Time synchronization of LED excitation source and dual-camera for quantitative FRET imaging

Multi-wavelength LED light source has been widely used as excitation source in fluorescence microscopy. This study presents a six-wavelength LED excitation source that integrates an internal time synchronization control mechanism, enabling high-precision synchronization between LED excitation and dual-camera imaging without requiring additional control hardware. The control system is built around an STM32F429 microcontroller and employs a hardware-triggered synchronization strategy specifically designed for quantitative fluorescence resonance energy transfer (FRET) imaging. By incorporating this excitation source into a dual-channel fluorescence microscope, we minimized the excitation-exposure timing discrepancy to $10\mu s$, effectively reducing unnecessary exposure and mitigating photobleaching. To assess system performance, we conducted quantitative FRET analysis in living cells expressing different standard FRET plasmids. Experimental results demonstrated that during time-lapse FRET analysis, the synchronization system reduced photobleaching-induced deviations in FRET efficiency ($E_D$) and acceptor-to-donor ratio ($R_c$) from 18.8% and 15.8% to 5.6% and 2.6%, respectively, thereby enhancing the reliability of FRET quantification. As FRET is widely used to study protein interactions, conformational changes, and signal transduction pathways, this system improves the accuracy of dynamic molecular measurements in live-cell imaging.