We have designed, constructed, and are utilizing a system that allows living cells to be followed by high resolution differential interference contrast (DIC) video-enhanced time-lapse light microscopy while selected organelles are subjected to laser microsurgery and/or manipulation with an optical force trap. This system couples the output from two different Neodymium-YAG lasers to the same inverted light microscope equipped with de Senarmont compensation DIC optics, a motorized stage, and a high-resolution low-light-level CCD camera. Unlike similar systems using phase-contrast optics, our unique DIC-based system can image living cells in thin optical sections without contamination due to phase halos or out-of-focus object information. These advantages greatly facilitate laser-based light microscopic studies on mitotic organelles and components, including spindle poles (centrosomes) and kinetochores, which are at or below the resolution limit of the light microscope and buried within a large complex structure. When used in conjunction with image processing and high-resolution object-tracking techniques, our system is already providing new information on the roles that kinetochores and spindle microtubules play during chromosome segregation in plant and animal cells. This development was published in 1995 in the Journal of the Microscopy Society of America (with a cover photograph), and its first application led to a Journal of Cell Biology paper. Both are featured in the highlights.
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