Multiple-color fluorescence imaging of chromosomes and microtubules in living cells

Microscopic observation of fluorescently-stained intracellular molecules wi thin a living cell provides a straightforward approach to understanding the ir temporal and spatial relationships. However, exposure to the excitation light used to visualize these fluorescently-stained molecules can be toxic to the cells. Here we describe several important considerations in microsco pe instrumentation and experimental conditions for avoiding the toxicity as sociated with observing living fluorescently-stained cells. Using a compute r-controlled fluorescence microscope system designed for live observation, we recorded time-lapse, multi-color images of chromosomes and microtubules in living human and fission yeast cells. In HeLa cells, a human cell line, microtubules were stained with rhodamine-conjugated tubulin, and chromosome s were stained with a DNA-specific fluorescent dye, Hoechst33342, or with r hodamine-conjugated histone. In fission yeast cells, microtubules were stai ned with alpha-tubulin fused with the jellyfish green fluorescent protein ( GFP), and chromosomes were stained with Hoechst33342.