Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification
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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.
KeywordsFörster 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.
- 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
- 19.Leavesley SJ, Gao Y, Nakhmani A (submitted) Spectral image cytometry for automated subcellular cyclic nucleotide measurements. Front Physiol Vasc PhysiolGoogle Scholar
- 24.Sommer C, Straehle C, Kothe U et al (2011) Ilastik: interactive learning and segmentation toolkit. In: IEEE. ISBN: 1424441277, pp 230–233Google Scholar