Stimulated emission–depletion-based point-scanning structured illumination microscopy

Wide-field linear structured illumination microscopy (LSIM) extends resolution beyond the diffraction limit by moving unresolvable high-frequency information into the passband of the microscopy in the form of moiré fringes. However, due to the diffraction limit, the spatial frequency of the structured illumination pattern cannot be larger than the microscopy cutoff frequency, which results in a twofold resolution improvement over wide-field microscopes. This Letter presents a novel approach in point-scanning LSIM, aimed at achieving higher-resolution improvement by combining stimulated emission depletion (STED) with point-scanning structured illumination microscopy (psSIM) (STED-psSIM). The according structured illumination pattern whose frequency exceeds the microscopy cutoff frequency is produced by scanning the focus of the sinusoidally modulated excitation beam of STED microscopy. The experimental results showed a 1.58-fold resolution improvement over conventional STED microscopy with the same depletion laser power.