Multicolor Timelapse Luminescence Microscopy: Optimizing Luciferases to Track Fast Gene Dynamics in Single Yeast Cells

Timelapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics during cell differentiation, cell cycle, and circadian clocks. However, fluorescent proteins are limited by high cellular auto-fluorescence, photo-toxicity, and slow chromophore maturation times. This problem becomes acute when the goal is to measure multiple, weakly transcribed genes and faithfully track fast dynamics. A promising alternative is luciferase, which is an enzyme that produces photons from a chemical substrate and does not require a maturation step. The drawback is that photon flux is much lower than fluorescent proteins. To date, timelapse luminescence microscopy has been successfully used to track gene dynamics in larger organisms and/or for slower dynamics (i.e. circadian oscillation), where more total photons can be collected.We have designed novel green, yellow, and red beetle luciferases for optimized expression in budding yeast, a small eukaryote. By combining timelapse luminescence microscopy with a microfluidic device, we are able to track gene expression in single cells with one-minute time resolution. This is an order of magnitude faster than previous studies, and opens the door for tracking fast dynamics (e.g. cell cycle) in single-cell microbes.