Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.

Published

Journal Article

Time-lapse 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 autofluorescence or phototoxicity that arises 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 that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which 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 time-lapse 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.

Full Text

Cited Authors

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

Published Date

  • November 2014

Published In

Volume / Issue

  • 25 / 22

Start / End Page

  • 3699 - 3708

PubMed ID

  • 25232010

Pubmed Central ID

  • 25232010

Electronic International Standard Serial Number (EISSN)

  • 1939-4586

International Standard Serial Number (ISSN)

  • 1059-1524

Digital Object Identifier (DOI)

  • 10.1091/mbc.E14-07-1187

Language

  • eng