Fluorescent speckle microscopy (FSM) is a technique we initially developed for measuring the movements and sites of polymerization/depolymerization of individual microtubules (MTs) and arrays of actin filaments in motile tissue culture cells and the poleward flux of MTs within spindle fibers during mitosis. Assembly of these polymers from a pool containing a low percentage of fluorescently labeled subunits (about 1% or less) produces a random distribution of fluorophores along the polymer lattice that produces """"""""fluorescent speckle"""""""" fiduciary marks varying from zero to several fluorophores (5-8) within the diffraction limited resolution of the microscope. The major focus of this application is on the further development of the FSM method for the analysis of MT function in spindle mechanics. In particular, how MT and kinetochore proteins function in spindle assembly, chromosome alignment and accurate chromosome segregation. This requires the development of new FSM microscope technology for the rapid recording of multi-wavelength and 3-D time-lapse images of MT fluorescent speckles relative to fluorescent marks or speckles at kinetochores, poles, MT associated proteins (MAPs), motor proteins and MT ends. A major next step for FSM to become a powerful analytical tool for these systems is the development of new Computer Vision methods for obtaining quantitative information about polymer movement and turnover in 2-D and 3-D at high resolution relative to the other molecular fluorescent markers in the spindle. To study protein function, we are particularly interested in optimizing FSM for genetic model organisms including budding yeast, for the biochemically accessible Xenopus egg extracts and for siRNA with mammalian tissue cells. Experience gained in the course of these studies will be used to direct and refine hardware and software development. ? ?
Showing the most recent 10 out of 17 publications
