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Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission
Published on: April 22, 2021
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Automated E-FRET microscope for dynamical live-cell FRET imaging
1College of Biophotonics & MOE Lab. of Laser Life Science, South China Normal University, Guangzhou, China.
Journal of Microscopy
|January 29, 2019
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.
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.
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