OSA's Digital Library

Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editor: Gregory W. Faris
  • Vol. 5, Iss. 1 — Jan. 4, 2010

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)

View Full Text Article

Enhanced HTML    Acrobat PDF (814 KB) Open Access

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



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:

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

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)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. A. D. Elder, A. Domin, G. S. Kaminski-Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by FRET-sensitized fluorescence emission,” J. R. Soc. Interface 6, S59–S81 (2009). [CrossRef]
  2. A. Esposito, T. Tiffert, J. M. A. Mauritz, S. Schlachter, L. H. Bannister, C. F. Kaminski, V. L. Lew, and J. M. Schnur, “FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells,” PLoS ONE 3(11), e3780 (2008). [CrossRef] [PubMed]
  3. X. W. Dai, M. E. Eccleston, Z. L. Yue, N. K. H. Slater, and C. F. Kaminski, “A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(L-lysine iso-phthalamide),” Polymer (Guildf.) 47(8), 2689–2698 (2006). [CrossRef]
  4. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed. (Plenum, New York, 1999).
  5. S. M. Matthews, A. D. Elder, K. Yunus, C. F. Kaminski, C. M. Brennan, and A. C. Fisher, “Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging,” Anal. Chem. 79(11), 4101–4109 (2007). [CrossRef] [PubMed]
  6. A. Periasamy, P. Wodnicki, X. Wang, S. Kwon, G. Gordon, and B. Herman, “Time-resolved fluorescence lifetime imaging microscopy using a picosecond pulsed tunable dye laser system,” Rev. Sci. Instrum. 67(10), 3722–3731 (1996). [CrossRef]
  7. W. Becker, H. Hickl, C. Zander, K. H. Drexhage, S. S. M. Sauer, and J. Wolfrum, “Time-resolved detection and identification of single analyte molecules in microcapillaries by timecorrelated single-photon counting (TCSPC),” Rev. Sci. Instrum. 70(3), 1835–1841 (1999). [CrossRef]
  8. J. R. Lakowicz and K. W. Berndt, “Lifetime-selective fluorescence imaging using an rf phase-sensitive camera,” Rev. Sci. Instrum. 62(7), 1727–1734 (1991). [CrossRef]
  9. P. Schneider and R. M. Clegg, “Rapid acquisition, analysis, and display of fuorescence lifetime-resolved images for real-time applications,” Rev. Sci. Instrum. 68(11), 4107 (1997). [CrossRef]
  10. J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, “Fluorescence lifetime imaging,” Anal. Biochem. 202(2), 316–330 (1992). [CrossRef] [PubMed]
  11. T. W. J. Gadella, T. M. Jovin, and R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM) - spatial resolution of microstructures on the nanosecond time-scale,” Biophys. Chem. 48(2), 221–239 (1993). [CrossRef]
  12. A. D. Elder, J. H. Frank, J. Swartling, X. Dai, and C. F. Kaminski, “Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources,” J. Microsc. 224(2), 166–180 (2006). [CrossRef] [PubMed]
  13. A. Squire, P. J. Verveer, and P. I. H. Bastiaens, “Multiple frequency fluorescence lifetime imaging microscopy,” J. Microsc. 197(2), 136–149 (2000). [CrossRef] [PubMed]
  14. Q. S. Hanley and A. H. A. Clayton, “AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers,” J. Microsc. 218(1), 62–67 (2005). [CrossRef] [PubMed]
  15. G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15(5), 805–815 (2005). [CrossRef] [PubMed]
  16. M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), 14–16 (2008). [CrossRef] [PubMed]
  17. G. J. Kremers, E. B. van Munster, J. Goedhart, and T. W. J. Gadella., “Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy,” Biophys. J. 95(1), 378–389 (2008). [CrossRef] [PubMed]
  18. S. Schlachter, A. D. Elder, A. Esposito, G. S. Kaminski, J. H. Frank, L. K. van Geest, and C. F. Kaminski, “mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy,” Opt. Express 17(3), 1557–1570 (2009). [CrossRef] [PubMed]
  19. A. D. Elder, S. Schlachter, and C. F. Kaminski, “Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy,” J. Opt. Soc. Am. A 25(2), 452–462 (2008). [CrossRef]
  20. J. Philip and K. Carlsson, “Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging,” J. Opt. Soc. Am. A 20(2), 368–379 (2003). [CrossRef]
  21. E. B. Van Munster and T. W. J. Gadella., “phiFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy,” J. Microsc. 213(1), 29–38 (2004). [CrossRef] [PubMed]
  22. C. F. Kaminski, R. S. Watt, A. D. Elder, J. H. Frank, and J. Hult, “Supercontinuum radiation for applications in chemical sensing and microscopy,” Appl. Phys. B 92(3), 367–378 (2008). [CrossRef]
  23. J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging,” J. Microsc. 227(3), 203–215 (2007). [CrossRef] [PubMed]
  24. Q. S. Hanley, V. Subramaniam, D. J. Arndt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging: Multi-point calibration, minimum resolvable differences, and artifact suppression,” Cytometry 43(4), 248–260 (2001). [CrossRef] [PubMed]
  25. T. B. Settersten, A. Dreizler, and R. L. Farrow, “Temperature- and species-dependent quenching of CO B probed by two-photon laser-induced fluorescence using a picosecond laser,” J. Chem. Phys. 117(7), 3173–3179 (2002). [CrossRef]
  26. S. E. D. Webb, Y. Gu, S. Leveque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juskaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, “A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning,” Rev. Sci. Instrum. 73(4), 1898–1907 (2002). [CrossRef]
  27. C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys. 37(23), 3296–3303 (2004). [CrossRef]
  28. R. K. P. Benninger, O. Hofmann, J. McGinty, J. Requejo-Isidro, I. Munro, M. A. A. Neil, A. J. Demello, and P. M. W. French, “Time-resolved fluorescence imaging of solvent interactions in microfluidic devices,” Opt. Express 13(16), 6275–6285 (2005). [CrossRef] [PubMed]
  29. D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellet, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, “Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,” N. J. Phys. 6, 180–192 (2004). [CrossRef]
  30. K. K. Sharman, A. Periasamy, H. Ashworth, J. N. Demas, and N. H. Snow, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71(5), 947–952 (1999). [CrossRef] [PubMed]
  31. O. Holub, M. J. Seufferheld, C. Gohlke, Govindjee, and R. M. Clegg, “Fluorescence lifetime imaging in real-time - a new technique in photosynthesis research,” Photosynthetica 38(4), 581–599 (2000). [CrossRef]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited