Full Field Nonlinear Structured Illumination Microscopy with STED

Abstract

Structured illumination microscopy (SIM) allows fast full-field imaging of biological samples. Linear SIM improves the lateral resolution limit by a factor of two. Once combined with a nonlinear effect of the fluorescence emission, SIM can further suppress the resolution limit to <100 nm, as reported by Photoswitchable and excitation saturated SIM (SSIM) [1, 2]. Here, we present a novel nonlinear SIM method named STED-SIM, which utilizes the stimulated emission depletion (STED) effect to achieve 4-fold resolution enhancement [3].STED-SIM utilizes a 2D grating to generate a 2D structured illumination pattern. The grating is shifted at high speed by piezo stages. Therefore, the imaging speed of 2D STED-SIM in only limited by the speed of the camera. Compared with previously reported nonlinear SIM approaches, STED-SIM has the advantage of fast switching response, negligible stochastic noise in switching. In addition, the achievable resolution of STED-SIM in theory is unlimited.STED-SIM microscope was first tested on fluorescent beads samples and achieved full field imaging over 10×10 [μm]ˆ2 at the speed of 2s/frame with 4-fold improved resolution. Imaging experiments of biological samples are under way.1. Gustafsson M. G. L. (2005). Proc. Natl. Acad. Sci. U.S.A. 10213081-13086.2. Rego E. H., Shao L., Macklin J. J., Winoto L., Johansson G. A., Kamps-Hughes N., et al. (2012). Proc. Natl. Acad. Sci. U.S.A. 109 E135-E143.3. Zhang H., Zhao M. and Peng L. (2011). Optics Express, 19, 24783-24794.