Fluorescence Lifetime Imaging Microscopy (FLIM)

描述（由申请人提供）：我们正在申请资金购买Alba荧光寿命成像显微镜（FLIM），该显微镜将被安置在马里兰大学巴尔的摩荧光光谱中心（CFS），这是一个校园范围的资源。FLIM是一种高度先进的光谱方法，用于成像细胞和组织，其中图像对比度基于寿命而不是荧光强度。FLIM现在广泛用于细胞功能的定量研究，包括组织形态和高密度蛋白质阵列。细胞内结构的分辨率是可能的图像在高时间和空间分辨率使用荧光标记的组件。我们向位于伊利诺斯州尚佩恩的ISS公司订购Alba-FLIM仪器。我们选择了Alba- FLIM仪器作为适合多用户环境的主力仪器，它坚固耐用且易于使用。重要的是，它是唯一可以收集时域（TD）或频域（FD）寿命图像的仪器。激光二极管光源提供的波长适合于各种实验，并且我们能够使用CFS中使用的其他激光器，使用仪器的访问端口，包括我们的Ti：蓝宝石系统用于双光子FLIM功能和脉冲染料激光器。激光扫描能力将提供高空间分辨率。使用相角测量（FD模式），帧速率可以更快，因为光子通量可以更高，我们可以在每个像素上花费更少的时间。停留时间为4微秒即可获得图像；在1秒内获得512x512像素的FLIM图像。时间相关单光子计数（TCSPC）采集电子器件允许复杂强度衰减的分辨率。来自巴尔的摩马里兰大学医学院的一群令人印象深刻的用户（14个主要用户使用R01、P01和R21； 5个次要用户使用R01、R21）将使用FLIM。研究项目（共22个）研究课题多样；其中11个项目与FRET在细胞内蛋白质-蛋白质相互作用研究中的应用有关，5个项目与细胞内离子成像、pH值和蛋白质共定位有关，6个项目与表面等离子体-荧光团相互作用及其在生物和临床系统中的应用研究有关。该仪器将对我们美国国立卫生研究院资助的研究产生重大的积极影响，细胞功能的定量结果也将对美国国立卫生研究院资助的研究产生重大影响。我们认为，对于UMB的学生和博士后来说，将FLIM作为研究生物分子相互作用的重要方法是很重要的。的确，提供使用最先进仪器的机会是UMB培训任务的一个关键方面。新的ITC将在NIH培训补助金的支持下加强对学生的培训。

DESCRIPTION (provided by applicant): We are requesting funds to purchase an Alba fluorescence lifetime imaging microscopy (FLIM) that will be housed in the University of Maryland-Baltimore Center for Fluorescence Spectroscopy (CFS), a campus-wide resource. The FLIM is a highly advanced spectroscopic method to image cells and tissues in which the image contrast is based on lifetime rather than fluorescence intensity. FLIM is now widely used for quantitative studies of cell function including tissue morphology and high density protein arrays. Resolution of intracellular structures is possible in images at high temporal and spatial resolution using fluorescently labeled components. We are requesting the Alba-FLIM instrument from ISS, Inc in Champaign, Illinois. We selected the Alba- FLIM instrument as a workhorse instrument suitable for multi-users environment that is robust and easy to use. Importantly, it is the only available instrument which can collect either time-domain (TD) or frequency-domain (FD) lifetime images. The wavelengths provided by laser diode sources are suitable for various experiments and we are able to use the other lasers available in the CFS using the access port of the instrument, including our Ti:Sapphire system for two-photon FLIM capabilities and pulsed dye lasers. The laser scanning capabilities will provide high spatial resolution. The frame rates can be faster using the phase angles measurement (FD mode) because the photon flux can be higher and we can spend less time at each pixel. Images can be acquired with a 4 microsec dwell time; a 512x512 pixel FLIM image is acquired in 1 sec. The time- correlated single photon counting (TCSPC) acquisition electronics allows resolution of complex intensity decays. An impressive group of users (14 major users with R01, P01 and R21; 5 minor users with R01, R21) from the School of Medicine University of Maryland at Baltimore will use the FLIM. The research topics of research projects (22 total) are diverse; including 11 projects related to the use of FRET in studies of intracellular protein-protein interactions, 5 projects related to intracellular imaging of ions, pH and protein co-localization, and 6 projects related to studies of surface plasmon-fluorophore interactions and their applications to biological and clinical systems. This instrument will have a major positive impact on our NIH-sponsored research and also quantitative results on cellular functions will have a significant impact on the NIH-funded research. We believe it is important for students and postdoctoral associates at UMB to gain exposure to FLIM as an important method for investigating biomolecular interactions. Indeed, providing access to state-of-the-art instrumentation is a key aspect of the training mission at UMB. The new ITC will enhance the training of students supported by a number of NIH training grants.