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Applied Optics

Applied Optics


  • Editor: Joseph N. Mait
  • Vol. 48, Iss. 32 — Nov. 10, 2009
  • pp: 6120–6131

Around-the-objective total internal reflection fluorescence microscopy

Thomas P. Burghardt, Andrew D. Hipp, and Katalin Ajtai  »View Author Affiliations

Applied Optics, Vol. 48, Issue 32, pp. 6120-6131 (2009)

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Total internal reflection fluorescence (TIRF) microscopy uses the evanescent field on the aqueous side of a glass/aqueous interface to selectively illuminate fluorophores within 100 nm of the interface. Applications of the method include epi-illumination TIRF, where the exciting light is refracted by the microscope objective to impinge on the interface at incidence angles beyond critical angle, and prism-based TIRF, where exciting light propagates to the interface externally to the microscope optics. The former has higher background autofluorescence from the glass elements of the objective where the exciting beam is focused, and the latter does not collect near-field emission from the fluorescent sample. Around-the- objective TIRF, developed here, creates the evanescent field by conditioning the exciting laser beam to propagate through the submillimeter gap created by the oil immersion high numerical aperture objective and the glass coverslip. The approach eliminates background light due to the admission of the laser excitation to the microscopic optics while collecting near-field emission from the dipoles excited by the 50 nm deep evanescent field.

© 2009 Optical Society of America

OCIS Codes
(110.0180) Imaging systems : Microscopy
(170.0180) Medical optics and biotechnology : Microscopy
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(180.2520) Microscopy : Fluorescence microscopy
(260.6970) Physical optics : Total internal reflection
(180.4243) Microscopy : Near-field microscopy

ToC Category:

Original Manuscript: July 17, 2009
Revised Manuscript: September 30, 2009
Manuscript Accepted: October 2, 2009
Published: November 2, 2009

Virtual Issues
Vol. 4, Iss. 13 Virtual Journal for Biomedical Optics

Thomas P. Burghardt, Andrew D. Hipp, and Katalin Ajtai, "Around-the-objective total internal reflection fluorescence microscopy," Appl. Opt. 48, 6120-6131 (2009)

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