A Differential Interference Contrast-Based Light Microscopic System for Laser Microsurgery and Optical Trapping of Selected Chromosomes during Mitosis In Vivo

Abstract

Laser microsurgery and laser-generated optical force traps (optical tweezers) are both valuable light microscopic-based approaches for studying intra- and extracellular motility processes, including chromosome segregation during mitosis. Here we describe a system in use in our laboratory that allows living cells to be followed by high-resolution differential interference contrast (DIC) video-enhanced time-lapse light microscopy while selected mitotic organelles and spindle components 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 both phase-contrast and 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 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 provides new information on the roles that kinetochores and spindle microtubules play during chromosome segregation in plant and animal cells.