Advertisement

FRET Microscopy for Real-Time Visualization of Second Messengers in Living Cells

  • Axel E. Kraft
  • Viacheslav O. Nikolaev
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1563)

Abstract

Förster Resonance Energy Transfer (FRET) microscopy is a useful tool in molecular biology and medical research to monitor and quantify real-time dynamics of protein-protein interactions and biochemical processes. Using this well-established technique, many novel signaling mechanisms can be investigated in intact cells or tissues and even in various subcellular compartments. Here, we describe how to perform FRET measurements in living cells expressing FRET-based biosensors and how to evaluate these data. This general protocol can be applied for FRET measurements with various fluorescent biosensors.

Key words

FRET Microscopy Imaging Fluorescence Biosensor 

Notes

Acknowledgments

The work in authors’ laboratory is supported by the Deutsche Forschungsgemeinschaft (grants NI 1301/1, FOR 2060), DZHK, and the Gertraud und Heinz Rose-Stiftung.

References

  1. 1.
    Herbst KJ, Ni Q, Zhang J (2009) Dynamic visualization of signal transduction in living cells: from second messengers to kinases. IUBMB Life 61:902–908CrossRefPubMedGoogle Scholar
  2. 2.
    Zaccolo M (2004) Use of chimeric fluorescent proteins and fluorescence resonance energy transfer to monitor cellular responses. Circ Res 94:866–873CrossRefPubMedGoogle Scholar
  3. 3.
    Sprenger JU, Nikolaev VO (2013) Biophysical techniques for detection of cAMP and cGMP in living cells. Int J Mol Sci 14:8025–8046CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Chan YH, Wu C, Ye F et al (2011) Development of ultrabright semiconducting polymer dots for ratiometric pH sensing. Anal Chem 83:1448–1455CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Busch C, Schroter T, Grabolle M et al (2012) An in vivo spectral multiplexing approach for the cooperative imaging of different disease-related biomarkers with near-infrared fluorescent forster resonance energy transfer probes. J Nucl Med 53:638–646CrossRefPubMedGoogle Scholar
  6. 6.
    Froese A, Nikolaev VO (2015) Imaging alterations of cardiomyocyte cAMP microdomains in disease. Front Pharmacol 6:172CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Börner S, Schwede F, Schlipp A et al (2011) FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells. Nat Protoc 6:427–438CrossRefPubMedGoogle Scholar
  8. 8.
    Sprenger JU, Perera RK, Steinbrecher JH et al (2015) In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease. Nat Commun 6:6965CrossRefPubMedGoogle Scholar
  9. 9.
    Sprenger JU, Perera RK, Gotz KR et al (2012) FRET microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors. J Vis Exp:e4081Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Institute of Experimental Cardiovascular ResearchUniversity Medical Center Hamburg-EppendorfHamburgGermany

Personalised recommendations