Orientation imaging of single molecules by wide-field epifluorescence microscopy

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Abstract
A simple imaging method for direct determination of single-molecule orientations is presented that uses a wide-field epifluorescence microscope and a sensitive CCD camera. Imaging is performed with slight defocusing of the optics, allowing for direct determination of single-molecule orientation based on the characteristic intensity distribution of the defocused images. Exact wave-optical calculations of these defocused images are presented and are in good agreement with the measurements. These calculations represent what is to the authors’ knowledge the first complete wave-optical modeling of defocused imaging of dipole emitters at an interface; the peculiarities of dipole emission at an interface and the vector effects of that emission and of imaging with a high-numerical-aperture objective are taken into account.