Microscopic Observation of Living Cells Stained with Fluorescent Probes

Abstract

Fluorescence imaging of living cells provides a unique opportunity to follow dynamic behavior of specific molecules under physiological conditions. In the fission yeast Schizosaccharomyces pombe, expression of a target protein genetically fused with a fluorescent protein such as the jellyfish green fluorescent protein (GFP) is widely used. In addition, fluorescent chemical reagents are also used to stain specific molecules (e.g., Hoechst 33324 to stain DNA). Specimens of S. pombe cells for live cell imaging are prepared by either of two methods: sandwiching the cells between glass coverslips and by mounting the cells on a glass-bottom culture dish. For time-lapse observation, it is necessary to immobilize fission yeast cells on the glass surface of the glass-bottom dish because they are nonadherent and tend to move easily as a result of stage movement, convection flow of culture medium, and the contact and pushing of neighboring cells during cell growth. Either concanavalin A or soybean lectin, which bind to S. pombe cell walls, can be used for immobilization. Considerations for sample preparations and observation conditions are described.