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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 22, Iss. 1 — Jan. 13, 2014
  • pp: 885–899

The fast polarization modulation based dual-focus fluorescence correlation spectroscopy

Martin Štefl, Aleš Benda, Ingo Gregor, and Martin Hof  »View Author Affiliations

Optics Express, Vol. 22, Issue 1, pp. 885-899 (2014)

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We introduce two new alternative experimental realizations of dual focus fluorescence correlation spectroscopy (2fFCS), a method which allows for obtaining absolute diffusion coefficient of fast moving fluorescing molecules at nanomolar concentrations, based on fast polarization modulation of the excitation beam by a resonant electro-optical modulator. The first approach rotates every second linearly polarized laser pulse by 90 degrees to obtain independent intensity readout for both foci, similar to original design. The second approach combines polarization modulation of cw laser and fluorescence lifetime correlation spectroscopy (FLCS) like analysis to obtain clean correlation curves for both overlapping foci. We tested our new approaches with different lasers and samples, revealed a need for intensity cross-talk corrections by comparing the methods with each other and discussed experimental artifacts stemming from improper polarization alignment and detector afterpulsing. The advantages of our solutions are that the polarization rotation approach requires just one pulsed laser for each wavelength, that the polarization modulation approach even mitigates the need of pulsed lasers by using standard cw lasers and that it allows the DIC prism to be placed at an arbitrary angle. As a consequence the presented experimental solutions for 2fFCS can be more easily implemented into commercial laser scanning microscopes.

© 2014 Optical Society of America

OCIS Codes
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(180.1790) Microscopy : Confocal microscopy
(300.2530) Spectroscopy : Fluorescence, laser-induced

ToC Category:

Original Manuscript: October 30, 2013
Revised Manuscript: December 16, 2013
Manuscript Accepted: December 19, 2013
Published: January 8, 2014

Virtual Issues
Vol. 9, Iss. 3 Virtual Journal for Biomedical Optics

Martin Štefl, Aleš Benda, Ingo Gregor, and Martin Hof, "The fast polarization modulation based dual-focus fluorescence correlation spectroscopy," Opt. Express 22, 885-899 (2014)

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