OSA's Digital Library

Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 8, Iss. 6 — Jun. 27, 2013

High speed multispectral fluorescence lifetime imaging

Farzad Fereidouni, Keimpe Reitsma, and Hans C. Gerritsen  »View Author Affiliations

Optics Express, Vol. 21, Issue 10, pp. 11769-11782 (2013)

View Full Text Article

Enhanced HTML    Acrobat PDF (1452 KB) Open Access

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



We report a spectrally resolved fluorescence lifetime imaging system based on time gated single photon detection with a fixed gate width of 200 ps and 7 spectral channels. Time gated systems can operate at high count rates but usually have large gate widths and sample only part of the fluorescence decay curve. In the system presented in this work, the fluorescence signal is sampled using a high speed transceiver. An error analysis is carried out to characterize the performance of both lifetime and spectral detection. The effect of gate width and spectral channel width on the accuracy of estimated lifetimes and spectral widths is described. The performance of the whole instrument is evaluated at count rates of up to 12 MHz. Accurate fluorescence lifetimes (error < 2%) are recorded at count rates as high as 5 MHz. This is limited by the PMT performance, not by the electronics. Analysis of the large spectral lifetime image sets is challenging and time-consuming. Here, we demonstrate the use of lifetime and spectral phasors for analyzing images of fibroblast cells with 2 different labeled components. The phasor approach provides a fast and intuitive way of analyzing the results of spectrally resolved fluorescence lifetime imaging experiments.

© 2013 OSA

OCIS Codes
(030.5260) Coherence and statistical optics : Photon counting
(110.2960) Imaging systems : Image analysis
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.6920) Medical optics and biotechnology : Time-resolved imaging
(110.4234) Imaging systems : Multispectral and hyperspectral imaging

ToC Category:
Imaging Systems

Original Manuscript: March 15, 2013
Revised Manuscript: April 25, 2013
Manuscript Accepted: April 26, 2013
Published: May 7, 2013

Virtual Issues
Vol. 8, Iss. 6 Virtual Journal for Biomedical Optics

Farzad Fereidouni, Keimpe Reitsma, and Hans C. Gerritsen, "High speed multispectral fluorescence lifetime imaging," Opt. Express 21, 11769-11782 (2013)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. R. A. Neher, M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, “Blind source separation techniques for the decomposition of multiply labeled fluorescence images,” Biophys. J.96(9), 3791–3800 (2009).
  2. F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express20(12), 12729–12741 (2012). [CrossRef] [PubMed]
  3. M. R. Hight, D. D. Nolting, E. T. McKinley, A. D. Lander, S. K. Wyatt, M. Gonyea, P. Zhao, and H. C. Manning, “Multispectral fluorescence imaging to assess pH in biological specimens,” J. Biomed. Opt.16(1), 016007–016007 (2011). [CrossRef] [PubMed]
  4. Y. Chen, J. P. Mauldin, R. N. Day, and A. Periasamy, “Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions,” J. Microsc.228(2), 139–152 (2007). [CrossRef] [PubMed]
  5. A. V. Agronskaia, L. Tertoolen, and H. C. Gerritsen, “Fast fluorescence lifetime imaging of calcium in living cells,” J. Biomed. Opt.9(6), 1230–1237 (2004). [CrossRef] [PubMed]
  6. A. Celli, S. Sanchez, M. Behne, T. Hazlett, E. Gratton, and T. Mauro, “The epidermal Ca2 gradient: Measurement using the phasor representation of fluorescent lifetime imaging,” Biophys. J.98(5), 911–921 (2010). [CrossRef] [PubMed]
  7. R. Sanders, A. Draaijer, H. C. Gerritsen, P. M. Houpt, and Y. K. Levine, “Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy,” Anal. Biochem.227(2), 302–308 (1995). [CrossRef] [PubMed]
  8. J. Lakowicz, Principles of Fluorescence Spectroscopy, (Kluwer Academic/Plenum Publisher, 2006)
  9. J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L. Johnson, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. U.S.A.89(4), 1271–1275 (1992). [CrossRef] [PubMed]
  10. F. Fereidouni, A. Esposito, G. A. Blab, and H. C. Gerritsen, “A modified phasor approach for analyzing time-gated fluorescence lifetime images,” J. Microsc.244(3), 248–258 (2011). [CrossRef] [PubMed]
  11. V. E. Centonze, M. Sun, A. Masuda, H. Gerritsen, and B. Herman, “Fluorescence resonance energy transfer imaging microscopy,” Methods Enzymol.360, 542–560 (2003). [CrossRef] [PubMed]
  12. R. Clegg, Fluorescence Resonance Energy Transfer, Fluorescence Imaging Spectroscopy and Microscopy vol. 137, (John Wiley & Sons, 1996), 155–156.
  13. Q. S. Hanley, D. J. Arndt-Jovin, and T. M. Jovin, “Spectrally resolved fluorescence lifetime imaging microscopy,” Appl. Spectrosc.56(2), 155–166 (2002). [CrossRef]
  14. D. K. Bird, K. W. Eliceiri, C. H. Fan, and J. G. White, “Simultaneous two-photon spectral and lifetime fluorescence microscopy,” Appl. Opt.43(27), 5173–5182 (2004). [CrossRef] [PubMed]
  15. W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, and K. Benndorf, ”Multi-wavelength TCSPC lifetime imaging” in Proc. SPIEAnonymous 79–84, (2002).
  16. M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan, “Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells,” Biophys. J.83(6), 3570–3577 (2002). [CrossRef] [PubMed]
  17. D. K. Nair, M. Jose, T. Kuner, W. Zuschratter, and R. Hartig, “FRET-FLIM at nanometer spectral resolution from living cells,” Opt. Express14(25), 12217–12229 (2006). [CrossRef] [PubMed]
  18. M. Jose, D. K. Nair, C. Reissner, R. Hartig, and W. Zuschratter, “Photophysics of Clomeleon by FLIM: discriminating excited state reactions along neuronal development,” Biophys. J.92(6), 2237–2254 (2007). [CrossRef] [PubMed]
  19. P. D. Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. Mcginty, R. K. P. Benninger, D. S. Elson, I. Munro, M. John Lever, P. Anand, M. A. A. Neil, and P. M. W. French, “Rapid hyperspectral fluorescence lifetime imaging,” Microsc. Res. Tech.70(5), 481–484 (2007). [CrossRef] [PubMed]
  20. S. Shrestha, B. E. Applegate, J. Park, X. Xiao, P. Pande, and J. A. Jo, “High-speed multispectral fluorescence lifetime imaging implementation for in vivo applications,” Opt. Lett.35(15), 2558–2560 (2010). [CrossRef] [PubMed]
  21. D. Strat, F. Dolp, B. von Einem, C. Steinmetz, C. A. F. von Arnim, and A. Rueck, “Spectrally resolved fluorescence lifetime imaging microscopy: Forster resonant energy transfer global analysis with a one- and two-exponential donor model,” J. Biomed. Opt.16(2), 026002 (2011). [CrossRef] [PubMed]
  22. Y. C. Chen and R. M. Clegg, “Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency,” J. Microsc.244(1), 21–37 (2011). [CrossRef] [PubMed]
  23. S. Schlachter, S. Schwedler, A. Esposito, G. S. Kaminski Schierle, G. D. Moggridge, and C. F. Kaminski, “A method to unmix multiple fluorophores in microscopy images with minimal a priori information,” Opt. Express17(25), 22747–22760 (2009). [CrossRef] [PubMed]
  24. A. Rück, Ch. Hülshoff, I. Kinzler, W. Becker, and R. Steiner, “SLIM: a new method for molecular imaging,” Microsc. Res. Tech.70(5), 485–492 (2007). [CrossRef] [PubMed]
  25. R. W. K. Leung, S. C. A. Yeh, and Q. Fang, “Effects of incomplete decay in fluorescence lifetime estimation,” Biomed. Opt. Express2(9), 2517–2531 (2011). [CrossRef] [PubMed]
  26. D. Bebelaar, “Time response of various types of photomultipliers and its wavelength dependence in time‐correlated single‐photon counting with an ultimate resolution of 47 ps FWHM,” Rev. Sci. Instrum.57(6), 1116–1125 (1986). [CrossRef]
  27. D. O'Connor, W. Ware, and J. Andre, “Deconvolution of fluorescence decay curves. A critical comparison of techniques,” J. Phys. Chem.83(10), 1333–1343 (1979). [CrossRef]
  28. A. Gafni, R. L. Modlin, and L. Brand, “Analysis of fluorescence decay curves by means of the Laplace transformation,” Biophys. J.15(3), 263–280 (1975). [CrossRef] [PubMed]
  29. J. A. Jo, Q. Fang, T. Papaioannou, and L. Marcu, “Fast model-free deconvolution of fluorescence decay for analysis of biological systems,” J. Biomed. Opt.9(4), 743–752 (2004). [CrossRef] [PubMed]
  30. M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008). [CrossRef] [PubMed]
  31. B. Q. Spring and R. M. Clegg, “Image analysis for denoising full-field frequency-domain fluorescence lifetime images,” J. Microsc.235(2), 221–237 (2009). [CrossRef] [PubMed]
  32. H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, “Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution,” J. Microsc.206(3), 218–224 (2002). [CrossRef] [PubMed]
  33. M. Köllner and J. Wolfrum, “How many photons are necessary for fluorescence-lifetime measurements?” Chem. Phys. Lett.200(1-2), 199–204 (1992). [CrossRef]
  34. J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. Sterenborg, and H. C. Gerritsen, “In vivo nonlinear spectral imaging in mouse skin,” Opt. Express14(10), 4395–4402 (2006). [CrossRef] [PubMed]
  35. D. U. Li, J. Arlt, J. Richardson, R. Walker, A. Buts, D. Stoppa, E. Charbon, and R. Henderson, “Real-time fluorescence lifetime imaging system with a 32× 32 0.13 microm CMOS low dark-count single-photon avalanche diode array,” Opt. Express18(10), 10257–10269 (2010). [CrossRef] [PubMed]

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