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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 2, Iss. 3 — Mar. 1, 2011
  • pp: 431–439

Spectroscopic detection improves multi-color quantification in fluorescence tomography

Giannis Zacharakis, Rosy Favicchio, Maria Simantiraki, and Jorge Ripoll  »View Author Affiliations

Biomedical Optics Express, Vol. 2, Issue 3, pp. 431-439 (2011)

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Simultaneous detection of several biological processes in vivo is a common requirement in biomedical and biological applications, and in order to address this issue the use of multiple fluorophores is usually the method of choice. Existing methodologies however, do not provide quantitative feedback of multiple fluorophore concentrations in small animals in vivo when their spectra overlap, especially when imaging the whole body in 3D. Here we present an approach where a spectroscopic module has been implemented into a custom-built Fluorescence Molecular Tomography (FMT) system. In contrast with other multispectral approaches, this multimodal imaging system is capable of recording the fluorescence spectra from each illumination point during a tomographic measurement. In situ spectral information can thus be extracted and used to improve the separation of overlapping signals associated with different fluorophores. The results of this new approach tested on both in vitro and in vivo experiments are presented, proving that accurate recovery of fluorophore concentrations can be obtained from multispectral tomography data even in the presence of high autofluorescence.

© 2011 OSA

OCIS Codes
(170.0110) Medical optics and biotechnology : Imaging systems
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(170.6510) Medical optics and biotechnology : Spectroscopy, tissue diagnostics
(170.6960) Medical optics and biotechnology : Tomography

ToC Category:
Molecular Imaging and Probe Development

Original Manuscript: November 1, 2010
Revised Manuscript: December 30, 2010
Manuscript Accepted: January 25, 2011
Published: January 31, 2011

Giannis Zacharakis, Rosy Favicchio, Maria Simantiraki, and Jorge Ripoll, "Spectroscopic detection improves multi-color quantification in fluorescence tomography," Biomed. Opt. Express 2, 431-439 (2011)

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  1. S. R. Cherry, “In vivo molecular and genomic imaging: new challenges for imaging physics,” Phys. Med. Biol. 49(3), R13–R48 (2004). [CrossRef] [PubMed]
  2. R. Weissleder and V. Ntziachristos, “Shedding light onto live molecular targets,” Nat. Med. 9(1), 123–128 (2003). [CrossRef] [PubMed]
  3. N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. G. Giepmans, A. E. Palmer, and R. Y. Tsien, “Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein,” Nat. Biotechnol. 22(12), 1567–1572 (2004). [CrossRef] [PubMed]
  4. V. V. Verkhusha and K. A. Lukyanov, “The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins,” Nat. Biotechnol. 22(3), 289–296 (2004). [CrossRef] [PubMed]
  5. J. P. Culver, A. M. Siegel, J. J. Stott, and D. A. Boas, “Volumetric diffuse optical tomography of brain activity,” Opt. Lett. 28(21), 2061–2063 (2003). [CrossRef] [PubMed]
  6. J. P. Culver, T. Durduran, D. Furuya, C. Cheung, J. H. Greenberg, and A. G. Yodh, “Diffuse optical tomography of cerebral blood flow, oxygenation, and metabolism in rat during focal ischemia,” J. Cereb. Blood Flow Metab. 23(8), 911–924 (2003). [CrossRef] [PubMed]
  7. B. W. Pogue, S. P. Poplack, T. O. McBride, W. A. Wells, K. S. Osterman, U. L. Osterberg, and K. D. Paulsen, “Quantitative hemoglobin tomography with diffuse near-infrared spectroscopy: pilot results in the breast,” Radiology 218(1), 261–266 (2001). [PubMed]
  8. D. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J. Gaudette, and Q. Zhang, “Imaging the body with diffuse optical tomography,” IEEE Signal Process. Mag. 18(6), 57–75 (2001). [CrossRef]
  9. V. Ntziachristos, “Fluorescence molecular imaging,” Annu. Rev. Biomed. Eng. 8(1), 1–33 (2006). [CrossRef] [PubMed]
  10. V. Ntziachristos, J. Ripoll, L. V. Wang, and R. Weissleder, “Looking and listening to light: the evolution of whole-body photonic imaging,” Nat. Biotechnol. 23(3), 313–320 (2005). [CrossRef] [PubMed]
  11. J. Ripoll and V. Ntziachristos, “Imaging scattering media from a distance: Theory and applications of non-contact optical tomography,” Mod. Phys. Lett. B 18(28 & 29), 1403–1431 (2004). [CrossRef]
  12. V. Ntziachristos, C. H. Tung, C. Bremer, and R. Weissleder, “Fluorescence molecular tomography resolves protease activity in vivo,” Nat. Med. 8(7), 757–761 (2002). [CrossRef] [PubMed]
  13. V. Ntziachristos, E. A. Schellenberger, J. Ripoll, D. Yessayan, E. E. Graves, A. Bogdanov, L. Josephson, and R. Weissleder, “Visualization of antitumor treatment by means of fluorescence molecular tomography with an annexin V-Cy5.5 conjugate,” Proc. Natl. Acad. Sci. U.S.A. 101(33), 12294–12299 (2004). [CrossRef] [PubMed]
  14. X. Montet, J. L. Figueiredo, H. Alencar, V. Ntziachristos, U. Mahmood, and R. Weissleder, “Tomographic fluorescence imaging of tumor vascular volume in mice,” Radiology 242(3), 751–758 (2007). [CrossRef] [PubMed]
  15. G. Zacharakis, H. Kambara, H. Shih, J. Ripoll, J. Grimm, Y. Saeki, R. Weissleder, and V. Ntziachristos, “Volumetric tomography of fluorescent proteins through small animals in vivo,” Proc. Natl. Acad. Sci. U.S.A. 102(51), 18252–18257 (2005). [CrossRef] [PubMed]
  16. A. Martin, J. Aguirre, A. Sarasa-Renedo, D. Tsoukatou, A. Garofalakis, H. Meyer, C. Mamalaki, J. Ripoll, and A. M. Planas, “Imaging changes in lymphoid organs in vivo after brain ischemia with three-dimensional fluorescence molecular tomography in transgenic mice expressing green fluorescent protein in T lymphocytes,” Mol. Imaging 7(4), 157–167 (2008). [PubMed]
  17. R. N. Germain, M. J. Miller, M. L. Dustin, and M. C. Nussenzweig, “Dynamic imaging of the immune system: progress, pitfalls and promise,” Nat. Rev. Immunol. 6(7), 497–507 (2006). [CrossRef] [PubMed]
  18. R. Weissleder and M. J. Pittet, “Imaging in the era of molecular oncology,” Nature 452(7187), 580–589 (2008). [CrossRef] [PubMed]
  19. T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003). [CrossRef] [PubMed]
  20. T. Zimmermann, “Spectral imaging and linear unmixing in light microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 245–265 (2005). [PubMed]
  21. G. Themelis, J. S. Yoo, and V. Ntziachristos, “Multispectral imaging using multiple-bandpass filters,” Opt. Lett. 33(9), 1023–1025 (2008). [CrossRef] [PubMed]
  22. A. Papadakis, E. Stathopoulos, G. Delides, K. Berberides, G. Nikiforidis, and C. Balas, “A novel spectral microscope system: application in quantitative pathology,” IEEE Trans. Biomed. Eng. 50(2), 207–217 (2003). [CrossRef] [PubMed]
  23. J. R. Mansfield, K. W. Gossage, C. C. Hoyt, and R. M. Levenson, “Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging,” J. Biomed. Opt. 10(4), 041207 (2005). [CrossRef] [PubMed]
  24. J. R. Mansfield, “Distinguished photons: a review of in vivo spectral fluorescence imaging in small animals,” Curr. Pharm. Biotechnol. 11(6), 628–638 (2010). [CrossRef] [PubMed]
  25. A. D. Zacharopoulos, P. Svenmarker, J. Axelsson, M. Schweiger, S. R. Arridge, and S. Andersson-Engels, “A matrix-free algorithm for multiple wavelength fluorescence tomography,” Opt. Express 17(5), 3042–3052 (2009). [CrossRef] [PubMed]
  26. M. Simantiraki, R. Favicchio, S. Psycharakis, G. Zacharakis, and J. Ripoll, “Multispectral unmixing of fluorescence molecular tomography data,” J. Innovative Optical Health Science 2(4), 353–364 (2009). [CrossRef]
  27. B. Brooksby, S. Srinivasan, S. Jiang, H. Dehghani, B. W. Pogue, K. D. Paulsen, J. Weaver, C. Kogel, and S. P. Poplack, “Spectral priors improve near-infrared diffuse tomography more than spatial priors,” Opt. Lett. 30(15), 1968–1970 (2005). [CrossRef] [PubMed]
  28. S. Srinivasan, B. W. Pogue, S. Jiang, H. Dehghani, and K. D. Paulsen, “Spectrally constrained chromophore and scattering near-infrared tomography provides quantitative and robust reconstruction,” Appl. Opt. 44(10), 1858–1869 (2005). [CrossRef] [PubMed]
  29. V. Ntziachristos and R. Weissleder, “Experimental three-dimensional fluorescence reconstruction of diffuse media by use of a normalized Born approximation,” Opt. Lett. 26(12), 893–895 (2001). [CrossRef] [PubMed]
  30. C. Kak, and M. Slaney, Principles of Computerized Tomographic Imaging (IEEE, New York, 1988).
  31. T. Durduran, R. Choe, J. P. Culver, L. Zubkov, M. J. Holboke, J. Giammarco, B. Chance, and A. G. Yodh, “Bulk optical properties of healthy female breast tissue,” Phys. Med. Biol. 47(16), 2847–2861 (2002). [CrossRef] [PubMed]
  32. A. Garofalakis, G. Zacharakis, G. Filippidis, E. Sanidas, D. D. Tsiftsis, V. Ntziachristos, T. G. Papazoglou, and J. Ripoll, “Characterization of the reduced scattering coefficient for optically thin samples: theory and experiments,” J. Opt. A, Pure Appl. Opt. 6(7), 725–735 (2004). [CrossRef]
  33. A. Garofalakis, G. Zacharakis, G. Filippidis, E. Sanidas, D. D. Tsiftsis, and E. Stathopoulos, “M. kafousi, J. Ripoll and T. G. Papazoglou,” Phys. Med. Biol. 50(1), 1–11 (2005). [PubMed]
  34. J. Swartling, J. Svensson, D. Bengtsson, K. Terike, and S. Andersson-Engels, “Fluorescence spectra provide information on the depth of fluorescent lesions in tissue,” Appl. Opt. 44(10), 1934–1941 (2005). [CrossRef] [PubMed]
  35. S. C. Davis, B. W. Pogue, S. B. Tuttle, H. Dehghani, and K. D. Paulsen, “Spectral distortion in diffuse molecular luminescence tomography in turbid media,” J. Appl. Phys. 105(10), 102024 (2009). [CrossRef] [PubMed]

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