Two-dimensional nonlinear structured illumination microscopy with rsEGFP2

Nonlinear structured illumination microscopy has achieved resolutions down to 40 nm and imaging speeds up to 2.86 frames/s. However, relatively few realizations of NSIM have been published in the literature. Here, we demonstrate patterned depletion NSIM (PD-NSIM) with the fluorescent protein rsEGFP2. With its fast switching kinetics and large number of switching cycles, rsEGFP2 promises to be a useful fluorophore for NSIM, providing a combination of speed, resolution, and longevity that fills a gap in the imaging space. Here, we present the first demonstration of live 2D PD-NSIM using rsEGFP2. We demonstrate imaging of actin filaments in U2OS cells, achieving sub-80 nm resolution in live-cell imaging. We examine the images in both real-space and Fourier-space to support our claim of increased resolution.