Far-field fluorescence microscopy beyond the diffraction limit

A technique has been developed to obtain three-dimensional structural information on a length scale well below the Rayleigh length with conventional far-field optics. By spectrally selecting a single molecule with high-resolution laser spectroscopy and using a CCD camera to register the spatial distribution of the emitted photons in three dimensions, one can determine the position of a molecule with unprecedented accuracy. One can resolve details in the specimen with sub-diffraction-limited resolution in three dimensions by applying this procedure to as many molecules as are present in the same diffraction-limited volume and obtaining their mutual positions. The feasibility of this technique is demonstrated for the system of pentacene in p-terphenyl at cryogenic temperatures for which molecules were localized with an accuracy of better than 40 nm in the lateral and 100 nm in the axial directions.