S: Widefield Light Microscopy with 100-nm-scale Resolution in Three Dimensions

2 ABSTRACT A new type of widefield fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses. Illumination light is split by a grating and a beam splitter into six mutually coherent beams, three of which enter the specimen through each objective lens. The resulting illumination intensity pattern contains high spatial frequency components both axially and laterally. In addition, the emission is collected by both objective lenses coherently, and combined interferometrically on a single camera, resulting in a detection transfer function with axially extended support. These two effects combine to produce near-isotropic resolution. Experimental images of test samples and biological specimens confirm the theoretical predictions. INTRODUCTION A considerable number of techniques for enhancing the spatial resolution of light microscopy have been developed over the last two decades (1–20). The number of underlying concepts is much smaller, however, and most of the techniques can be seen as based on these three: (a) collecting light over a broader set of angles by combining separate apertures (1–3); (b) using nonuniform illumination light (either a focused beam (4, 5) or a periodic pattern (6–13)), by itself or combined with nonlinear photoresponses (4, 5, 14–16); or (c) precisely locating molecules or particles individually (18–20). Here we describe a combination of structured illumination microscopy, which is based on concept (b), with I