Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification

  • Silas J. LeavesleyEmail author
  • Arie Nakhmani
  • Yi Gao
  • Thomas C. Rich
Part of the Methods in Molecular Biology book series (MIMB, volume 1294)


A variety of FRET probes have been developed to examine cAMP localization and dynamics in single cells. These probes offer a readily accessible approach to measure localized cAMP signals. However, given the low signal-to-noise ratio of most FRET probes and the dynamic nature of the intracellular environment, there have been marked limitations in the ability to use FRET probes to study localized signaling events within the same cell. Here, we outline a methodology to dissect kinetics of cAMP-mediated FRET signals in single cells using automated image analysis approaches. We additionally extend these approaches to the analysis of subcellular regions. These approaches offer an unique opportunity to assess localized cAMP kinetics in an unbiased, quantitative fashion.


Förster resonance energy transfer Image cytometry Microscopy Cyclic nucleotide 



This work was supported by NIH grants P01 HL066299 and S10 RR027535, and the Abraham Mitchell Cancer Research Fund.


  1. 1.
    Förster T (1948) Zwischenmolekulare energiewanderung und fluoreszenz. Ann Phys 437:55–75CrossRefGoogle Scholar
  2. 2.
    Clegg RM (1995) Fluorescence resonance energy transfer. Curr Opin Biotechnol 6:103–110CrossRefPubMedGoogle Scholar
  3. 3.
    Zaccolo M (2004) Use of chimeric fluorescent proteins and fluorescence resonance energy transfer to monitor cellular responses. Circ Res 94:866–873CrossRefPubMedGoogle Scholar
  4. 4.
    Mongillo M, McSorley T, Evellin S et al (2004) Fluorescence resonance energy transfer–based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ Res 95:67–75CrossRefPubMedGoogle Scholar
  5. 5.
    Ponsioen B, Zhao J, Riedl J et al (2004) Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep 5:1176–1180CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Nikolaev VO, Bünemann M, Hein L et al (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215–37218CrossRefPubMedGoogle Scholar
  7. 7.
    Nikolaev VO, Gambaryan S, Lohse MJ (2006) Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Methods 3:23–25CrossRefPubMedGoogle Scholar
  8. 8.
    Honda A, Adams SR, Sawyer CL et al (2001) Spatiotemporal dynamics of guanosine 3′, 5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci 98:2437–2442CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Leavesley SJ, Britain A, Cichon LK et al (2013) Assessing FRET using spectral techniques. Cytometry A 83:898–912PubMedGoogle Scholar
  10. 10.
    Rich TC, Britain AL, Stedman T et al (2013) Hyperspectral imaging of FRET-based cGMP probes. In: Krieg T, Lukowski R (eds) Guanylate cyclase and cyclic GMP: methods and protocols, vol 1020, 1st edn, Methods in molecular biology. Springer Science+Business Media, LLC, New York. ISBN 1627034587Google Scholar
  11. 11.
    Zimmermann T, Rietdorf J, Girod A et al (2002) Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2–YFP FRET pair. FEBS Lett 531:245–249CrossRefPubMedGoogle Scholar
  12. 12.
    Carpenter AE, Jones TR, Lamprecht MR et al (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7:R100CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Leavesley SJ, Annamdevula N, Boni J et al (2012) Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently-labeled cells in highly autofluorescent tissue. J Biophotonics 5:67–84CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Favreau PF, Hernandez C, Lindsey AS et al (2014) Tunable thin-film optical filters for hyperspectral microscopy. J Biomed Opt 19:011017-1–011017-11CrossRefGoogle Scholar
  15. 15.
    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
  16. 16.
    Gordon GW, Berry G, Liang XH et al (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74:2702–2713CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Sun Y, Hays NM, Periasamy A et al (2012) Monitoring protein interactions in living cells with fluorescence lifetime imaging microscopy. Methods Enzymol 504:371CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Gu Y, Di W, Kelsell D et al (2004) Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing. J Microsc 215:162–173CrossRefPubMedGoogle Scholar
  19. 19.
    Leavesley SJ, Gao Y, Nakhmani A (submitted) Spectral image cytometry for automated subcellular cyclic nucleotide measurements. Front Physiol Vasc PhysiolGoogle Scholar
  20. 20.
    Berney C, Danuser G (2003) FRET or no FRET: a quantitative comparison. Biophys J 84:3992–4010CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Klarenbeek JB, Goedhart J, Hink MA et al (2011) A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range. PLoS One 6:e19170CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Novo D, Grégori G, Rajwa B (2013) Generalized unmixing model for multispectral flow cytometry utilizing nonsquare compensation matrices. Cytometry A 83A:508–520CrossRefGoogle Scholar
  23. 23.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682CrossRefPubMedGoogle Scholar
  24. 24.
    Sommer C, Straehle C, Kothe U et al (2011) Ilastik: interactive learning and segmentation toolkit. In: IEEE. ISBN: 1424441277, pp 230–233Google Scholar
  25. 25.
    Rich TC, Webb KJ, Leavesley SJ (2014) Can we decipher the information content contained within cyclic nucleotide signals? J Gen Physiol 143:17–27CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Feinstein WP, Zhu B, Leavesley SJ et al (2012) Assessment of cellular mechanisms contributing to cAMP compartmentalization in pulmonary microvascular endothelial cells. Am J Physiol Cell Physiol 302:C839–C852CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Beavo J, Bechtel P, Krebs E (1974) Activation of protein kinase by physiological concentrations of cyclic AMP. Proc Natl Acad Sci 71:3580–3583CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Ljosa V, Carpenter AE (2009) Introduction to the quantitative analysis of two-dimensional fluorescence microscopy images for cell-based screening. PLoS Comput Biol 5:e1000603CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Silas J. Leavesley
    • 1
    • 2
    • 3
    Email author
  • Arie Nakhmani
    • 4
  • Yi Gao
    • 6
  • Thomas C. Rich
    • 2
    • 3
    • 5
  1. 1.Department of Chemical and Biomolecular EngineeringUniversity of South AlabamaMobileUSA
  2. 2.Department of PharmacologyUniversity of South AlabamaMobileUSA
  3. 3.Center for Lung BiologyUniversity of South AlabamaMobileUSA
  4. 4.Department of Electrical and Computer EngineeringUniversity of Alabama at BirminghamBirminghamUSA
  5. 5.College of EngineeringUniversity of South AlabamaMobileUSA
  6. 6.Department of Biomedical InformaticsStony Brook UniversityStony BrookUSA

Personalised recommendations