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Journal of Biomedical Optics

Super-resolution measurements with evanescent-wave fluorescence-excitation using variable beam incidence
Author(s): Dinah Loerke; Beate Preitz; Walter Stuhmer; Martin Oheim
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Paper Abstract

The evanescent wave (EW) elicited by total internal reflection of light selectively excites fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epiillumination microscopy, and—close to the coverslip—can offer a fivefold enhancement of axial optical sectioning compared to confocal and two-photon microscopy. The decay length of the evanescent field is a function of the refractive indices and light wavelength involved, and is modulated by the beam angle. EW microscopy was used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution. We modified an upright microscope to accommodate the condenser optics needed for EW excitation. Systematic variations of the angle of incidence were attained using an acousto-optical deflector, telecentric optics, and a hemicylindrical prism. The three-dimensional reconstruction of the fluorophore distribution from angle-resolved image stacks results in topographical information with an axial resolution of tens of nanometers. We applied this technique to study the axial position of dye-labeled subcellular storage organelles (’vesicles’) of ?300 nm diameter in the ‘‘footprint’’ region of living neuroendocrine cells grown on the interface.

Paper Details

Date Published: 1 January 2000
PDF: 8 pages
J. Biomed. Opt. 5(1) doi: 10.1117/1.429964
Published in: Journal of Biomedical Optics Volume 5, Issue 1
Show Author Affiliations
Dinah Loerke, Max-Planck Institute (Germany)
Beate Preitz, Max-Planck Institute (Germany)
Walter Stuhmer, Max-Planck Institute (Germany)
Martin Oheim, Ecole Superieure de Physique et Chimie Industriell (France)

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