Luminescence Imaging: Molecules, Cells and Tissues

描述(申请人提供)：本申请的目的是获得一种光子计数相机和附件，以允许在培养细胞内和整个动物中实现高时间和空间分辨率的发光；附件包括优化体外光蛋白表达和激活所需的多孔板和单管发光仪，以及将在其中进行整个动物成像的成像站。爱因斯坦医学院目前没有这样高灵敏度的相机或全动物成像仪，在肯尼迪中心神经科学系所在的九层楼里，目前也没有这两种类型的光度计，拟议中的研究将在那里进行。建议的研究都是NIH资助的九名研究人员项目的自然延伸，这些项目将使用相对较新的空间分辨发光记录技术，由于新一代载体构建，光蛋白在光输出的强度和波长方面得到了优化，而在基因表达报告方面则变得不稳定，因此这种技术变得更容易获得。本提案中概述的用户研究内容多种多样，包括用于确定连接蛋白分子二聚化和结合伙伴的生物发光共振能量转移，用于定位星形胶质细胞和星形细胞瘤细胞内ATP释放的发光报告分子，用于确定移植到小鼠体内后其组织分布的发光标记骨髓细胞，以及与发光报告分子融合的基因启动子，以确定细胞因子刺激的星形胶质细胞中基因激活的时间历程，并建立一种中等高通量的方法，用于确定连接蛋白特异性缝隙连接通道阻滞剂。该提案还包括每两个月举行一次用户小组会议的计划，通过该会议将吸引新的用户使用该仪器，并将讨论新的分析工具和协议以及可能出现的问题。它还包括培训计划以及在研究生课程中纳入相关方法。

DESCRIPTION (provided by applicant): The purpose of this application is to obtain a photon-counting camera and accessories to allow high temporal and spatial resolution of luminescence within cultured cells and in whole animals; accessories include both multiwell plate and single tube luminometers required for optimization of expression and activation of the photoproteins in vitro and an imaging station in which whole animal imaging will be performed. There is no such high sensitivity camera or whole animal imager currently at Einstein College of Medicine, and there is currently neither type of luminometer in the nine-floor Kennedy Center housing the Department of Neuroscience, where the proposed studies will be performed. The studies that are proposed are all natural extensions of NIH-funded projects of nine investigators that will use the relatively new technique of spatially resolved luminescence recording that has become more accessible due to new generations of vector constructs in which the photoproteins have been optimized with regard to intensity and wavelengths of light output and destabilized with regard to gene expression reporting. The studies of users outlined in this proposal are diverse, including bioluminescent resonance energy transfer to determine dimerization and binding partners for connexin molecules, luminescent reporter molecules to localize ATP release from within astrocytes and astrocytoma cells, luminescence-tagged bone marrow cells in order to characterize their tissue distribution following transplantation into mice, and gene promoters fused to luminescent reporter molecules to identify time course of gene activation in cytokine-stimulated astrocytes and to develop a moderately high throughput assay by which to identify connexin-specific gap junction channel blockers. The proposal also includes the plan for a bimonthly user group meeting, through which new users will be attracted to the instrument and new analytical tools and protocols will be discussed, as well as problems that may arise. It also includes plans for training as well as inclusion of relevant methods in a graduate student course.