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

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.