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A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium

Author

Listed:
  • Franka H. Linden

    (University of Amsterdam)

  • Eike K. Mahlandt

    (University of Amsterdam)

  • Janine J. G. Arts

    (University of Amsterdam
    University of Amsterdam)

  • Joep Beumer

    (Royal Netherlands Academy of Arts and Sciences and University Medical Center)

  • Jens Puschhof

    (Royal Netherlands Academy of Arts and Sciences and University Medical Center)

  • Saskia M. A. Man

    (University of Amsterdam)

  • Anna O. Chertkova

    (University of Amsterdam)

  • Bas Ponsioen

    (University Medical Centre Utrecht)

  • Hans Clevers

    (Royal Netherlands Academy of Arts and Sciences and University Medical Center)

  • Jaap D. Buul

    (University of Amsterdam
    University of Amsterdam)

  • Marten Postma

    (University of Amsterdam)

  • Theodorus W. J. Gadella

    (University of Amsterdam)

  • Joachim Goedhart

    (University of Amsterdam)

Abstract

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2–9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids.

Suggested Citation

  • Franka H. Linden & Eike K. Mahlandt & Janine J. G. Arts & Joep Beumer & Jens Puschhof & Saskia M. A. Man & Anna O. Chertkova & Bas Ponsioen & Hans Clevers & Jaap D. Buul & Marten Postma & Theodorus W., 2021. "A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium," Nature Communications, Nature, vol. 12(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-27249-w
    DOI: 10.1038/s41467-021-27249-w
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    References listed on IDEAS

    as
    1. Niels Heemskerk & Lilian Schimmel & Chantal Oort & Jos van Rijssel & Taofei Yin & Bin Ma & Jakobus van Unen & Bettina Pitter & Stephan Huveneers & Joachim Goedhart & Yi Wu & Eloi Montanez & Abigail Wo, 2016. "F-actin-rich contractile endothelial pores prevent vascular leakage during leukocyte diapedesis through local RhoA signalling," Nature Communications, Nature, vol. 7(1), pages 1-17, April.
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    Cited by:

    1. Dorothy Koveal & Paul C. Rosen & Dylan J. Meyer & Carlos Manlio Díaz-García & Yongcheng Wang & Li-Heng Cai & Peter J. Chou & David A. Weitz & Gary Yellen, 2022. "A high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors," Nature Communications, Nature, vol. 13(1), pages 1-14, December.

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