With the advent of digital cameras and motorization of mechanical components, computer control of microscopes has become increasingly important. Software for microscope image acquisition should not only be easy to use, but also enable and encourage novel approaches. The open‐source software package µManager aims to fulfill those goals. This unit provides step‐by‐step protocols describing how to get started working with µManager, as well as some starting points for advanced use of the software. Curr. Protoc. Mol. Biol. 92:14.20.1‐14.20.17. © 2010 by John Wiley & Sons, Inc.

Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtubules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluorescence and immunoelectron microscopy (immunoEM). We show that Drosophila embryonic spindles at metaphase and anaphase contain abundant bundles of MTs running between the spindle poles. These interpolar MT bundles are parallel near the poles and antiparallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphorylated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of organizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle midzone. Thus we propose that bipolar kinesin motors and MTs interact by a “sliding filament mechanism” during the formation and function of the mitotic spindle.