Super-resolution imaging and detection of fluorescence from single      molecules by scanning near-field optical microscopy

Alfred J. Meixner; Dieter Zeisel; A. Martin Bopp; Guido Tarrach

doi:10.1117/12.200620

1 August 1995 Super-resolution imaging and detection of fluorescence from single molecules by scanning near-field optical microscopy

Alfred J. Meixner, Dieter Zeisel, A. Martin Bopp, Guido Tarrach

Author Affiliations +

Optical Engineering, Vol. 34, Issue 8, (August 1995). https://doi.org/10.1117/12.200620

Abstract

Scanning near-field microscopy (SNOM or NSOM) is a versatile and attractive scanning probe technique for imaging with subdiffraction-limited spatial resolution using visible light. At least three different types of images can be recorded simultaneously of the selected sample area, such as the topography, the near-field optical transmission, and the fluorescence from excited chromophores. We have built such a microscope, especially designed for achieving the high resolution and the sensitivity needed for single molecule detection. We report on optical near-field investigations of surface structures and thin polymer films that are doped with fluorescent dye molecules. The effective aperture diameters of the fiber tips used in the SNOM experiments were determined by a photon-scanning tunneling microscope (PSTM) giving values between 70 and 160 nm. The transmission imaging of transparent polymer phase gratings reveals the existence of different contrast mechanisms, which are either based on the inherent distance dependence of the optical near field or on the periodic change of boundary conditions for the electric field component of the light between the aperture and the sample. Furthermore, we demonstrate selective irreversible photobleaching of dye molecules at moderate concentration (10^-5 M) induced locally by the subwavelength-sized probe tip. Finally, we present fluorescence images showing single molecule detection in a thin solid film. The chromophores (rhodamine 6G) were embedded at low concentration (10^-7 M) in a 25-nm thin polyvinylbutyral film. A lateral resolution of 160 nm was achieved. We find that the signal strengths of the brightest fluorescent features vary considerably in a sequence of images (a typical single-molecule behavior), whereas the fluorescence background exhibits the usual photobleaching behavior of a large ensemble.

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Alfred J. Meixner, Dieter Zeisel, A. Martin Bopp, and Guido Tarrach "Super-resolution imaging and detection of fluorescence from single molecules by scanning near-field optical microscopy," Optical Engineering 34(8), (1 August 1995). https://doi.org/10.1117/12.200620

Published: 1 August 1995

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