In this chapter we describe the development of a high-resolution, multimode digital imaging
system based on a wide-field epifluorescent and transmitted light microscope and a cooled
charge-coupled device (CCD) camera. Taylor and colleagues (Farkas et al., 1993; Taylor et
al., 1992) have reviewed the advantages of using multiple optical modes to obtain
quantitative information about cellular processes and described instrumentation they have
developed for multimode digital imaging. The instrument described here is somewhat
specialized for our microtubule and mitosis studies, but it is also applicable to a variety of
problems in cellular imaging including tracking proteins fused to the green fluorescent
protein (GFP) in live cells (Cubitt et al., 1995; Heim and Tsien, 1996; Olson et al., 1995).
For example, the instrument has been valuable for correlating the assembly dynamics of
individual cytoplasmic microtubules (labeled by conjugating X-rhodamine to tubulin) with
the dynamics of membranes of the endoplasmic reticulum (ER, labeled with DiOC6) and the
dynamics of the cell cortex [by differential interference contrast (DIC)] in migrating
vertebrate epithelial cells (Waterman-Storer and Salmon, 1997). The instrument has also
been important in the analysis of mitotic mutants in the powerful yeast genetic system
Saccharo-myces cerevisiae. Yeast cells are a major challenge for high-resolution imaging of
nuclear or microtubule dynamics because the preanaphase nucleus is only about 2 μm wide
in a cell about 6 μm wide. We have developed methods for visualizing nuclear and spindle
dynamics during the cell cycle using high-resolution digitally enhanced DIC (DE-DIC)
imaging (Yang et al., 1997; Yeh et al., 1995). Using genetic and molecular techniques.
Bloom and coworkers (Shaw et al., 1997a,b) have been able to label the cytoplasmic astral
microtubules in dividing yeast cells by expression of cytoplasmic dynein fused to GFP.
Overlays of GFP and DIC images of dividing cells have provided the opportunity to see for
the first time the dynamics of cytoplasmic microtubules in live yeast cells and how these
dynamics and microtubule interactions with the cell cortex change with mitotic cell cycle
events in wild-type and in mutant strains (Shaw et al., 1997a,b)
