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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 7, Iss. 10 — Oct. 5, 2012

flatFLIM: enhancing the dynamic range of frequency domain FLIM

Klaus C. Schuermann and Hernán E. Grecco  »View Author Affiliations

Optics Express, Vol. 20, Issue 18, pp. 20730-20741 (2012)

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Fluorescence Lifetime Imaging Microscopy (FLIM) is a quantitative technique to probe the nanoenvironment of fluorescent molecules. It is the most robust way to quantify Förster Resonance Energy Transfer (FRET) as it allows reliable differentiation between concentration changes and quenching. In this way, molecular interactions can be imaged in single living cells. The most common wide-field implementation is homodyne Frequency Domain (FD) FLIM, which determines the fluorescence lifetime by measuring the phase and modulation changes of the fluorescence in each pixel upon excitation with a light source modulated at a high frequency. The fluorescence lifetimes are derived from a stack of images acquired at different phase shifts between excitation and detection. In this work we describe a simple method to enhance the dynamic range of FD-FLIM based on precompensating the expected fluorescence modulation by varying the laser power through the phase stack. We show theoretically and experimentally that most of the dynamic range of the camera can be recovered to quantify cells with different intensities. This improvement can be added to any FD-FLIM setup with minimal modifications, enhancing the throughput of information content.

© 2012 OSA

OCIS Codes
(170.1420) Medical optics and biotechnology : Biology
(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: June 18, 2012
Revised Manuscript: July 25, 2012
Manuscript Accepted: August 1, 2012
Published: August 24, 2012

Virtual Issues
Vol. 7, Iss. 10 Virtual Journal for Biomedical Optics

Klaus C. Schuermann and Hernán E. Grecco, "flatFLIM: enhancing the dynamic range of frequency domain FLIM," Opt. Express 20, 20730-20741 (2012)

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