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Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and λ) Hyperspectral FRET Imaging and Analysis
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Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and λ) Hyperspectral FRET Imaging and Analysis

Article DOI: 10.3791/61720-v • 08:22 min • October 27th, 2020
October 27th, 2020 •
1,2, 3, 1, 3, 1,2, 1,2, 1,2,3
1Department of Pharmacology, University of South Alabama, 2Center for Lung Biology, University of South Alabama, 3Department of Chemical and Biomolecular Engineering, University of South Alabama

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Summary
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Due to inherent low signal-to-noise ratio (SNR) of Fӧrster resonance energy transfer (FRET) based sensors, measurement of cAMP signals has been challenging, especially in three spatial dimensions. Here, we describe a hyperspectral FRET imaging and analysis methodology that allows measurement of cAMP distribution in three spatial dimensions.

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3-Dimensional CAMP Distributions Living Cells 4-Dimensional X Y Z λ Hyperspectral FRET Imaging Analysis Protocol Measuring Visualizing FRET Cyclic AMP Signals Spatial Dimensions Individual Cells Axially Laterally Acquire FRET Cyclic AMP Image Data Three Dimensions Sample Distributions Imaging Warm Tyrodes Buffer Water Bath Cover Slip Seated Transfected Cells Cell Chamber Mounting Gasket Leaking Prevention Delicate Task Wiper Excess Medium Removal Adherent Cells Removal Working Buffer Addition Nuclear Label Addition Gentle Rocking Of The Chamber For Incubation Aluminum Foil Covering For Incubation Period At Room Temperature
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