Time-lapse fluorescence imaging of yeast cells allows the study of multiple fluorescent targets in single cells, but is often hampered by the tedious cultivation using agar pads or glass bottom wells. Here, we describe the fabrication and operation of a microfluidic device for long-term imaging of yeast cells under constant or changing media conditions. The device allows acquisition of high quality images as cells are fixed in a two-dimensional imaging plane. Four yeast strains can be analyzed simultaneously over several days while up to four different media can be flushed through the chip. The microfluidic device does not rely on specialized equipment for its operation. To illustrate the use of the chip in DNA damage research, we show how common readouts for DNA damage or genomic instability behave upon induction with genotoxic chemicals (MMS, HU) or induction of a single double-strand break using induced CRISPR-Cas9 expression.
Keywords: CRISPR-Cas9; DNA damage; Fluorescence microscopy; Genomic instability; Hydroxyurea; Long-term imaging; MMS; Microfluidic; Perfusion; S. cerevisiae; Single-cell analysis.