3-D reconstruction and analysis of mammalian mitotic spindle structure

Mammalian spindles are generally large and may contain over a thousand microtubules (MTs). For this reason they are difficult to reconstruct in three dimensions and many researchers have chosen to study the smaller and simpler spindles of lower eukaryotes. Nevertheless, the mammalian spindle is used for many experimental studies and it would be useful to know its detailed structure.We have been using serial cross sections and computer reconstruction methods to analyze MT distributions in mitotic spindles of PtK cells, a mammalian tissue culture line. Images from EM negatives are digtized on a light box by a Dage MTI video camera containing a black and white Saticon tube. The signal is digitized by a Parallax 1280 graphics device in a MicroVax III computer. Microtubules are digitized at a magnification such that each is 10-12 pixels in diameter.