Several microtubule-related structures in mitotic spindles and kinetochores have been studied using serial block-face imaging, a technique that combines a 3D light microscope with serial SEM imaging (Kubista et al., 2005). This method uses the tip of an ultramicrotome to expose the surface of a resin-embedded cellular structure and then the resin block is imaged with a 3D-stacking electron microscope. The final image stack is then used for 3D reconstruction of the object. The 3D reconstruction workflow includes automated segmentation of the structure and its division into several subvolumes. The volume data are then represented in different formats, depending on the question to be answered. These formats include isosurfaces and iso-surfaces for volume rendering, triangle meshes for animation and 3D printing, as well as segmentation of the whole structure or particular microtubules or organelles. In addition to microtubules and their postmitotic counterparts, the most commonly used segmented objects for analysis are sister chromatids and centromeres. The main limitation of serial block-face imaging is the size of the structures that can be imaged. The largest structures that have been imaged to date are the kinetochores, which include centromeres and major proteins. The largest kinetochore in humans has a diameter of around 200 nm and there are about 50 microtubules attached to it (Kubista et al., 2008). There is still much room for improvement in this area. Recently, the first microtubules decorated with a kinesin motor were visualized in serial block-face imaging (Kanchanawong et al., 2011).
One of the most notable achievements in this area was the use of serial block-face imaging to study human chromosomes (Winey et al., 2012). These 3D models allowed quantification of the numbers of microtubules attached to a chromosome. Another type of analysis includes the visualization of 3D reconstructions of a metaphase chromosome. The reconstruction of a whole chromosome is an isosurface model, generated by a mathematical algorithm that integrates volume data from multiple serial sections into a single 3D mesh. This model can then be visualized as an isosurface in different colours or as a mesh for further analysis.
The following figure is a composite of serial section micrographs from three different angles. It was constructed using the software program IMOD (Kremer et al., 1996). The program allows the location and measurement of the curved portion of the spindle (red line in the top micrograph). This curved region is very characteristic of the curved spindles, and is clearly visible in the two sections that are shown in the uppermost micrographs (section series II-IV). The micrographs were then used as a reference for the two intermediate sections (section series V-VII). The angle of the section series with respect to the spindle axis can be altered by varying the precise position of the virtual cutting plane within the stack. 827ec27edc