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

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

DOI:
10.3791/61720-v

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08:22 min

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October 27, 2020

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, , , , , ,
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

Chapitres

  • 00:04Introduction
  • 00:34Imaging Preparation
  • 01:16Image Acquisition
  • 04:00Image Analysis
  • 05:22FRET Efficiency Calculation and Mapping
  • 06:26Results: Representative 3D cAMP Distribution and Hyperspectral FRET Imaging and Analysis
  • 07:57Conclusion

Summary

Traduction automatique

Traduction automatique

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.

Tags

Keywords: 3D CAMP Distribution4D Hyperspectral FRET ImagingCyclic AMPFRETConfocal MicroscopySpectral Imaging3D Cell ImagingLive Cell ImagingTyrodes BufferNuclear LabelingLaser SettingsZ-stack Acquisition

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