Quantitative Fluorescence Microscopy by Digital Image Analysis

一个视频图像存储系统，采集处理器和数字化仪，以及一个光标发生器将被收购，以完成一个分布式的，但集成的，成像设施的荧光研究中心在生物医学科学。交互式视频图像采集和分析，以及用于数据采集和还原的超小型计算机也是成像设施的一部分。四个独立的实验室联网，允许多个用户应用。在细胞生物学、细胞生理学、发育生物学、调控生物学和生物化学方面的各种各样的项目将由仪器支持。支持的项目包括对静止细胞的生长因子刺激和巨噬细胞趋化性的分析。比值成像将作为一种光谱方法，用于确定（细胞）生理参数（如pH和pCa）的时空变化。两个或多个可分离荧光探针的多参数分析也将用于关联各种细胞功能的时空动态。将对活细胞中的肌动蛋白网络进行三维重建。一种双波长全内反射图像分析方法将用于定量从细胞底物到选定分子的距离。在活细胞中，适当标记的蛋白质的荧光寿命和荧光各向异性的时空变化将被开发。第一个目标是钙调蛋白。总的来说，这些设备，加上中心的其他资源，将允许最先进的测量，极大地扩展我们对细胞功能的理解。

A video image memory system, acquistion procesor and digitizer, and a cursor generator will be acquired to complete a distributed, yet integrated, imaging facilty for the Center for Fluorescence Research in Biomedical Sciences. Interactive video image acquisition and analysis, and supermini computers for data acquisition and reduction are also part of the imaging facility. Four separate laboratories are networked to allow multiple user applications. A wide variety of projects in cell biology, cell physiology, developmental biology, regulatory biology, and biological chemistry will be supported by in the instrumentation. Among the projects to be supported are analyses of the growth factor stimulation of quiescent cells and macrophage chemotaxis. Ratio imaging will be used as a spectroscopic method for determining the spatial and temporal changes in (cellular) physiological parameters such as pH and pCa. Multiple parameter analysis of two or more separable fluorescent probes will also be used to correlate spatial and temporal dynamics of a variety of cellular funcitons. Three dimensional reconstruction will be carried out on actin networks in living cells. A method of two wavelength total internal reflection image analysis will be used to quantitate the distance from the cell substrate to selected molecules. Methods for mapping the spatial and temporal variations in fluorescence lifetime and fluorescence anisotropy of suitably labeled proteins in living cells will be developed. The first target for mapping with be calmodulin. Overall, the equipment, coupled with other resources in the Center, will permit state-of-the-art measurements greatly extending our understanding of cellular function.