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Related Experiment Video

Updated: Jan 30, 2026

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission
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Automated E-FRET microscope for dynamical live-cell FRET imaging.

C Zhang1, Y Liu1, H Sun1

  • 1College of Biophotonics & MOE Lab. of Laser Life Science, South China Normal University, Guangzhou, China.

Journal of Microscopy
|January 29, 2019
PubMed
Summary

An automated microscope speeds up quantitative live-cell FRET imaging from 12 to 3 seconds. This advancement enables dynamic monitoring of cellular processes like Bax redistribution during apoptosis.

Keywords:
Biochemical signal transductionFRETmicroscopesystem design

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • Acceptor-sensitised 3-cube fluorescence resonance energy transfer (FRET) imaging, or E-FRET, is a key method for quantifying FRET in cells.
  • Current manual E-FRET measurements are time-consuming (12 s), require skilled operators, and pose challenges for dynamic live-cell imaging.
  • There is a need for faster, more accessible methods for quantitative live-cell FRET imaging to study dynamic cellular processes.

Purpose of the Study:

  • To develop and implement an automated E-FRET microscope for rapid, quantitative live-cell FRET imaging.
  • To reduce the time required for E-FRET measurements and improve user-friendliness.
  • To enable dynamic online quantitative live-cell FRET imaging for studying cellular signal transduction.

Main Methods:

  • An automated E-FRET microscope with a user-friendly interface was constructed.
  • The system automates cell location, microscope calibration, and E-FRET image acquisition with a single 'Capture' click.
  • Live cells expressing FRET tandem constructs (e.g., CFP-Bax and YFP-Bax) were imaged dynamically.

Main Results:

  • The automated microscope reduced quantitative E-FRET imaging time from 12 seconds to 3 seconds.
  • Dynamic imaging of CFP-Bax and YFP-Bax coexpressing cells treated with staurosporine revealed three distinct Bax redistribution stages.
  • These stages included translocation to mitochondria (10 min), mitochondrial membrane insertion/conformational change (30 min), and oligomerization (10 min).

Conclusions:

  • The automated E-FRET microscope offers a convenient and stable platform for quantitative FRET imaging of living cells.
  • Its user-friendly interface and speed facilitate dynamic FRET measurements, crucial for understanding cellular signaling.
  • This technology enhances the application of FRET for mapping biochemical signal transduction pathways in real-time.