REAL TIME DUAL FLUORESCENCE IMAGING OF LIVING CELLS

描述(改编自申请人的摘要)：摘录 来自活细胞中荧光探针的定量信息 视频显微镜已经成为一种使细胞可视化的令人兴奋的新方法 动力学。同时检测阳离子和阴离子变化的探头 浓度、pH和膜电位使时间成为可能 这些过程的可视化。一个主要的例子是离子的量化。 用荧光比率成像法测定活细胞内的通量。潜在的更多 令人兴奋的应用是多种染料在同一活细胞中的动力学 定量测定细胞膜和细胞质成分的动态变化。这个 调查人员建议扩展现有技术以开发一种实时 一种用于快速采集和分析的视频显微镜图像处理系统 动力学数据。它将同时捕获两个独立的荧光 以视频帧速率发射图像。调查人员将(I) 同时采集和存储由两种不同发射产生的图像 标准视频帧速率下来自两个独立视频通道的波长 (30帧/秒)。调查人员将使用一种新型的光学列车，没有 能够同时进行双重激励和双重发射的移动部件 这些图像。每个发射波长将由一个人同时成像 对两台摄像机进行数字化、背景校正和合成 标准视频帧速率。新的可编程格式的CCD摄像机可能会允许 以每秒60张图像的速度拍摄。数据将存储在高分辨率视频上 用胶带进行进一步的离线分析。(Ii)第二个目标是改善 单个图像的质量(信噪比)和空间分辨率 允许对单个框架进行分析。光动力损伤与光漂白 规定使用低激励电平。发射光级将为 通过优化光学元件和滤光片的吞吐量而增加。 最先进的图像增强器板，连接到ccd摄像机 将提高敏感度，消除滞后和开花。(Iii) 演示同时获取两种不同类型的动态变化 在同一个单元格中，研究人员将应用新的 显微镜对一种生物相关的动力学荧光的研究 模型：流感血凝素(HA)促进细胞-细胞融合。首字母 融合事件可能涉及即时的脂质双层融合：相反 广泛的蛋白质相互作用和细胞质混合可能发生在 建立了双层邻接性。膜的同时测量 而融合过程中的细胞质运动可能解决这一基本问题 问题。融合将通过重新分布一个 膜结合染料DiI和细胞质染料Calcein，两者最初都是 包含在一个单元格中。拟议的研究将极大地改善 调查人员能够获取和处理来自 使用廉价机器的活细胞系统的数量。因此，它将 为其他生物医学研究人员提供一套可供应用的新工具 一些不同的细胞动力学问题，如：细胞内离子 细胞内膜组分的运动、形成和回收 贩运，细胞骨架和细胞外组织的动力学 基质成分和病毒入侵宿主细胞，仅举几例。

DESCRIPTION (Adapted from the applicant's abstract): The extraction of quantitative information from fluorescent probes in living cells using videomicroscopy has become an exciting new approach to visualizing cell dynamics. Probes which sense changes in both cation and anion ion concentrations, pH and membrane potential have made possible temporal visualization of these processes. A leading example is quantitation of ion fluxes in living cells by fluorescence ratio imaging. A potentially more exciting application is kinetics of multiple dyes in the same living cell to quantitate dynamics of membrane and cytoplasmic constituents. The investigators propose to extend existing technology to develop a real-time video microscope image processing system for rapid acquisition and analysis of kinetic data. It will simultaneously capture two separate fluorescence emission images at video frame rates. The investigators will (I) simultaneously acquire and store images generated by two different emission wave lengths from two separate video channels at standard video-frame rates (30 frames/sec). The investigators will use a novel optical train with no moving parts capable of both dual excitation and dual emission to create the images. Each emission wavelength will be imaged simultaneously by one of two cameras then digitized, background corrected and combined at standard video frame rates. New programmable format CCD cameras may allow capture at 60 images/sec. Data will be stored on high resolution video tape for further off-line analysis. (II) A second goal is to improve the quality (signal/noise ratio) and spatial resolution of individual images to allow analysis of single frames. Photodynamic damage and photobleaching dictate use of low excitation levels. Emission light levels will be increased by optomizing throughput of the optical elements and filters. State of the art image intensifier plates, coupled to CCD video cameras will increase sensitivity and eliminate lag and bloom. (III) To demonstrate simultaneous acquisition of dynamic changes in two different fluorophores in the same cell, the investigators will apply the new microscope to the study of a biologically relevant kinetic fluorescent model: cell-cell fusion promoted by influenza hemagglutinin (HA). Initial fusion events may involve immediate lipid bilayer fusion: conversely extensive protein interactions and cytoplasmic mixing may take place before bilayer contiguity is established. Simultaneous measurements of membrane and cytoplasm movements during fusion may resolve this fundamental question. The fusion will be visualized by the redistribution of a membrane bound dye, DiI, and a cytoplasmic dye, calcein, both originally contained in one cell. The proposed research will greatly improve the investigators ability to acquire and process dual emission data from a number of living cell systems using inexpensive machinery. Thus it will provide other biomedical researchers with a new set of tools to be applied to a number of different problems of cell dynamics, eg; intracellular ion movements, formation and retrieval of membrane components, intracellular trafficking, dynamics of organization of cytoskeletal and extracellular matrix components and virus invasion of host cells, to name just a few.