Visualization of spindle behavior using confocal microscopy

Numerous comparisons have shown that confocal imaging of fluorescently labeled samples has superior image clarity compared to traditional epifluorescence microscopy, especially when imaging through thick specimens. Nevertheless, one limitation of confocal microscopy is the speed at which these systems acquire images. Newer confocal technologies, however, have overcome this drawback by rapidly scanning images while still maintaining the precision and resolution of a confocal microscope. One example is the spinning disk confocal, which can rapidly acquire images with minimal photobleaching. The following protocol provides methodology using spinning disk technology to visualize spindle microtubule behavior in live breast cancer cells expressing green fluorescent protein:alpha-tubulin. It begins with sample preparation, covers equipment setup and image acquisition, and ends with image processing and archiving. Image acquisition is subdivided into two categories- imaging the complete mitotic cycle and imaging rapid mitotic events, to address the varying parameters required for the each experiment.