First we will consider our design criteria for the instrument, then consider separately the major features of the microscope components, the cooled CCD camera, and the MetaMorph digital imaging system. There are three main parts to the system: a Nikon FXA microscope, a Hamamatsu C4880 cooled CCD camera, and a MetaMorph digital imaging system. 2 provides model numbers and sources of the key components of the instrument. Our high-resolution multimode digital imaging system is shown in Fig. 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). 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. 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). 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. The instrument has also been important in the analysis of mitotic mutants in the powerful yeast genetic system Saccharo-myces cerevisiae. 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 DiOC 6) and the dynamics of the cell cortex in migrating vertebrate epithelial cells ( Waterman-Storer and Salmon, 1997).

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). 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. 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.

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