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Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification

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cAMP Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1294))

Abstract

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.

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Acknowledgement

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

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of South Alabama, 150 Jaguar Drive, SH 4129, Mobile, AL, 36688, USA

    Silas J. Leavesley

  2. Department of Pharmacology, University of South Alabama, Mobile, AL, USA

    Silas J. Leavesley & Thomas C. Rich

  3. Center for Lung Biology, University of South Alabama, Mobile, AL, USA

    Silas J. Leavesley & Thomas C. Rich

  4. Department of Electrical and Computer Engineering, University of Alabama at Birmingham, Birmingham, AL, USA

    Arie Nakhmani

  5. College of Engineering, University of South Alabama, Mobile, AL, USA

    Thomas C. Rich

  6. Department of Biomedical Informatics, Stony Brook University, Stony Brook, NY, USA

    Yi Gao

Authors
  1. Silas J. Leavesley
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  2. Arie Nakhmani
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  3. Yi Gao
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  4. Thomas C. Rich
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Corresponding author

Correspondence to Silas J. Leavesley .

Editor information

Editors and Affiliations

  1. Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, United Kingdom

    Manuela Zaccolo

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© 2015 Springer Science+Business Media New York

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Leavesley, S.J., Nakhmani, A., Gao, Y., Rich, T.C. (2015). Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification. In: Zaccolo, M. (eds) cAMP Signaling. Methods in Molecular Biology, vol 1294. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2537-7_5

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  • DOI: https://doi.org/10.1007/978-1-4939-2537-7_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2536-0

  • Online ISBN: 978-1-4939-2537-7

  • eBook Packages: Springer Protocols

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