Fluorescence Speckle Microscopy (FSM) is a new technique we have discovered for visualizing movement, assembly, and turnover of macromolecular assemblies in living cells. When a small fraction of subunits have fluorescent label, these polymers are seen to acquire a fluorescent speckle distribution along their lattice during their assembly. The highest speckle contrast is achieved for fractions of about 1% or less where the fluorescence of each speckle corresponds to a variable number of only a few fluorophores per resolvable unit (about 0.25 um) in the microscope. We have shown that speckles containing 1 or 2 fluorophores can be detected and recorded using a wide-field fluorescence light microscope, and digital imaging with a low noise cooled CCD camera. We are at very early stages in the development of SM and there are numerous improvements needed to take FSM much more powerful and open up new applications. These include improving the sensitivity, stability and resolution in 4-D (3D plus time) of FSM, combining FSM with other modes of light microscopy, determining new ways of measuring the kinetics of speckle motions and life-times from FSM images, and testing the developments in important collaborative projects. To achieve these goals, we have the following Specific Aims: 1) Assemble a stable, precision, multi-mode, multi-wavelength digital microscope and computer system for 4-D for FSM; 2) Use in vitro and living cell models for microtubles and actin filament assembly to determine the optimal fraction of fluorescently labeled subunits for FSM using fluorescent dyes and GFP. Apply ways of produce multiple fluorophores per subunit that are reduced in the problems of quenching and photobleaching, and explore their advantages; 3) trajectories and lifetimes in 2D and 3D from stacks of time-lapse FSM images; and 4) Test new FSM developments in biomedical projects investigating the motility and dynamics of microtuble and actin filament arrays in mitotic and migrating cells as well as in assaying microtuble rotation by microtuble motor proteins.
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