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Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Published on: April 14, 2015

Calibration of FRET-based biosensors using multiplexed biosensor barcoding.

Jhen-Wei Wu, Jr-Ming Yang, Chao-Cheng Chen

Biorxiv : the Preprint Server for Biology

|September 16, 2024

Summary

This summary is machine-generated.

This study introduces a calibration method for Förster resonance energy transfer (FRET) biosensors, ensuring reliable measurements across diverse imaging conditions. The new approach enables accurate, long-term monitoring of cellular activities using FRET imaging.

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Published on: April 7, 2023

Area of Science:

Biophysics
Cell Biology
Molecular Imaging

Background:

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is crucial for genetically encoded biosensors used in live-cell imaging.
The FRET ratio, a proxy for FRET efficiency, is sensitive to imaging conditions, hindering cross-experiment comparisons.
Existing methods require correction for signal crosstalk, limiting practical applications.

Purpose of the Study:

To develop a robust calibration strategy for FRET biosensor imaging.
To enable accurate and reproducible measurements of biochemical dynamics in live cells.
To facilitate highly multiplexed and long-term FRET imaging studies.

Main Methods:

Theoretical analysis of FRET ratio dependence on excitation intensity.
Development of "FRET-ON" and "FRET-OFF" calibration standards.
Introduction of calibration standards into barcoded cells for multiplexed imaging.
Validation of calibrated FRET ratio independence from imaging settings.

Main Results:

Theoretical predictions regarding FRET ratio changes at high excitation intensity were confirmed.
Calibrated FRET ratios demonstrated independence from varying imaging conditions.
A strategy for calculating FRET efficiency was successfully implemented.
The method allows for reliable, multiplexed FRET biosensor imaging.

Conclusions:

A simple and effective strategy for calibrated FRET biosensor imaging has been established.
This approach enhances the reliability and comparability of FRET measurements across experiments.
The findings support advanced applications in live-cell dynamics monitoring and long-term imaging.