Sub-Diffraction-Limit Imaging with Stochastic Optical Reconstruction Microscopy

Abstract

Light microscopy is a widely used imaging method in biomedical research. However, the resolution of conventional optical microcopy is limited by the diffraction of light, making structures smaller than 200 nm difficult to resolve. To overcome this limit, we have developed a new form of fluorescence microscopy - Stochastic Optical Reconstruction Microscopy (STORM). STORM makes use of single-molecule imaging methods and photo-switchable fluorescent probes to temporally separate the otherwise spatially overlapping images of individual molecules. An STORM image is acquired over a number of imaging cycles, and in each cycle only a subset of the fluorescent labels is switched on such that each of the active fluorophores is optically resolvable from the rest. This allows the position of these fluorophores to be determined with nanometer accuracy. Over the course of many such cycles, the positions of numerous fluorophores are determined and used to construct a super-resolution image. Using this method, we have demonstrated multi-color, three-dimensional (3D) imaging of biomolecules and cells with ∼ 20 nm lateral and ∼ 50 nm axial resolutions. In principle, the resolution of this technique can reach the molecular scale.