Visualization of kinetochores and analysis of their refractility in crane-fly spermatocytes after aldehyde fixation

Glutaraldehyde and formaldehyde were used to fix crane-fly spermatocytes for observation with differential interference contrast (DIC) microscopy. In aldehyde-fixed cells, kinetochores exhibit contrast not normally observed in living cells. Although the mechanism underlying this result is not understood, the visualization of kinetochores as distinct refractile objects opens the way for analysis of unstained kinetochores with the light microscope. The analysis of kinetochore refractility reported in this paper is made possible by the finding that the refractility of chromosomes in formaldehyde-fixed cells decreases as the concentration of formaldehyde is increased. In 4% formaldehyde, the refractility of chromosomes is matched with that of its surround, chromosomes appear invisible, and kinetochores may be analyzed as if chromosomes were not present. Kinetochores were imaged with DIC optics, and then 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 optical path gradient. Curves from autosomal kinetochores imaged in anaphase had slopes approximately one-half those recorded at metaphase under identical optical conditions. By contrast, kinetochore thicknesses (defined as the distance between the peak and the valley of a gray-level scan) at those two stages were not significantly different. These data suggest a loss of dry mass from autosomal kinetochores during anaphase. Neither the refractility nor thickness of lagging sex kinetochores varied as autosomes went through anaphase. The conclusion drawn from these findings is that the decreased refractility of autosomal kinetochores in anaphase is movement-related.