Abstract

We demonstrate an integrated optical microscope to image the living epithelial tissue by simultaneously exciting multiple endogenous nonlinear optical signals. By employing the spectral lifetime detection capability, this technology provides a unique approach to sensing the fine structure, the protein distribution, and the cellular metabolism of epithelial tissue in vivo. In particular, we investigated the two-photon excitation fluorescence (TPEF) of tryptophan, an essential amino acid serving as the building block of protein. Our findings show that the TPEF of cellular tryptophan produces a good contrast to reveal the morphology of the epithelial cell layer, and the contrast can be further enhanced by applying low-concentration acetic acid.

© 2009 Optical Society of America

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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]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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2009 (1)

2008 (2)

2007 (1)

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

2006 (1)

2005 (5)

Y. Wu and J. Y. Qu, Opt. Lett. 30, 3045 (2005).
[CrossRef] [PubMed]

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. So, J. Biomed. Opt. 10, 024016 (2005).
[CrossRef] [PubMed]

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

M. C. Skala, J. M. Squirrell, K. M. Vrotsos, J. C. Eickoff, A. G. Fitzpatrick, K. W. Eliceiri, and N. Ramanujam, Cancer Res. 65, 1180 (2005).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

2004 (1)

A. B. MacLean, Gynecol. Oncol. 95, 691 (2004).
[CrossRef] [PubMed]

2003 (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]

2000 (1)

1997 (1)

A. Hickel, M. Graupner, D. Lehner, A. Hermetter, O. Glatter, and H. Griengl, Enzyme Microb. Technol. 21, 361 (1997).
[CrossRef]

1990 (1)

J. D. Schaechter and R. J. Wurtman, Brain Res. 532, 203 (1990).
[CrossRef] [PubMed]

1981 (1)

E. Gudgin, R. L. Delgado, and W. R. Ware, Can. J. Chem. 59, 1037 (1981).
[CrossRef]

Alkilani, A.

Anvari, B.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Bird, D. K.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Boryskina, O. P.

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]

Collier, T.

Delgado, R. L.

E. Gudgin, R. L. Delgado, and W. R. Ware, Can. J. Chem. 59, 1037 (1981).
[CrossRef]

Diagaradjane, P.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Eickhoff, J.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

Eickoff, J. C.

M. C. Skala, J. M. Squirrell, K. M. Vrotsos, J. C. Eickoff, A. G. Fitzpatrick, K. W. Eliceiri, and N. Ramanujam, Cancer Res. 65, 1180 (2005).
[CrossRef] [PubMed]

Eliceiri, K. W.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

M. C. Skala, J. M. Squirrell, K. M. Vrotsos, J. C. Eickoff, A. G. Fitzpatrick, K. W. Eliceiri, and N. Ramanujam, Cancer Res. 65, 1180 (2005).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Fitzpatrick, A. G.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

M. C. Skala, J. M. Squirrell, K. M. Vrotsos, J. C. Eickoff, A. G. Fitzpatrick, K. W. Eliceiri, and N. Ramanujam, Cancer Res. 65, 1180 (2005).
[CrossRef] [PubMed]

Fleury, V.

Follen, M.

Glatter, O.

A. Hickel, M. Graupner, D. Lehner, A. Hermetter, O. Glatter, and H. Griengl, Enzyme Microb. Technol. 21, 361 (1997).
[CrossRef]

Grand, Y. L.

Graupner, M.

A. Hickel, M. Graupner, D. Lehner, A. Hermetter, O. Glatter, and H. Griengl, Enzyme Microb. Technol. 21, 361 (1997).
[CrossRef]

Griengl, H.

A. Hickel, M. Graupner, D. Lehner, A. Hermetter, O. Glatter, and H. Griengl, Enzyme Microb. Technol. 21, 361 (1997).
[CrossRef]

Gudgin, E.

E. Gudgin, R. L. Delgado, and W. R. Ware, Can. J. Chem. 59, 1037 (1981).
[CrossRef]

Guilbert, T.

Hancewicz, T. M.

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. So, J. Biomed. Opt. 10, 024016 (2005).
[CrossRef] [PubMed]

Hermetter, A.

A. Hickel, M. Graupner, D. Lehner, A. Hermetter, O. Glatter, and H. Griengl, Enzyme Microb. Technol. 21, 361 (1997).
[CrossRef]

Hickel, A.

A. Hickel, M. Graupner, D. Lehner, A. Hermetter, O. Glatter, and H. Griengl, Enzyme Microb. Technol. 21, 361 (1997).
[CrossRef]

Huang, H. Y.

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]

Kaplan, P. D.

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. So, J. Biomed. Opt. 10, 024016 (2005).
[CrossRef] [PubMed]

Keely, P. J.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Kortum, R. R.

Laiho, L. H.

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. So, J. Biomed. Opt. 10, 024016 (2005).
[CrossRef] [PubMed]

Lee, W. J.

Lehner, D.

A. Hickel, M. Graupner, D. Lehner, A. Hermetter, O. Glatter, and H. Griengl, Enzyme Microb. Technol. 21, 361 (1997).
[CrossRef]

Li, D.

MacLean, A. B.

A. B. MacLean, Gynecol. Oncol. 95, 691 (2004).
[CrossRef] [PubMed]

Malpica, A.

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]

Odin, C.

Pelet, S.

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. So, J. Biomed. Opt. 10, 024016 (2005).
[CrossRef] [PubMed]

Pradier, B.

Qu, J. Y.

Ramanujam, N.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

M. C. Skala, J. M. Squirrell, K. M. Vrotsos, J. C. Eickoff, A. G. Fitzpatrick, K. W. Eliceiri, and N. Ramanujam, Cancer Res. 65, 1180 (2005).
[CrossRef] [PubMed]

Riching, K. M.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

Schaechter, J. D.

J. D. Schaechter and R. J. Wurtman, Brain Res. 532, 203 (1990).
[CrossRef] [PubMed]

Shen, P.

Shieh, D. B.

Skala, M. C.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

M. C. Skala, J. M. Squirrell, K. M. Vrotsos, J. C. Eickoff, A. G. Fitzpatrick, K. W. Eliceiri, and N. Ramanujam, Cancer Res. 65, 1180 (2005).
[CrossRef] [PubMed]

So, P. T. C.

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. So, J. Biomed. Opt. 10, 024016 (2005).
[CrossRef] [PubMed]

Squirrell, J. M.

M. C. Skala, J. M. Squirrell, K. M. Vrotsos, J. C. Eickoff, A. G. Fitzpatrick, K. W. Eliceiri, and N. Ramanujam, Cancer Res. 65, 1180 (2005).
[CrossRef] [PubMed]

Sun, C. K.

Sung, K. B.

Tai, S. P.

Vaughan, E. M.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Vrotsos, K. M.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

M. C. Skala, J. M. Squirrell, K. M. Vrotsos, J. C. Eickoff, A. G. Fitzpatrick, K. W. Eliceiri, and N. Ramanujam, Cancer Res. 65, 1180 (2005).
[CrossRef] [PubMed]

Ware, W. R.

E. Gudgin, R. L. Delgado, and W. R. Ware, Can. J. Chem. 59, 1037 (1981).
[CrossRef]

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]

White, J. G.

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

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]

Wong, M. S.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Wu, P. C.

Wu, Y.

Wurtman, R. J.

J. D. Schaechter and R. J. Wurtman, Brain Res. 532, 203 (1990).
[CrossRef] [PubMed]

Yan, L.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Yaseen, M. A.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Yu, C. H.

Yu, J.

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Zheng, W.

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]

Brain Res. (1)

J. D. Schaechter and R. J. Wurtman, Brain Res. 532, 203 (1990).
[CrossRef] [PubMed]

Can. J. Chem. (1)

E. Gudgin, R. L. Delgado, and W. R. Ware, Can. J. Chem. 59, 1037 (1981).
[CrossRef]

Cancer Res. (2)

M. C. Skala, J. M. Squirrell, K. M. Vrotsos, J. C. Eickoff, A. G. Fitzpatrick, K. W. Eliceiri, and N. Ramanujam, Cancer Res. 65, 1180 (2005).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, Cancer Res. 65, 8766 (2005).
[CrossRef] [PubMed]

Enzyme Microb. Technol. (1)

A. Hickel, M. Graupner, D. Lehner, A. Hermetter, O. Glatter, and H. Griengl, Enzyme Microb. Technol. 21, 361 (1997).
[CrossRef]

Gynecol. Oncol. (1)

A. B. MacLean, Gynecol. Oncol. 95, 691 (2004).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. So, J. Biomed. Opt. 10, 024016 (2005).
[CrossRef] [PubMed]

Lasers Surg. Med. (1)

P. Diagaradjane, M. A. Yaseen, J. Yu, M. S. Wong, and B. Anvari, Lasers Surg. Med. 37, 382 (2005).
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (3)

Proc. Natl. Acad. Sci. USA (2)

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]

M. C. Skala, K. M. Riching, A. G. Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, Proc. Natl. Acad. Sci. USA 104, 19494 (2007).
[CrossRef] [PubMed]

Supplementary Material (2)

» Media 1: MOV (3955 KB)     
» Media 2: MOV (3901 KB)     

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Figures (2)

Fig. 1
Fig. 1

Representative TPEF and SHG images of hamster oral epithelium excited at 600 and 760 nm. B band, U band, and SHG images are presented in left, middle, and right columns, respectively: (a1)–(a3) SC; (b1)–(b3) SG; (c1)–(c3) stratum spinosum (SS); (d1), (d2) SB; (e1)–(e3) stroma (S); (f1)–(f3) soft muscle (M). (a3)–(d3) are U-band images of hamster cheek pouch exposed to 6% AA. Multimedia supplements: stack movies formed with the TPEF and SHG images simultaneously excited at 600 and 760 nm from tissues without (Media 1) and with (Media 2) 6% AA treatment. All images are reconstructed from raw data without any processing.

Fig. 2
Fig. 2

Representative TPEF spectra and lifetime histograms. (a) TPEF spectra excited at 600 nm from SC, SB, S, M, and tryptophan solution (Try); (b) corresponding spectra excited at 760 nm; (c) histograms of tryptophan fluorescence lifetime from SC, SG, SS, SB, and S layers without AA treatment; (d) corresponding histograms with 6% AA treatment.

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