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Journal of Biomedical Optics

Parallel two-channel near- and far-field fluorescence microscopy
Author(s): Doriel Verdes; Thomas Ruckstuhl; Stefan Seeger
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Paper Abstract

We report a new two-channel fluorescence microscopy technique for surface-generated fluorescence. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic mirror objective that is used to collect the fluorescence at large surface angles above the critical angle of the water/glass interface. An aspheric lens, incorporated into the solid parabolic element, is used for diffraction-limited laser focusing and for collecting fluorescence at low angles with respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and subcritical angles, two detection volumes strongly differing in their axial resolution are generated at the surface of a glass cover slip. The collection of supercritical angle fluorescence (SAF) results in a strict surface confinement of the detection volume, whereas collecting below the critical angle allows gathering the fluorescence emitted several microns deep inside the sample. Consequently, the signals from surface-bound and unbound diffusing fluorescent molecules can be obtained simultaneously.

Paper Details

Date Published: 1 May 2007
PDF: 7 pages
J. Biomed. Opt. 12(3) 034012 doi: 10.1117/1.2747627
Published in: Journal of Biomedical Optics Volume 12, Issue 3
Show Author Affiliations
Doriel Verdes, Univ. of Zurich (Switzerland)
Thomas Ruckstuhl, Univ. of Zürich (Switzerland)
Stefan Seeger, Univ. of Zürich (Switzerland)

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