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