Nonlinear structured illumination microscopy with surface plasmon resonance enhanced stimulated emission depletion

Abstract

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 protein. 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 cells.