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Updated: Jan 20, 2026

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Triple Fluorescence Anisotropy Reporter Imaging in Living Cells.

Brian Ross1,2, Shenq Huey Wong3, Nicole E Snell3

  • 1Department of Pharmacology, University of California, San Diego, La Jolla, California, United States.

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|August 20, 2019
PubMed
Summary
This summary is machine-generated.

New FLuorescence Anisotropy REporters (FLAREs) enable multicolor imaging of three intracellular signals simultaneously. This polarization-based multiplexing advances live-cell biosensing by freeing up the optical spectrum for additional tools.

Keywords:
AnisotropyFRETGFPHomotransferImagingPolarization

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Area of Science:

  • Cellular Biology
  • Biophysics
  • Molecular Imaging

Background:

  • Genetically encoded biosensors typically use two different fluorescent proteins for ratiometric measurements.
  • This approach limits spectral availability for additional sensors or optogenetic tools.
  • Homotransfer biosensors, like FLAREs, use same-color proteins to overcome spectral crowding.

Purpose of the Study:

  • To present a protocol for multicolor imaging using FLuorescence Anisotropy REporters (FLAREs).
  • To demonstrate simultaneous measurement of three independent intracellular signals in live cells.
  • To enable advanced multiplexed biosensing applications.

Main Methods:

  • Configuration and calibration of a widefield microscope for multicolor FLARE imaging.
  • Co-transfection of three FLARE sensors (calcium, MAPK, PKA) into HEK293 cells.
  • Triple Förster Resonance Energy Transfer (FRET) imaging using polarization measurements.

Main Results:

  • Successful triple FRET imaging of independent intracellular signals.
  • Demonstration of FLAREs' capability for multicolor live-cell imaging.
  • Comparison showing polarization-based multiplexing is advantageous over heterotransfer FRET biosensors.

Conclusions:

  • FLAREs offer a powerful method for multiplexed live-cell biosensing.
  • Polarization-based imaging expands spectral capacity for simultaneous measurements.
  • This technique allows concurrent tracking of multiple signaling activities in targeted cell populations.