Abstract

In vivo two-photon imaging of intrinsic contrast can provide valuable information about structural tissue elements such as collagen and elastin and fluorescent metabolites such as nicotinamide adenine dinucleotide. Yet low signal and overlapping emission spectra can make it difficult to identify and delineate these species in vivo. We present a novel approach that combines excitation scanning with spectrally resolved emission two-photon microscopy, allowing distinct structures to be delineated based on their characteristic spectral fingerprints. The amounts of intrinsic fluorophores present in each voxel can also be evaluated. We demonstrate our method using in vivo imaging of nude mouse skin.

© 2008 Optical Society of America

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References

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  1. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
    [CrossRef] [PubMed]
  2. S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
    [CrossRef] [PubMed]
  3. N. Ramanujam, in Encyclopedia of Analytical Chemistry, R.A.Meyers, ed. (Wiley, 2000), Vol. 1, pp. 20-56.
  4. J. A. Palero, H. S. de Bruijn, A. van der Ploeg-van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, Opt. Express 14, 4395 (2006).
    [CrossRef] [PubMed]
  5. J. R. Mansfield, K. W. Gossage, C. C. Hoyt, and R. M. Levenson, J. Biomed. Opt. 10, 041207 (2005).
    [CrossRef]
  6. A. J. Radosevich, M. B. Bouchard, S. A. Burgess, R. Stolpher, B. Chen, and E. M. C. Hillman, in Biomedical Optics, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BW67.
  7. M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, and S. E. Fraser, J. Biomed. Opt. 8, 329 (2003).
    [CrossRef] [PubMed]
  8. P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, J. Biomed. Opt. 6, 277 (2001).
    [CrossRef] [PubMed]
  9. B. Han, B. Jung, J. S. Nelson, and E.-H. Choi, J. Biomed. Opt. 12, 014006 (2007).
    [CrossRef] [PubMed]
  10. M. A. Albota, C. Xu, and W. W. Webb, Appl. Opt. 37, 7352 (1998).
    [CrossRef]

2007 (1)

B. Han, B. Jung, J. S. Nelson, and E.-H. Choi, J. Biomed. Opt. 12, 014006 (2007).
[CrossRef] [PubMed]

2006 (1)

2005 (1)

J. R. Mansfield, K. W. Gossage, C. C. Hoyt, and R. M. Levenson, J. Biomed. Opt. 10, 041207 (2005).
[CrossRef]

2003 (2)

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, and S. E. Fraser, J. Biomed. Opt. 8, 329 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

2002 (1)

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

2001 (1)

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, J. Biomed. Opt. 6, 277 (2001).
[CrossRef] [PubMed]

1998 (1)

Albota, M. A.

Bouchard, M. B.

A. J. Radosevich, M. B. Bouchard, S. A. Burgess, R. Stolpher, B. Chen, and E. M. C. Hillman, in Biomedical Optics, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BW67.

Burgess, S. A.

A. J. Radosevich, M. B. Bouchard, S. A. Burgess, R. Stolpher, B. Chen, and E. M. C. Hillman, in Biomedical Optics, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BW67.

Campagnola, P. J.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, J. Biomed. Opt. 6, 277 (2001).
[CrossRef] [PubMed]

Chen, B.

A. J. Radosevich, M. B. Bouchard, S. A. Burgess, R. Stolpher, B. Chen, and E. M. C. Hillman, in Biomedical Optics, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BW67.

Choi, E.-H.

B. Han, B. Jung, J. S. Nelson, and E.-H. Choi, J. Biomed. Opt. 12, 014006 (2007).
[CrossRef] [PubMed]

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Clark, H. A.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, J. Biomed. Opt. 6, 277 (2001).
[CrossRef] [PubMed]

de Bruijn, H. S.

Dickinson, M. E.

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, and S. E. Fraser, J. Biomed. Opt. 8, 329 (2003).
[CrossRef] [PubMed]

Fraser, S. E.

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, and S. E. Fraser, J. Biomed. Opt. 8, 329 (2003).
[CrossRef] [PubMed]

Gerritsen, H. C.

Gossage, K. W.

J. R. Mansfield, K. W. Gossage, C. C. Hoyt, and R. M. Levenson, J. Biomed. Opt. 10, 041207 (2005).
[CrossRef]

Han, B.

B. Han, B. Jung, J. S. Nelson, and E.-H. Choi, J. Biomed. Opt. 12, 014006 (2007).
[CrossRef] [PubMed]

Heikal, A. A.

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

Hillman, E. M. C.

A. J. Radosevich, M. B. Bouchard, S. A. Burgess, R. Stolpher, B. Chen, and E. M. C. Hillman, in Biomedical Optics, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BW67.

Hoyt, C. C.

J. R. Mansfield, K. W. Gossage, C. C. Hoyt, and R. M. Levenson, J. Biomed. Opt. 10, 041207 (2005).
[CrossRef]

Huang, S.

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Jung, B.

B. Han, B. Jung, J. S. Nelson, and E.-H. Choi, J. Biomed. Opt. 12, 014006 (2007).
[CrossRef] [PubMed]

Levenson, R. M.

J. R. Mansfield, K. W. Gossage, C. C. Hoyt, and R. M. Levenson, J. Biomed. Opt. 10, 041207 (2005).
[CrossRef]

Lewis, A.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, J. Biomed. Opt. 6, 277 (2001).
[CrossRef] [PubMed]

Loew, L. M.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, J. Biomed. Opt. 6, 277 (2001).
[CrossRef] [PubMed]

Mansfield, J. R.

J. R. Mansfield, K. W. Gossage, C. C. Hoyt, and R. M. Levenson, J. Biomed. Opt. 10, 041207 (2005).
[CrossRef]

Mohler, W. A.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, J. Biomed. Opt. 6, 277 (2001).
[CrossRef] [PubMed]

Nelson, J. S.

B. Han, B. Jung, J. S. Nelson, and E.-H. Choi, J. Biomed. Opt. 12, 014006 (2007).
[CrossRef] [PubMed]

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Palero, J. A.

Radosevich, A. J.

A. J. Radosevich, M. B. Bouchard, S. A. Burgess, R. Stolpher, B. Chen, and E. M. C. Hillman, in Biomedical Optics, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BW67.

Ramanujam, N.

N. Ramanujam, in Encyclopedia of Analytical Chemistry, R.A.Meyers, ed. (Wiley, 2000), Vol. 1, pp. 20-56.

Simbuerger, E.

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, and S. E. Fraser, J. Biomed. Opt. 8, 329 (2003).
[CrossRef] [PubMed]

Sterenborg, H. J. C. M.

Stolpher, R.

A. J. Radosevich, M. B. Bouchard, S. A. Burgess, R. Stolpher, B. Chen, and E. M. C. Hillman, in Biomedical Optics, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BW67.

van der Ploeg-van den Heuvel, A.

Waters, C. W.

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, and S. E. Fraser, J. Biomed. Opt. 8, 329 (2003).
[CrossRef] [PubMed]

Webb, W. W.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

M. A. Albota, C. Xu, and W. W. Webb, Appl. Opt. 37, 7352 (1998).
[CrossRef]

Williams, R. M.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Xu, C.

Zimmermann, B.

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, and S. E. Fraser, J. Biomed. Opt. 8, 329 (2003).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biophys. J. (1)

S. Huang, A. A. Heikal, and W. W. Webb, Biophys. J. 82, 2811 (2002).
[CrossRef] [PubMed]

J. Biomed. Opt. (4)

J. R. Mansfield, K. W. Gossage, C. C. Hoyt, and R. M. Levenson, J. Biomed. Opt. 10, 041207 (2005).
[CrossRef]

M. E. Dickinson, E. Simbuerger, B. Zimmermann, C. W. Waters, and S. E. Fraser, J. Biomed. Opt. 8, 329 (2003).
[CrossRef] [PubMed]

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, J. Biomed. Opt. 6, 277 (2001).
[CrossRef] [PubMed]

B. Han, B. Jung, J. S. Nelson, and E.-H. Choi, J. Biomed. Opt. 12, 014006 (2007).
[CrossRef] [PubMed]

Opt. Express (1)

Proc. Natl. Acad. Sci. USA (1)

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Other (2)

A. J. Radosevich, M. B. Bouchard, S. A. Burgess, R. Stolpher, B. Chen, and E. M. C. Hillman, in Biomedical Optics, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BW67.

N. Ramanujam, in Encyclopedia of Analytical Chemistry, R.A.Meyers, ed. (Wiley, 2000), Vol. 1, pp. 20-56.

Supplementary Material (4)

» Media 1: AVI (630 KB)     
» Media 2: AVI (2215 KB)     
» Media 3: JPG (988 KB)     
» Media 4: AVI (3662 KB)     

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Figures (3)

Fig. 1
Fig. 1

In vivo 3D hyperspectral unmixing using seed regions. Rows (a), (b), and (c) show different depth slices in the skin. Left, 350 505 nm emission images for 800 nm excitation with seed regions indicated; center and right, color merges of the six unmixed component images. (d) Nine-element basis spectra of the seed regions (shown on linear colormaps). (e) 3D isosurface rendering of unmixed data (yellow, blue, cyan, and red = gray components shown). Multimedia supplements: RGB–CMY stack movie ( Media 1 ); raw data, fit, and fit residuals movie (Media 2 ); conventional histology (Media 3).

Fig. 2
Fig. 2

In vivo maps of intrinsic fluorophore concentration. Each voxel was calculated using nonnegative least-squares fitting to basis spectra of pure samples of intrinsic fluorophores. Below, Max, maximum fit contrast for each map; SG, sebaceous gland; H, hair; BG, background. Amplitudes are fractions with respect to the concentrations of the in vitro solutions. Multimedia supplement: raw wavelength scan movie ( Media 4 ).

Fig. 3
Fig. 3

In vitro validation. (a), (b) Single- and (uncorrected) two-photon excitation spectra of NADH and FAD for three emission bands. (c) Results of nonnegative least-squares fits for in vitro mixtures.

Equations (1)

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F mix = K n ϕ n η n σ n C n ,

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