title: Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.
abstract: 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.
authors.affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.
authors.forename: Anyimilehidi
authors.initials: A
authors.lastname: Mazo-Vargas
authors.affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710 Department of Physics, Duke University, Durham, NC 27710.
authors.forename: Heungwon
authors.initials: H
authors.lastname: Park
authors.affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.
authors.forename: Mert
authors.initials: M
authors.lastname: Aydin
authors.affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710 Department of Physics, Duke University, Durham, NC 27710 nicolas.buchler@duke.edu.
authors.forename: Nicolas E
authors.initials: NE
authors.lastname: Buchler
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D001517:Coleoptera / Q000737:chemistry / Q000201:enzymology; D048888:Fireflies / Q000737:chemistry / Q000201:enzymology; D015966:Gene Expression Regulation, Fungal; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D046210:Microfluidic Analytical Techniques; D008856:Microscopy, Fluorescence; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods; D059008:Time-Lapse Imaging
chemical_list: D018797:Cell Cycle Proteins; D019476:Insect Proteins; D029701:Saccharomyces cerevisiae Proteins; D008156:Luciferases