Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification

  • Silas J. Leavesley
  • Arie Nakhmani
  • Yi Gao
  • Thomas C. Rich
Protocol
Part of the Methods in Molecular Biology book series (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.

Keywords

Förster resonance energy transfer Image cytometry Microscopy Cyclic nucleotide 

Notes

Acknowledgement

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

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Silas J. Leavesley
    • 1
    • 2
    • 3
  • 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

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