biomedical research and strongly support this application. NIH-funded users are from multiple departments at UCSD and from the neighboring La Jolla Institute for Allergy and Immunology (LIAI). Whereas standard fluorescence microscopes only image fluorescence intensity, a FLIMscope also images the fluorescence lifetime of fluorophores enabling the discrimination of spectrally overlapping fluorophores, the use of fluorescence lifetime environment-sensitive probes, robust measurement of Forster Resonance Energy Transfer (FRET) with FLIM FRET, and Fluorescence Lifetime Correlation Spectroscopy (FLCS). The users' research projects will be dramatically enhanced by access to a FLIMscope applying these techniques to a broad variety of topics. For example, discriminating autofluorescence from fluorescent proteins and probes; measuring the microenvironment pH, superoxide level, metallomatrix proteinase activity, and cell membrane potential; and monitoring protein-protein interactions, nanoparticle accumulation or cleavage, FRET-quenching of fluorescent microbubbles, caspase-8 kinetics, and neutrophil adhesion in sickle cell disease. The FLIMscope we chose is the MicroTime 200 by Picoquant. The configuration has 4 pulsed laser diodes from ~400 nm to ~800nm allowing a broad range of fluorophores to be excited. Time correlated Single Photon Counting (TCSPC) technology with two Single Photon Avalanche Diode (SPAD) detectors allows simultaneous fluorescence lifetime measurement of two different wavelength fluorophores. These are integrated into a research grade inverted confocal microscope capable of three-dimensional scanning over both a small (80 micron) and large (10 cm) range. The host computer controls data acquisition and has fluorescence lifetime analysis software. The MicroTime 200 is developed by Picoquant, the world leaders in fluorescence time domain technology, and has been designed and manufactured specifically and solely for FLIM. Consequently, the MicroTime 200 is currently the best choice for a FLIMscope. The IVISR, supported by the In Vivo Cancer and Molecular Imaging Center (ICMIC) at UCSD, is a comprehensive imaging facility for users at UCSD and local institutions. It has an established infrastructure for resource administration, recharge mechanisms, training IVISR imaging staff, and supporting users. This application is also endorsed by the Institute of Engineering and Medicine, the Center for Multiscale Imaging of Living Systems, the UCSD Cancer Center Microscopy core, and the UCSD Moores Cancer Center.

描述（由申请人提供）：本申请要求购买荧光寿命成像显微镜（FLIMSCOPE）作为在加利福尼亚大学圣地亚哥分校（UCSD）Moores癌症中心的体内成像共享资源（IVISR）的共享工具。在UCSD或本地机构中没有专用的，共享的Flimscope，这是一个明显的未满足需求。我们召集了包括Roger Tsien博士（诺贝尔奖获得者）在内的十几种杰出用户，这些用户都表达了Flimscope将带给其生物医学研究并强烈支持此应用的重大好处。 NIH资助的用户来自UCSD的多个部门，以及邻近的La Jolla Allergy and Immunology研究所（LIAI）。虽然仅仅图像荧光强度，但flimscope还图像荧光团的荧光寿命可实现谱系重叠的荧光团的区分，而荧光寿命敏感探针的使用，将FORSTER RESONACE ENEMATION（FLIM转移（FRET）与FLIM FRETERCERCTIONT和FLC FRESS CROPFERISTINCE和FLEC CREPTISTIME cOPPERTERTITY cOPCERTITION）一起使用。通过将这些技术应用于各种各样的主题，用户的研究项目将大大增强。例如，将自荧光与荧光蛋白和探针区分开；测量微环境pH值，超氧化物水平，Metallomomatrix蛋白酶活性和细胞膜电位；并监测蛋白质 - 蛋白质相互作用，纳米颗粒的积累或裂解，荧光微泡的有点淬火，caspase-8动力学以及镰状细胞疾病中的中性粒细胞粘附。 我们选择的Flimscope是PicoQuant的Microtime 200。该构型具有4个脉冲激光二极管，从〜400 nm到〜800nm，可以激发大量的荧光团。时间相关的单个光子计数（TCSPC）技术与两个单个光子雪崩二极管（SPAD）检测器允许同时荧光寿命测量两个不同的波长荧光团。这些集成到研究级倒置的共聚焦显微镜中，能够在小（80微米）和大（10 cm）范围内进行三维扫描。主机计算机控制数据采集并具有荧光寿命分析软件。 Microtime 200是由荧光时间域技术的世界领导者Picquant开发的，并且是专门设计和制造的。因此，Microtime 200目前是Flimscope的最佳选择。 IVISR在UCSD的体内癌和分子成像中心（ICMIC）的支持下，是UCSD和本地机构用户的全面成像设施。它具有既定的基础架构，用于资源管理，充电机制，培训IVISR成像人员和支持用户。该应用也得到了工程与医学研究所，生活系统多尺度成像，UCSD癌症中心显微镜核心和UCSD Moores癌症中心的认可。