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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 17, Iss. 8 — Apr. 13, 2009
  • pp: 6493–6508

Global analysis of time correlated single photon counting FRET-FLIM data

Hernan E. Grecco, Pedro Roda-Navarro, and Peter J. Verveer  »View Author Affiliations

Optics Express, Vol. 17, Issue 8, pp. 6493-6508 (2009)

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Fluorescence lifetime imaging microscopy (FLIM) can be used to quantify molecular reactions in cells by detecting fluorescence resonance energy transfer (FRET). Confocal FLIM systems based on time correlated single photon counting (TCSPC) methods provide high spatial resolution and high sensitivity, but suffer from poor signal to noise ratios (SNR) that complicate quantitative analysis. We extend a global analysis method, originally developed for single frequency domain FLIM data, with a new filtering method optimized for FRET-FLIM data and apply it to TCSPC data. With this approach, the fluorescent lifetimes and relative concentrations of free and interacting molecules can be reliably estimated, even if the SNR is low. The required calibration values of the impulse response function are directly estimated from the data, eliminating the need for reference samples. The proposed method is efficient and robust, and can be routinely applied to analyze FRET-FLIM data acquired in intact cells.

© 2009 Optical Society of America

OCIS Codes
(170.1420) Medical optics and biotechnology : Biology
(170.1790) Medical optics and biotechnology : Confocal microscopy
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.3650) Medical optics and biotechnology : Lifetime-based sensing
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: January 23, 2009
Revised Manuscript: February 26, 2009
Manuscript Accepted: March 10, 2009
Published: April 3, 2009

Virtual Issues
Vol. 4, Iss. 6 Virtual Journal for Biomedical Optics

Hernan E. Grecco, Pedro Roda-Navarro, and Peter J. Verveer, "Global analysis of time correlated single photon counting FRET-FLIM data," Opt. Express 17, 6493-6508 (2009)

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