Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification

Silas J Leavesley; Naga Annamdevula; Santina Johnson; D J Pleshinger; Thomas C Rich

doi:10.1007/978-1-0716-2245-2_10

Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification

Methods Mol Biol. 2022:2483:167-180. doi: 10.1007/978-1-0716-2245-2_10.

Authors

Silas J Leavesley^{1

2

3}, Naga Annamdevula^{4

5

6}, Santina Johnson^{4

5}, D J Pleshinger^{4

5}, Thomas C Rich^{4

5

6

7}

Affiliations

¹ Department of Chemical and Biomolecular Engineering, University of South Alabama, Mobile, AL, USA. leavesley@southalabama.edu.
² Department of Pharmacology, University of South Alabama, Mobile, AL, USA. leavesley@southalabama.edu.
³ Center for Lung Biology, University of South Alabama, Mobile, AL, USA. leavesley@southalabama.edu.
⁴ Department of Pharmacology, University of South Alabama, Mobile, AL, USA.
⁵ Center for Lung Biology, University of South Alabama, Mobile, AL, USA.
⁶ Department of Physiology, University of South Alabama, Mobile, AL, USA.
⁷ College of Engineering, University of South Alabama, Mobile, AL, USA.

PMID: 35286675
DOI: 10.1007/978-1-0716-2245-2_10

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 a unique opportunity to assess localized cAMP kinetics in an unbiased, quantitative fashion.

Keywords: Cyclic nucleotide; Förster resonance energy transfer; Image cytometry; Microscopy.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't

MeSH terms

Cyclic AMP*
Fluorescence Resonance Energy Transfer* / methods
Image Processing, Computer-Assisted / methods
Kinetics
Signal-To-Noise Ratio

Substances

Cyclic AMP