OSA's Digital Library

Journal of the Optical Society of America A

Journal of the Optical Society of America A


  • Vol. 20, Iss. 2 — Feb. 1, 2003
  • pp: 368–379

Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging

Johan Philip and Kjell Carlsson  »View Author Affiliations

JOSA A, Vol. 20, Issue 2, pp. 368-379 (2003)

View Full Text Article

Enhanced HTML    Acrobat PDF (236 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



We deduce the signal-to-noise ratio for fluorescence lifetime imaging when using frequency-domain methods. We assume mono-exponential decay and quantum-noise-limited performance. The results are compared with Monte Carlo simulations with good agreement. We also compare our results with previous investigations of time-domain methods for fluorescence lifetime imaging. For a given number of detected photons, we find that frequency-domain and time-domain methods are equally good. The correct choice of detection technique and its parameters is important for obtaining good results.

© 2003 Optical Society of America

OCIS Codes
(000.5490) General : Probability theory, stochastic processes, and statistics
(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
(270.5290) Quantum optics : Photon statistics

Original Manuscript: May 28, 2002
Revised Manuscript: September 18, 2002
Manuscript Accepted: September 18, 2002
Published: February 1, 2003

Johan Philip and Kjell Carlsson, "Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging," J. Opt. Soc. Am. A 20, 368-379 (2003)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. C. G. Morgan, A. C. Mitchell, J. G. Murray, “Nanosecond time-resolved fluorescence microscopy: principles and practice,” Trans. R. Microsc. Soc. 1, 463–466 (1990).
  2. E. P. Buurman, R. Sanders, A. Draaijer, H. C. Gerritsen, J. J. F. van Veen, P. M. Houpt, Y. K. Levine, “Fluorescence lifetime imaging using a confocal laser scanning microscope,” Scanning 14, 155–159 (1992). [CrossRef]
  3. D. W. Piston, D. R. Sandison, W. W. Webb, “Time-resolved fluorescence imaging and background rejection by two-photon excitation in laser scanning microscopy,” in Time-Resolved Laser Spectroscopy in Biochemistry III, J. R. Lakowicz, ed., Proc. SPIE1640, 379–389 (1992). [CrossRef]
  4. T. W. J. Gadella, T. M. Jovin, R. M. Clegg, “Fluorescence lifetime imaging microscopy (FLIM): spatial resolution of microstructures on the nanosecond timescale,” Biophys. Chem. 48, 221–239 (1993). [CrossRef]
  5. R. Müller, C. Zander, M. Sauer, M. Deimel, D.-S. Ko, S. Siebert, J. Arden-Jacob, G. Deltau, N. J. Marx, K. H. Drexhage, J. Wolfrum, “Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser,” Chem. Phys. Lett. 262, 716–722 (1996). [CrossRef]
  6. A. H. Buist, M. Müller, E. J. Gijsbers, G. J. Brakenhoff, T. S. Sosnowski, T. B. Norris, J. Squier, “Double-pulse fluorescence lifetime measurements,” J. Microsc. (Oxford) 186, 212–220 (1997). [CrossRef]
  7. M. Sauer, J. Arden-Jacob, K. H. Drexhage, F. Göbel, U. Lieberwirth, K. Mühlegger, R. Müller, J. Wolfrum, C. Zander, “Time-resolved identification of individual mononucleotide molecules in aqueous solution with pulsed semiconductor lasers,” Bioimaging 6, 14–24 (1998). [CrossRef]
  8. J. Sytsma, J. M. Vroom, C. J. de Grauw, H. C. Gerritsen, “Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation,” J. Microsc. (Oxford) 191, 39–51 (1998). [CrossRef]
  9. C. J. de Grauw, H. C. Gerritsen, “Multiple time-gate module for fluorescence lifetime imaging,” Appl. Spectrosc. 55, 670–678 (2001). [CrossRef]
  10. T. Oida, Y. Sako, A. Kusumi, “Fluorescence lifetime imaging microscopy (flimscopy),” Biophys. J. 64, 676–685 (1993). [CrossRef] [PubMed]
  11. C. J. R. van der Oord, H. C. Gerritsen, F. F. G. Rommerts, D. A. Shaw, I. H. Munro, Y. K. Levine, “Micro-volume time-resolved fluorescence spectroscopy using a confocal synchrotron radiation microscope,” Appl. Spectrosc. 49, 1469–1473 (1995). [CrossRef]
  12. P. T. C. So, T. French, W. M. Yu, K. M. Berland, C. Y. Dong, E. Gratton, “Time-resolved fluorescence microscopy using two-photon excitation,” Bioimaging 3, 49–63 (1995). [CrossRef]
  13. A. Draaijer, R. Sanders, H. C. Gerritsen, “Fluorescence lifetime imaging, a new tool in confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Plenum, New York, 1995), pp. 491–505.
  14. K. König, P. T. C. So, W. W. Mantulin, B. J. Tromberg, E. Gratton, “Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress,” J. Microsc. (Oxford) 183, 197–204 (1996).
  15. H. Brismar, B. Ulfhake, “Fluorescence lifetime measurements in confocal microscopy of neurons labeled with multiple fluorophores,” Nat. Biotechnol. 15, 373–377 (1997). [CrossRef] [PubMed]
  16. T. French, P. T. C. So, D. J. Weaver, T. Coelho-Sampaio, E. Gratton, “Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing,” J. Microsc. (Oxford) 185, 339–353 (1997). [CrossRef]
  17. A. Squire, P. I. H. Bastiaens, “Three dimensional image restoration in fluorescence lifetime imaging microscopy,” J. Microsc. (Oxford) 193, 36–49 (1999). [CrossRef]
  18. K. Carlsson, A. Liljeborg, R. M. Andersson, H. Brismar, “Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance,” J. Microsc. (Oxford) 199, 106–114 (2000). [CrossRef]
  19. J. R. Lakowicz, H. Szmacinski, “Fluorescence lifetime-based sensing of pH, Ca+2,K+ and glucose,” Sens. Actuators B 11, 133–143 (1993). [CrossRef]
  20. G. Krishnamoorthy, A. Srivastava, “Intracellular dynamics seen through time-resolved fluorescence microscopy,” Curr. Sci. 72, 835–845 (1997).
  21. R. D. Spencer, G. Weber, “Measurements of subnanosecond fluorescence lifetimes with a cross-correlation phase fluorometer,” Ann. N.Y. Acad. Sci. 158, 361–376 (1969). [CrossRef]
  22. D. V. O’Connor, D. Phillips, Time-Correlated Single Photon Counting (Academic, New York, 1984).
  23. M. Köllner, J. Wolfrum, “How many photons are necessary for fluorescence lifetime measurements?” Chem. Phys. Lett. 200, 199–204 (1992). [CrossRef]
  24. R. M. Ballew, J. N. Demas, “An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays,” Anal. Chem. 61, 30–33 (1989). [CrossRef]
  25. E. Gaviola, “Ein Fluorometer. Apparat zur Messung von Fluoreszenzabklingungszeiten,” Z. Phys. 42, 853–861 (1927). [CrossRef]
  26. K. Carlsson, A. Liljeborg, “Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique,” J. Microsc. (Oxford) 191, 119–127 (1998). [CrossRef]
  27. K. Carlsson, J. Philip, “Theoretical investigation of the signal-to-noise ratio for different fluorescence lifetime imaging techniques,” in Optical Diagnostics of Living Cells V, D. L. Farkas, R. C. Leif, eds., Proc. SPIE4622, 70–78 (2002). [CrossRef]
  28. K. Carlsson, A. Liljeborg, “Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation,” J. Microsc. (Oxford) 185, 37–46 (1997). [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