REAL TIME DUAL FLUORESCENCE IMAGING OF LIVING CELLS

描述（改编自申请人摘要）：提取

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.