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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 17, Iss. 25 — Dec. 7, 2009
  • pp: 22747–22760

A method to unmix multiple fluorophores in microscopy images with minimal a priori information

S. Schlachter, S. Schwedler, A. Esposito, G.S. Kaminski Schierle, G.D. Moggridge, and C.F. Kaminski  »View Author Affiliations

Optics Express, Vol. 17, Issue 25, pp. 22747-22760 (2009)

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The ability to quantify the fluorescence signals from multiply labeled biological samples is highly desirable in the life sciences but often difficult, because of spectral overlap between fluorescent species and the presence of autofluorescence. Several so called unmixing algorithms have been developed to address this problem. Here, we present a novel algorithm that combines measurements of lifetime and spectrum to achieve unmixing without a priori information on the spectral properties of the fluorophore labels. The only assumption made is that the lifetimes of the fluorophores differ. Our method combines global analysis for a measurement of lifetime distributions with singular value decomposition to recover individual fluorescence spectra. We demonstrate the technique on simulated datasets and subsequently by an experiment on a biological sample. The method is computationally efficient and straightforward to implement. Applications range from histopathology of complex and multiply labelled samples to functional imaging in live cells.

© 2009 Optical Society of America

OCIS Codes
(170.3650) Medical optics and biotechnology : Lifetime-based sensing
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: September 2, 2009
Revised Manuscript: November 9, 2009
Manuscript Accepted: November 14, 2009
Published: November 30, 2009

Virtual Issues
Vol. 5, Iss. 1 Virtual Journal for Biomedical Optics

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. Express 17, 22747-22760 (2009)

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  1. 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, 203-215 (2007). [CrossRef] [PubMed]
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  15. F. S. Wouters and A. Esposito, "Quantitative analysis of fluorescence lifetime imaging made easy," HFSP J. 2(1), 7-11 (2008). [CrossRef]
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  17. A. D. Elder, S. C. Schlachter, and C. F. Kaminski, "Theoretical investigation of the photon efficiency in frequency-domain FLIM," J. Opt. Soc. Am. A 25, 452-462 (2008). [CrossRef]
  18. D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. A. de Beule, C. Dunsby, M. A. A. Neil, and P. M. W. French, "Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source," Opt. Lett. 32(23), 3408-3410 (2007). [CrossRef]
  19. P. Serge, J. R. P. Michael, K. Daekeun, K. Ki Hean, J. S. Tsu-Te, and T. C. S. Peter, "Frequency domain lifetime and spectral imaging microscopy," Microsc. Res. Tech. 69(11), 861-874 (2006).
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  21. J. A. Levitt, D. R. Matthews, S. M. Ameer-Beg, and K. Suhling, "Fluorescence lifetime and polarization-resolved imaging in cell biology," Curr. Opin. Biotechnol. 20(1), 28-36 (2009). [CrossRef]
  22. N. P. Galletly, J. McGinty, C. Dunsby, F. Teixeira, J. Requejo-Isidro, I. Munro, D. S. Elson, M. A. A. Neil, A. C. Chu, P. M. French, and G. W. Stamp, "Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin." British Journal of Dermatology 159, 152-161 (2008). [CrossRef] [PubMed]
  23. A. D. Elder, S.M. Matthews, J. Swartling, K. Yunus, J. H. Frank, C. M. Brennan, A. C. Fisher, and C. F. Kaminski, "The application of frequency-domain fluorescence lifetime imaging microscopy as a quantitative analytical tool for microfluidic devices," Opt. Express 14(12), 5456-5467 (2006). [CrossRef]
  24. 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]
  25. X. Dai, Z. Yue, M. E. Eccleston, J. Swartling, N. K. H. Slater, and C. F. Kaminski, "Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells," Nanomed. Nanotechnol. Bio. Med. 4(1), 49-56 (2008). [CrossRef]
  26. 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 47(8), 2689-2698 (2006). [CrossRef]

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