Measuring fast gene dynamics in single cells with timelapse luminescence microscopy

Mazo-Vargas A, Park H, Aydin M, Buchler NE

Timelapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular auto-fluorescence or photo-toxicity that arise from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than fluorescent proteins. Thus, timelapse luminescence microscopy has only been successfully used to track gene dynamics in larger organisms and for slower processes, where more total photons can be collected in one exposure. Here, we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining timelapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.


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September, 2014



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