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Imaging Vacuolar Anthocyanins with Fluorescence Lifetime Microscopy (FLIM)

  • Alexandra Chanoca
  • Brian Burkel
  • Erich Grotewold
  • Kevin W. Eliceiri
  • Marisa S. OteguiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1789)

Abstract

Anthocyanins are intrinsically fluorescent pigments that accumulate in plant vacuoles. We have developed a platform to analyze the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), under in vitro and in vivo conditions. Fluorescence lifetime of a fluorophore can be influenced by temperature, pH, oxygen concentration, and other environmental conditions. Within plant cells, the anthocyanin fluorescence lifetime correlates with distinct subcellular compartments. Vacuolar anthocyanins exhibit shorter fluorescence lifetime than the cytoplasmic pool. Consistent with these observations, lower pH of anthocyanins solutions correlated with shorter fluorescence lifetimes. We discuss here the use of FLIM as a tool for analyzing the subcellular distribution of anthocyanins and estimating variation in vacuolar pH in intact cells.

Key words

Fluorescence lifetime microscopy (FLIM) Anthocyanins Arabidopsis thaliana Plant vacuole pH 

Notes

Acknowledgments

This work was supported by NSF grant MCB-1048847 to EG and MSO.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Alexandra Chanoca
    • 1
    • 2
  • Brian Burkel
    • 3
  • Erich Grotewold
    • 4
  • Kevin W. Eliceiri
    • 2
    • 3
  • Marisa S. Otegui
    • 1
    • 2
    • 5
    Email author
  1. 1.Department of BotanyUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Laboratory of Molecular and Cellular BiologyUniversity of Wisconsin-MadisonMadisonUSA
  3. 3.Laboratory for Optical and Computational Instrumentation (LOCI)University of Wisconsin-MadisonMadisonUSA
  4. 4.Department of Biochemistry and Molecular BiologyMichigan State UniversityEast LansingUSA
  5. 5.Department of GeneticsUniversity of Wisconsin-MadisonMadisonUSA

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