Structured illumination microscopy (SIM) breaks the resolution limit caused by optical diffraction, and nonlinear SIM can further improve the resolution with nonlinear effect. However, current nonlinear SIM methods such as Saturated SIM and Photo-switching SIM are unsatisfactory in biomedical imaging. The stimulated emission depletion (STED) effect is considered as a great nonlinear effect with fast switching response, negligible stochastic noise during switching, low shot noise and theoretical unlimited resolution. We propose an original nonlinear structured illumination microscopy based on both patterned excitation illumination and structured STED field (SSTED-SIM). Theoretical study and simulation results demonstrated that SSTED-SIM is capable of providing the ability of fast imaging speed, and low imaging noise at the same time compared with other nonlinear SIM techniques.
Nonlinear structured illumination microscopy (SIM) allows full-field imaging at resolutions <100 nm. Two
nonlinear effects, excitation saturation (SSIM) and the photo-switching of protein had been applied to nonlinear
SIM. We report a new SIM technique which utilizes the nonlinearity of STED effect. Resolution and signal noise
ratio simulation shows that STED-SIM may serve as a better alternative to SSIM and SIM with photo-switchable
SIM requires a strong nonlinear effect in a large area. We use Surface Plasmon Resonant to enhance of evanescence
field near a dielectric-metal-dielectric interface. An 8 times STED effect enhancement is achieved on an optimized
glass-silver-glass-water planar structure. We further use the interference of two SPR-enhanced STED fields
propagating at opposite direction to generate a 1D structured STED field. Combined with a uniform excitation field,
the structure STED field allows full field total internal reflection imaging with an enhanced resolution along the
structured dimension. Less than 50 nm resolution is demonstrated.
A STED-SIM microscope with 2D structured STED field is under development. Future research will apply the
microscope to superresolution imaging of membrane resident or near membrane structure at super-resolution in live