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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 16, Iss. 22 — Oct. 27, 2008
  • pp: 18495–18504

A programmable light engine for quantitative single molecule TIRF and HILO imaging

Marcel van’t Hoff, Vincent de Sars, and Martin Oheim  »View Author Affiliations

Optics Express, Vol. 16, Issue 22, pp. 18495-18504 (2008)

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We report on a simple yet powerful implementation of objective-type total internal reflection fluorescence (TIRF) and highly inclined and laminated optical sheet (HILO, a type of dark-field) illumination. Instead of focusing the illuminating laser beam to a single spot close to the edge of the microscope objective, we are scanning during the acquisition of a fluorescence image the focused spot in a circular orbit, thereby illuminating the sample from various directions. We measure parameters relevant for quantitative image analysis during fluorescence image acquisition by capturing an image of the excitation light distribution in an equivalent objective back-focal plane (BFP). Operating at scan rates above 1 MHz, our programmable light engine allows directional averaging by circular spinning the spot even for sub-millisecond exposure times. We show that restoring the symmetry of TIRF/HILO illumination reduces scattering and produces an evenly lit field-of-view that affords on-line analysis of evanescent-field excited fluorescence without pre-processing. Utilizing crossed acousto-optical deflectors, our device generates arbitrary intensity profiles in BFP, permitting variable-angle, multi-color illumination, or objective lenses to be rapidly exchanged.

© 2008 Optical Society of America

OCIS Codes
(110.0180) Imaging systems : Microscopy
(170.1530) Medical optics and biotechnology : Cell analysis
(180.2520) Microscopy : Fluorescence microscopy
(240.6690) Optics at surfaces : Surface waves

ToC Category:

Original Manuscript: September 5, 2008
Revised Manuscript: October 14, 2008
Manuscript Accepted: October 19, 2008
Published: October 24, 2008

Virtual Issues
Vol. 3, Iss. 12 Virtual Journal for Biomedical Optics

Marcel van 't Hoff, Vincent de Sars, and Martin Oheim, "A programmable light engine for quantitative single molecule TIRF and HILO imaging," Opt. Express 16, 18495-18504 (2008)

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