Video digitizer analysis of birefringence along the lengths of single chromosomal spindle fibres. I. Description of the system and general results.

A new system, based on a video digitizer interfaced to a microcomputer, has been developed to quantify birefringence of individual chromosomal spindle fibres from videotaped images of spindles. (The system also can be used for any other purpose that requires the analysis of video intensities.) Retardations along the lengths of single chromosomal spindle fibres have been studied throughout metaphase and anaphase in cells kept at constant temperatures. The instrumental readings are accurate to within less than 0.06 nm retardation, but operationally the retardation values along a single chromosomal spindle fibre can vary by up to 0.15 nm, primarily because of variation in operator definition of the spindle fibre. Retardations vary with position along the fibre. During anaphase the retardations along a given chromosomal spindle fibre do not move poleward, but rather change as if the oriented material is disorganized at the kinetochore. The retardation at the kinetochore of a chromosomal spindle fibre does not change during anaphase, except for nonpredictable jumps of 20-30% that sometimes occur. Thus there is no 'decay of birefringence' during anaphase, such as has been described in other species. In this regard our data, that pertain only to single chromosomal spindle fibres, differ from those previously published; we argue that this is because the published data deal with mixtures of chromosomal and continuous spindle fibres, and because changes in birefringence can appear to occur, artefactually, when measurements of birefringence are made at a single spot in a spindle.