Super-Resolution Confocal Microscopy Enabled by Fluorescence Intensity Ratio Encoding and Difference

We present fluorescence intensity ratio encoding and difference (FIRED) microscopy, an innovative far-field super-resolution method capable of surpassing the optical diffraction barrier in fluorescence microscopy. By dynamically modulating the effective excitation spot in an optimized confocal microscope, this method uses the unique ratio of fluorescence intensity between two images generated via distinct excitation light paths to derive a weight matrix. The weight matrix encodes the original confocal image to generate the super-resolution FIRE image. Subsequent differential modulation between this FIRE image and a weighted confocal image produces the final FIRED image, suppressing artifacts while enhancing both resolution and background contrast. Experimental validations confirmed that FIRED achieves a resolution improvement of up to 4-fold over conventional confocal microscopy, attaining a lateral resolution of 71.2 nm. Additionally, FIRED exhibits an enhanced signal-to-background ratio, positioning it as a robust tool for pushing the limits of confocal microscopy in biological and materials science applications.