Dual-channel structured illumination super-resolution quantitative FRET imaging

The Structured Illumination (SI)-based Super Resolution Fluorescence Resonance Energy Transfer (SR-FRET) imaging technique, known as SISR-FRET, enables the investigation of molecular structures and functions in cellular organelles by resolving sub-diffraction FRET signals within living cells. FRET microscopy offers unique advantages for quantitatively detecting dynamic interactions and spatial distribution of biomolecules within living cells. The spatial resolution of conventional FRET microscopy is limited by the diffraction limit, and it can only capture the average behavior of these events within the resolution limits of conventional fluorescence microscopy. SISR-FRET performs sequential linear reconstruction of the three-channel SIM images followed by FRET quantitative analysis using a common localization mask-based filtering approach. This two-step process ensures the fidelity of the reconstructed SR-FRET signals while effectively removing false-positive FRET signals caused by SIM artifacts. However, the slow imaging speed resulting from the switching of excitation-emission channels in SISR-FRET imaging limits its application in fast imaging scenarios. To address this issue, this study proposes a dual-channel structured illumination super-resolution quantitative FRET imaging system and method. By incorporating a FRET dual-channel imaging and registration module into the imaging pathway, spatial switching and channel multiplexing of the SISR-FRET excitation-emission channels are achieved. Combining the image reconstruction algorithm with channel sub-pixel registration correction, the dual-channel SISR-FRET technique enhances the temporal resolution by 3.5 times while preserving the quantitative super-resolution FRET analysis. Experimental results were obtained using a multi-color SIM system to perform super-resolution imaging of living cells expressing mitochondria outer membrane FRET standard plasmids. These experiments validate the improved spatial and temporal resolution of dual-channel SISR-FRET and the fidelity of FRET quantification analysis. In summary, this research presents a novel dual-channel structured illumination super-resolution FRET imaging system and methodology. It overcomes the limitations of slow imaging speed in SISR-FRET by enabling spatial switching and channel multiplexing of excitation-emission channels. The proposed technique enhances the temporal resolution while maintaining quantitative analysis of super-resolution FRET. Experimental validation demonstrates the increased spatial and temporal resolution of dual-channel SISR-FRET and the accuracy of FRET quantification analysis. This advancement contributes to the study of molecular structures and functions in cellular organelles, providing valuable insights into the intricate mechanisms of living cells.