Time synchronization of LED excitation source and dual-camera for quantitative FRET imaging
Hai Wen1, Huan Zuo1, Xinghong Cai1
1MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou 510631, China; Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou 510631, China.
This study introduces a synchronized six-wavelength LED light source for fluorescence microscopy. It significantly reduces photobleaching and improves the accuracy of quantitative fluorescence resonance energy transfer (FRET) imaging in live cells.
Area of Science:
- Biophysics
- Microscopy
- Molecular Imaging
Background:
- Multi-wavelength LED light sources are crucial for fluorescence microscopy.
- Accurate timing between excitation and imaging is vital for quantitative fluorescence resonance energy transfer (FRET).
- Existing systems often require complex external hardware for synchronization.
Purpose of the Study:
- To develop a novel six-wavelength LED excitation source with integrated time synchronization.
- To enable high-precision synchronization between LED excitation and dual-camera imaging for quantitative FRET.
- To reduce photobleaching and improve the reliability of live-cell FRET analysis.
Main Methods:
- An STM32F429 microcontroller was used to build a hardware-triggered synchronization control system.
- The system was integrated into a dual-channel fluorescence microscope.
- Quantitative FRET analysis was performed in living cells using standard FRET plasmids.
Main Results:
- Excitation-exposure timing discrepancy was minimized to 10μs.
- Photobleaching-induced deviations in FRET efficiency (ED) and acceptor-to-donor ratio (Rc) were reduced from 18.8% and 15.8% to 5.6% and 2.6%.
- The reliability of FRET quantification was significantly enhanced.
Conclusions:
- The developed synchronized LED excitation source improves the accuracy of dynamic molecular measurements in live-cell imaging.
- This system offers a robust solution for quantitative FRET studies without additional control hardware.
- Enhanced FRET quantification aids in studying protein interactions and signaling pathways.
