VISUALIZATION OF KINETOCHORES AND ANALYSIS OF THEIR REFRACTILITY IN CRANE-FLY SPERMATOCYTES AFTER ALDEHYDE FIXATION

Glutaraldehyde and formaldehyde were used to fix crane-fly spermatocyt es for observation with differential interference contrast (DIC) micro scopy. In aldehyde-fixed cells, kinetochores exhibit contrast not norm ally observed in living cells. Although the mechanism underlying this result is not understood, the visualization of kinetochores as distinc t refractile objects opens the way for analysis of unstained kinetocho res with the light microscope. The analysis of kinetochore refractilit y reported in this paper is made possible by the finding that the refr actility of chromosomes in formaldehyde-fixed cells decreases as the c oncentration of formaldehyde is increased. In 4% formaldehyde, the ref ractility of chromosomes is matched with that of its surround, chromos omes appear invisible, and kinetochores may be analyzed as if chromoso mes were not present. Kinetochores were imaged with DIC optics, and th en digital image analysis was performed. Gray-level scans through the highlight and shadow of an individual kinetochore parallel to the axis of shear resulted in a curve having a slope proportional to the DIC o ptical path gradient. Curves from autosomal kinetochores imaged in ana phase had slopes approximately one-half those recorded at metaphase un der identical optical conditions. By contrast, kinetochore thicknesses (defined as the distance between the peak and the valley of a gray-le vel scan) at those two stages were not significantly different. These data suggest a loss of dry mass from autosomal kinetochores during ana phase. Neither the refractility nor thickness of lagging sex kinetocho res varied as autosomes went through anaphase. The conclusion drawn fr om these findings is that the decreased refractility of autosomal kine tochores in anaphase is movement-related. (C) 1998 Wiley-Liss, Inc.