Research Papers

Super-resolution measurements with evanescent-wave fluorescence excitation using variable beam incidence

[+] Author Affiliations
Dinah Loerke

Max-Planck Institute for Experimental Medicine, D-37075?Go¨ttingen, Germany

Beate Preitz

Max-Planck Institute for Experimental Medicine, D-37075?Go¨ttingen, Germany

Walter Stu¨hmer

Max-Planck Institute for Experimental Medicine, D-37075?Go¨ttingen, Germany

Martin Oheim

Max-Planck Institute for Experimental Medicine, Go¨ttingen, Germany, and Membrane Biology Group, University of Edinburgh, Scotland

J. Biomed. Opt. 5(1), 23-30 (Jan 01, 2000). doi:10.1117/1.429964
History: Received Mar. 8, 1999; Revised Oct. 20, 1999; Accepted Nov. 1, 1999
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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. © 2000 Society of Photo-Optical Instrumentation Engineers.

© 2000 Society of Photo-Optical Instrumentation Engineers

Citation

Dinah Loerke ; Beate Preitz ; Walter Stu¨hmer and Martin Oheim
"Super-resolution measurements with evanescent-wave fluorescence excitation using variable beam incidence", J. Biomed. Opt. 5(1), 23-30 (Jan 01, 2000). ; http://dx.doi.org/10.1117/1.429964


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