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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 3 — Feb. 11, 2013
  • pp: 3523–3539

Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy

René Schneider, Tilman Glaser, Michael Berndt, and Stefan Diez  »View Author Affiliations

Optics Express, Vol. 21, Issue 3, pp. 3523-3539 (2013)

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Illumination based on objective-type total internal reflection (TIR) is nowadays widely used in high-performance fluorescence microscopy. However, the desirable application of such setups for dark-field imaging of scattering entities is cumbersome due to the spatial overlap of illumination and detection light, which cannot be separated spectrally. Here, we report a novel TIR approach based on a parabolically shaped quartz prism that allows for the detection of single-molecule fluorescence as well as single-particle scattering with high signal-to-noise ratios. We demonstrate homogeneous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, we quantitatively compare the fluorescence performance of our setup to objective-type TIR and demonstrate sub-nanometer localization accuracies for the scattering of 40 nm gold nanoparticles (AuNPs). When bound to individual kinesin-1 motors, the AuNPs reliably report on the characteristic 8 nm stepping along microtubules.

© 2013 OSA

OCIS Codes
(110.0180) Imaging systems : Microscopy
(180.2520) Microscopy : Fluorescence microscopy
(260.6970) Physical optics : Total internal reflection
(290.5850) Scattering : Scattering, particles
(110.2945) Imaging systems : Illumination design

ToC Category:

Original Manuscript: January 14, 2013
Revised Manuscript: January 14, 2013
Manuscript Accepted: January 21, 2013
Published: February 5, 2013

Virtual Issues
Vol. 8, Iss. 3 Virtual Journal for Biomedical Optics

René Schneider, Tilman Glaser, Michael Berndt, and Stefan Diez, "Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy," Opt. Express 21, 3523-3539 (2013)

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