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Journal of the Optical Society of America A

Journal of the Optical Society of America A


  • Editor: Franco Gori
  • Vol. 27, Iss. 5 — May. 1, 2010
  • pp: 1145–1155

Statistical analysis and optimization of frequency-domain fluorescence lifetime imaging microscopy using homodyne lock-in detection

Yuxiang Lin and Arthur F. Gmitro  »View Author Affiliations

JOSA A, Vol. 27, Issue 5, pp. 1145-1155 (2010)

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Fluorescence lifetime imaging microscopy is used widely in biological research, but the accuracy and precision of lifetime measurements are limited. Photon noise is an inherent error source that cannot be eliminated. In this paper, we present a general approach to compute the probability density of the estimated lifetime for frequency-domain fluorescence lifetime imaging microscopy using homodyne lock-in detection. The analysis for commonly used excitation methods, including sinusoidal modulation, square-wave modulation, and a periodically pulsed light source, are given and compared to the results of Monte Carlo simulations. The optimum parameters of the excitation waveforms to minimize the variance of the estimated lifetimes are also derived and compared to previously published results.

© 2010 Optical Society of America

OCIS Codes
(000.5490) General : Probability theory, stochastic processes, and statistics
(030.4280) Coherence and statistical optics : Noise in imaging systems
(030.5290) Coherence and statistical optics : Photon 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

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: November 11, 2009
Manuscript Accepted: February 25, 2010
Published: April 28, 2010

Virtual Issues
Vol. 5, Iss. 9 Virtual Journal for Biomedical Optics

Yuxiang Lin and Arthur F. Gmitro, "Statistical analysis and optimization of frequency-domain fluorescence lifetime imaging microscopy using homodyne lock-in detection," J. Opt. Soc. Am. A 27, 1145-1155 (2010)

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  1. K. M. Hanson, M. J. Behne, N. P. Barry, T. M. Mauro, E. Gratton, and R. M. Clegg, “Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient,” Biophys. J. 83, 1682–1690 (2002). [CrossRef] [PubMed]
  2. H.-J. Lin, P. Herman, and J. R. Lakowicz, “Fluorescence lifetime-resolved pH imaging of living cells,” Cytometry, Part A 52A, 77–89 (2003). [CrossRef]
  3. M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104, 19494–19499 (2007). [CrossRef] [PubMed]
  4. T. Vo-Dinh, Biomedical Photonics Handbook (CRC, 2003). [CrossRef]
  5. 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, 302–308 (1995). [CrossRef] [PubMed]
  6. E. B. van Munster and T. W. J. Gadella, “Fluorescence lifetime imaging microscopy (FLIM),” Adv. Biochem. Eng./Biotechnol. 95, 143–175 (2005).
  7. A. Draaijer, R. Sanders, and H. C. Gerritsen, “Fluorescence lifetime imaging, a new tool in confocal microscopy” in Handbook of Biological Confocal Microscopy, J.Pawley, ed. (Plenum, 1995), pp. 491–505.
  8. J. Philip and K. Carlsson, “Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging,” J. Opt. Soc. Am. A 20, 368–379 (2003). [CrossRef]
  9. A. Esposito, H. C. Gerritsen, and F. S. Wouters, “Optimizing frequency-domain fluorescence lifetime sensing for high-throughput applications: photon economy and acquisition speed,” J. Opt. Soc. Am. A 24, 3261–3273 (2007). [CrossRef]
  10. B. R. Frieden, Probability, Statistical Optics, and Data Testing: A Problem Solving Approach (Springer, 2001). [CrossRef]
  11. J. Hayya and D. Armstrong, “A note on the ratio of two normally distributed variables,” Manage. Sci. 21, 1338–1341 (1975). [CrossRef]

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