Abstract

We present a multimodal nonlinear imaging approach to elucidate microstructures and spectroscopic features of oral mucosa and submucosa in vivo. The hamster buccal pouch was imaged using 3-D high resolution multiphoton and second harmonic generation microscopy. The multimodal imaging approach enables colocalization and differentiation of prominent known spectroscopic and structural features such as keratin, epithelial cells, and submucosal collagen at various depths in tissue. Visualization of cellular morphology and epithelial thickness are in excellent agreement with histological observations. These results suggest that multimodal nonlinear optical microscopy can be an effective tool for studying the physiology and pathology of mucosal tissue.

© 2004 Optical Society of America

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Acta Pathol. Microbiol. Scand. (1)

S.M. Sirsat and J.J. Pindborg, �??Subepithelial changes in oral submucous fibrosis,�?? Acta Pathol. Microbiol. Scand. 70, 161-173 (1967).
[CrossRef] [PubMed]

Arch. Otolaryngol. Head Neck Surg. (1)

A. Gillenwater, R. Jacob, R. Ganeshappa, B. Kemp, A.K. El-Naggar, J.L. Palmer, G. Clayman, M.M. Follen, R.R. Richards-Kortum, �??Noninvasive diagnosis of oral neoplasia based on fluorescence spectroscopy and native tissue autofluorescence,�?? Arch. Otolaryngol. Head Neck Surg. 124, 1251-1258 (1998).
[PubMed]

Biophy. J. (1)

S. Huang, A.A. Heikal, and W.W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophy. J. 82, 2811-2825 (2002).
[CrossRef]

Cancer (1)

M.G. Muller, T.A. Valdez, I. Georgakoudi, V. Backman, C. Fuentes, S. Kabani, N. Laver, Z.M. Wang, C.W. Boone, R.R. Dasari, S.M. Shapshay, and M.S. Feld, "Spectroscopic detection and evaluation of morphologic and biochemical changes in early human oral carcinoma," Cancer 97, 1681-1692 (2003).
[CrossRef] [PubMed]

Cancer Res. (1)

I. Georgakoudi, B.C. Jacobson, M.G. Muller, E.E. Sheets, K. Badizadegan, D.L. Carr-Locke, C.P. Crum, C.W. Boone, R.R. Dasari, J. Van Dam, and M.S. Feld, �??NADH and collagen as in vivo quantitatitive fluorescent biomarkers of epithelial precancerous changes,�?? Cancer Res. 62, 682-687 (2002).
[PubMed]

Clin. Cancer Res. (1)

A.L. Clark, A.M. Gillenwater, T.G. Collier, R. Alizadeh-Naderi, A.K. El-Naggar, and R.R. Richards-Kortum, "Confocal microscopy for real-time detection of oral cavity neoplasia," Clin. Cancer Res. 9, 4714-4721 (2003).
[PubMed]

Curr. Opin. Chem Biol. (1)

K. Sokolov, M. Follen, and R. Richards-Kortum, "Optical spectroscopy for detection of neoplasia," Curr. Opin. Chem. Biol. 6, 651-658 (2002).
[CrossRef] [PubMed]

Gastrointest. Endosc. (1)

A.A. Elfert, P. J. Pasricha, B. Bell, R. Johnigan, S.Y. Xiao, K.H. Calhoun, and M. Motamedi, "High resolution optical coherence tomography for early detection of epithelial neoplastic transformation," Gastrointest. Endosc. 53, AB117 (2001).
[CrossRef]

IEEE J. Sel. Top Quant. Electron. (1)

J.A. Izatt, M.D. Kulkarni, H.W. Wang, K. Kobayashi, M.V. Sivak, �??Optical coherence tomography and microscopy in gastrointestinal tissues,�?? IEEE J. Sel. Top. Quant. Electron. 2, 1017-1028 (1996).
[CrossRef]

J. Gastroenterol. (1)

C. Pitris, C. Jesser, S.A. Boppart, D. Stamper, M.E. Brezinski, J.G. Fujimoto, �??Feasibility of optical coherence tomography for high-resolution imaging of human gastrointestinal tract malignancies,�?? J. Gastroenterol. 35, 87-92 (2000).
[CrossRef] [PubMed]

J. Infect. Dis. (1)

S.M. Smith, G.B. Baskin, P.A. Marx, �??Estrogen protects against vaginal transmission of simian immunodeficiency virus,�?? J. Infect. Dis. 182, 708-715 (2000).
[CrossRef] [PubMed]

J. Oral Pathol. Med. (1)

S. Alberti, C.T. Spadella, T.R.C.G. Francischone, G.F. Assis, T.M. Cestari, L.A.A. Taveira, �??Exfoliative cytology of the oral mucosa in type II diabetic patients: morphology and cytomorphometry,�?? J. Oral Pathol. Med. 32, 538-543 (2003).
[CrossRef] [PubMed]

J. Oral. Pathol. Med. (1)

H.M. Chen, C.Y. Wang, C.T. Chen, H. Yang, Y.S. Kuo, W.H. Lan, M.Y.P. Kuo, and C.P. Chiang, �??Auto-fluorescence spectra of oral submucous fibrosis,�?? J. Oral. Pathol. Med. 32: 337-343 (2003).
[CrossRef] [PubMed]

Laryngoscope (1)

W.M. White, M. Rajadhyaksha, S. Gonzalez, R.L. Fabian, and R.R. Anderson, "Noninvasive imaging of human oral mucosa in vivo by confocal reflectance microscopy," Laryngoscope 109, 1709-1717 (1999).
[CrossRef] [PubMed]

Nat. Biotechnol. (2)

P.J. Campagnola and L.M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotechnol. 21, 1356-1360 (2003).
[CrossRef] [PubMed]

W.R. Zipfel, R.M. Williams, and W.W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1368-1376 (2003).
[CrossRef]

Nat. Med. (2)

E. Brown, T. Mckee, E. Ditomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, "Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation," Nat. Med. 9, 796-800 (2003).
[CrossRef] [PubMed]

E.B. Brown, R.B. Campbell, Y. Tsuzuki, L. Xu, P. Carmeliet, D. Fukumura, and R.K. Jain, "In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy," Nat. Med. 7, 864-868 (2001).
[CrossRef] [PubMed]

Neoplasia (1)

N. Ramanujam, "Fluorescence spectroscopy of neoplastic and non-neoplastic tissues," Neoplasia 2, 89-117 (2000).
[CrossRef] [PubMed]

Opt. Express (2)

Opt. Lett. (1)

Oral Oncol. (2)

K. Onizawa, N. Okamura, H. Saginoya, and H. Yoshida, "Characterization of autofluorescence in oral squamous cell carcinoma," Oral Oncol. 39, 150-156 (2003).
[CrossRef] [PubMed]

K. Onizawa, N. Okamura, H. Saginoya, and H. Yoshida, "Characterization of autofluorescence in oral squamous cell carcinoma," Oral Oncol. 39, 150-156 (2003).
[CrossRef] [PubMed]

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

W.R. Zipfel, R.M. Williams, R. Christie, A.Y. Nikitin, B.T. Hyman, and W.W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," P. Natl. Acad. Sci. USA 100, 7075-7080 (2003).
[CrossRef]

A. Zoumi, A. Yeh, and B.J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," P. Natl. Acad. Sci. USA 99, 11014-11019 (2002).
[CrossRef]

Photochem. Photobiol. (1)

R. Drezek, C. Brookner, I. Pavlova, I. Boiko, A. Malpica, R. Lotan, M. Follen, and R. Richards-Kortum, "Autofluorescence microscopy of fresh cervical-tissue sections reveals alterations in tissue biochemistry with dysplasia," Photochem. Photobiol. 73, 636-641 (2001).
[CrossRef] [PubMed]

Other (3)

L. Moss-Salentijn and M. Hendricks-Klyvert. Dental and Oral Tissues (Lea & Febiger, Philadelphia, 1985), Chap.4.

William O. Dobbins, �??Diagnostic Pathology of the Intestinal Mucosa: an Atlas and Review of Biopsy Interpretation,�?? (Springer-Verlag, New York, NY, 1990).

Histological Typing of Cancer and Precancer of the Oral Mucosa,�?? J.J. Pindborg, ed. (Springer-Verlag, New York, NY, 1997).

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Figures (4)

Fig. 1.
Fig. 1.

Experimental setup

Fig. 2.
Fig. 2.

MPM (first column) and SHGM (second column) x-y images of buccal cheek pouch at various depths (z) using 800 nm excitation. The first MPM image (9 µm) shows the keratin layer of the epithelium. The second shows cells located in the epithelium (see Fig. 3 for rationale). In the images taken at 42 µm, both cells and fiber striations can be seen in the MPM image, while SHGM confirms the banded/fibrillar structure is collagen. Collagen is seen in both the 81 µm and 90 µm images.

Fig. 3.
Fig. 3.

(a) Reconstructed MPM y-z cross-section of buccal pouch. The box at the lower left is a false color cross-section taken from the region outlined by the red box. (b) SHGM cross-section corresponding to (a). (c) Two color coregistered image of MPM (red) and SHGM (green). The main contributor to the SHG signal is collagen, which is not present in the epithelium (ep), but is present in the submucosa (sm). The topmost bright thin layer in (a) and (c) is attributed to the keratin layer of the epithelium according to analysis of individual x-y images near the surface. Assessment of these characteristics and comparison with histology (d) allow the identification of the epithelium (ep), consisting of a bright keratin layer followed by a dark cellular layer, and bright submucosa (sm). Horizontal scale bars: 40 µm. Vertical scale bars: 60 µm.

Fig. 4.
Fig. 4.

MPM images with 730 nm (a–d), 780 nm (e–h), and 800 nm (i–l) excitation and different emission filters: 700 nm short pass filter (a, e, i); 400–450 nm band pass filter (b, f, j); 450–500 nm band pass filter (c, g, k); 500–550 nm band pass filter (d, h, l). Image m was a SHG image taken with 800 nm excitation and a narrow band pass filter 400/14 nm. All images were taken at the same imaging depth 35 µm. Green bar represents the detector gain used to take the corresponding image, with full-filled bar representing normalized maximal gain.

Tables (1)

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Table 1. Quantitative measurement of morphologic features

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