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Applied Optics

Applied Optics

APPLICATIONS-CENTERED RESEARCH IN OPTICS

  • Vol. 28, Iss. 24 — Dec. 15, 1989
  • pp: 5237–5242

Evanescent field excitation of fluorescence by epi-illumination microscopy

Andrea L. Stout and Daniel Axelrod  »View Author Affiliations


Applied Optics, Vol. 28, Issue 24, pp. 5237-5242 (1989)
http://dx.doi.org/10.1364/AO.28.005237


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Abstract

By simple modification of the pattern of fluorescence excitation light in an epi-illumination inverted microscope, one can achieve conditions that produce total internal reflection fluorescence (TIRF) by evanescent wave excitation. Though traditionally requiring a collimated beam traversing through a special prism, TIRF also can be achieved by epi-illumination through the periphery of a 1.4 numerical aperture objective. An opaque disk of appropriate size is placed in the illumination path external to the microscope so as to cast a sharp, real-image shadow at the objective's back focal plane. This shadow allows a hollow cone of epi-illumination rays traveling at only super-critical angles to reach the glass/water interface at the sample plane. Three kinds of TIRF illumination patterns can be produced by variations of this scheme: (1) a small spot of illumination of 1.5 μm radius by use of a laser light source, (2) a large region of illumination by use of a laser-illuminated diffusing screen located upbeam from the opaque disk, and (3) a large region of illumination by use of a conventional mercury arc.

© 1989 Optical Society of America

History
Original Manuscript: May 30, 1989
Published: December 15, 1989

Citation
Andrea L. Stout and Daniel Axelrod, "Evanescent field excitation of fluorescence by epi-illumination microscopy," Appl. Opt. 28, 5237-5242 (1989)
http://www.opticsinfobase.org/ao/abstract.cfm?URI=ao-28-24-5237

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