Resolution Improvement of Structured Illumination Microscopy using Non‐Diffraction‐Limited Point‐Scanned Fluorescence Fringe

Abstract To further improve the resolving ability of structured illumination microscopy (SIM), a space‐time‐modulation–based laser‐scanning structured illumination microscopy (LSSIM) technique is presented in which a non‐diffraction‐limited non‐sinusoidal cumulative fluorescence fringe is generated in samples by point scanning the modulated excitation beam. The higher‐frequency information of samples can be mixed using the cumulative fringes with unlimited frequency into the passband of a confocal system. The non‐sinusoidal intensity‐modulated function used in LSSIM is specifically designed as a combination of cosine functions with different orders. The resolving ability of LSSIM experimentally by imaging fluorescent beads and bovine pulmonary artery endothelial cells with stained F‐actin is demonstrated. The results demonstrate three‐fold resolution enhancement over the confocal system, surpassing the resolving ability of conventional SIM. Moreover, the optical system is a simple modification of laser‐scanning confocal microscopy, making LSSIM more compatible with other techniques and suitable for biomedical research without the specific requirements for fluorescent dyes.