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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 17, Iss. 25 — Dec. 7, 2009
  • pp: 23181–23203

ϕ2FLIM: a technique for alias-free frequency domain fluorescence lifetime imaging

Alan D. Elder, Clemens F. Kaminski, and Jonathan H. Frank  »View Author Affiliations


Optics Express, Vol. 17, Issue 25, pp. 23181-23203 (2009)
http://dx.doi.org/10.1364/OE.17.023181


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Abstract

A new approach to alias-free wide-field fluorescence lifetime imaging in the frequency domain is demonstrated using a supercontinuum source for fluorescence excitation and a phase-modulated image intensifier for detection. This technique is referred to as phi-squared fluorescence lifetime imaging (ϕ2FLIM). The phase modulation and square-wave gating of the image intensifier eliminate aliasing by the effective suppression of higher harmonics. The ability to use picosecond excitation pulses without aliasing expands the range of excitation sources available for frequency-domain fluorescence lifetime imaging (fd-FLIM) and improves the modulation depth of conventional homodyne fd-FLIM measurements, which use sinusoidal intensity modulation of the excitation source. The ϕ2FLIM results are analyzed using AB-plots, which facilitate the identification of mono-exponential and multi-exponential fluorescence decays and provide measurements of the fluorophore fractions in two component mixtures. The rapid acquisition speed of the technique enables lifetime measurements in dynamic systems, such as temporally evolving samples and samples that are sensitive to photo-bleaching. Rapid ϕ2FLIM measurements are demonstrated by imaging the dynamic mixing of two different dye solutions at 5.5 Hz. The tunability of supercontinuum radiation enables excitation wavelength resolved FLIM measurements, which facilitates analysis of samples containing multiple fluorophores with different absorption spectra.

© 2009 OSA

OCIS Codes
(170.3650) Medical optics and biotechnology : Lifetime-based sensing
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:
Microscopy

History
Original Manuscript: September 11, 2009
Revised Manuscript: November 13, 2009
Manuscript Accepted: November 23, 2009
Published: December 3, 2009

Virtual Issues
Vol. 5, Iss. 1 Virtual Journal for Biomedical Optics

Citation
Alan D. Elder, Clemens F. Kaminski, and Jonathan H. Frank, "ϕ2FLIM: a technique for alias-free frequency domain fluorescence lifetime imaging," Opt. Express 17, 23181-23203 (2009)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-17-25-23181


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