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Journal of the Optical Society of America B

Journal of the Optical Society of America B


  • Vol. 20, Iss. 3 — Mar. 1, 2003
  • pp: 554–559

Orientation imaging of single molecules by wide-field epifluorescence microscopy

Martin Böhmer and Jörg Enderlein  »View Author Affiliations

JOSA B, Vol. 20, Issue 3, pp. 554-559 (2003)

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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.

© 2003 Optical Society of America

OCIS Codes
(260.1960) Physical optics : Diffraction theory
(260.2510) Physical optics : Fluorescence
(300.2530) Spectroscopy : Fluorescence, laser-induced

Martin Böhmer and Jörg Enderlein, "Orientation imaging of single molecules by wide-field epifluorescence microscopy," J. Opt. Soc. Am. B 20, 554-559 (2003)

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