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Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 12, Iss. 11 — May. 31, 2004
  • pp: 2478–2486
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In vivo multimodal nonlinear optical imaging of mucosal tissue

Ju Sun, Tuya Shilagard, Brent Bell, Massoud Motamedi, and Gracie Vargas  »View Author Affiliations


Optics Express, Vol. 12, Issue 11, pp. 2478-2486 (2004)
http://dx.doi.org/10.1364/OPEX.12.002478


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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

1. Introduction

The goal of this study is to investigate the application of a multimodal nonlinear imaging approach based on the integration of multiphoton microscopy (MPM) with second harmonic generation microscopy (SHGM) for in vivo evaluation of epithelial tissue microstructure. The combination of these imaging modalities allows deep visualization of tissue with high resolution capable of resolving cellular and subcellular microstructures, which has been demonstrated in several tissue types [21

21. 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]

23

23. A.T. Yeh, N. Nassif, A. Zoumi, and B.J. Tromberg, “Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence,” Opt. Lett. 27, 2082–2084 (2002). [CrossRef]

]. MPM is ideal for functional imaging of microscopic structures by either autofluorescence (e.g., NAD(P)H etc.) or use of functional fluorescent probes [24

24. 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]

26

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

]. SHGM has been used to visualize collagen in nonmucosal tumors, but was not combined with MPM [27

27. 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]

]. However, SHGM complements MPM by providing direct contrast for specific structural proteins such as collagen and can be coregistered with MPM. In this study, we used the combined nonlinear 3D optical imaging technique to visualize the morphological, spectroscopic, and microstructural features of mucosal tissue under in vivo conditions in hamster cheek pouch. This animal model is commonly used to study mucosal disorders and provides easy access for imaging and manipulation [28

28. L. Coghlan, U. Utzinger, R.A. Drezek, D. Heintzelmann, A.F. Zuluaga, C. Brookner, R.R. Richards-Kortum, I. Gimenez-Conti, and M. Follen, “Optimal fluorescence excitation wavelengths for detection of squamous intra-epithelial neoplasia: results from an animal model,” Opt. Express 7, 436–446 (2000), http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-12-436. [CrossRef] [PubMed]

].

2. Materials and methods

The combined multiphoton and SHG microscope setup is shown in Fig. 1. Illumination of the sample for both multiphoton excitation and SHG is accomplished using a Ti:sapphire femtosecond laser (100 fs, 82 MHz) that is tunable from 720 to 950 nm (Tsunami, Spectra-Physic, CA). Both multiphoton autofluorescence and SHG were collected in a backscattering (epi-illumination) geometry. Excitation wavelengths centered within 730–840 nm were used, while power incident on the sample was kept less than 20 mW in all experiments. A 690 nm short pass dichroic mirror in the detection path is used to separate fluorescence emission or SHG from reflected illumination. For SHG imaging, the laser is tuned to 800 nm and a bandpass filter centered at 400 nm with a 14 nm bandwidth was inserted to reject the fluorescence emission.

Fig. 1. Experimental setup

Approximately 10 minutes prior to imaging, hamsters were anesthetized with ketamine (150 mg/kg) and Xylazine (2.5 mg/kg) by intraperitoneal injection. The cheek pouch was then manually everted, rinsed with physiological saline solution, and pinned on a sample holder that was then fixed onto the microscope stage, allowing the cheek pouch to be imaged. Two objectives were used in this study: 40×, 1.2 NA, water immersion objective (C-Apochromat) and 10×, 0.3 NA, dry objective (Plan-Neofluar). The target area on the buccal pouch was centered with the aid of an aiming beam from the excitation source and then precisely located by scanning (1 s per frame with 512×512 pixels) using the low magnification objective (10×) in reflectance mode. Once the surface of the target area was located, the microscope setup was switched to multiphoton fluorescence mode with the 40× objective. Sequential depth-scans were taken using a typical z increment of 3 µm. SHGM was then performed on the same image volume using the same z increment. Thus, for each z-plane, corresponding MPM and SHGM images were acquired. To reduce noise and improve imaging quality, an averaging scanning scheme was used, resulting in a dwell time of 30–60 µs per pixel. A biopsy punch was taken at the center of the imaging area. The tissue sample was fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned perpendicular to the sample surface with a sectioning thickness of 5 µm. Several sections near the starting and end point were omitted from further processing. Alternate sections were stained in groups of four with hematoxylin-eosin (H&E) and Massons’ trichrome; another four sections were made following a 50 µm gap in sectioning. This method allowed for sections to be sampled across a 300–400 µm distance approximately centered in the imaging area (imaging field of view was 320×320 µm).

3. Results and discussion

Four buccal pouches were imaged in this study. Typical images of tissue sections visualized at various depths with MPM and SHGM are depicted in Fig. 2. The first column consists of multiphoton fluorescence images whereas the second column shows the corresponding SHG images. All of the MPM images shown in Fig. 2 were collected using the same detector gain. For SHG images, the detector gain was increased by 50%. MPM allows imaging of cellular structures within the epithelium up to approximately 40 µm and of collagen beyond this depth. There is no detected SHG signal in the superficial depths as expected, since the source of SHG in the cheek pouch is most likely due to collagen found in the submucosa [2

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

]. The first sign of collagen as revealed by SHG occurs at approximately 40 µm, confirming that the fibrillar structure in the corresponding autofluorescence image is mainly collagen. Collagen is the dominant component of the connective tissue below the epithelium in oral mucosa. These images demonstrate how dual imaging with MPM and SHGM helps differentiate signals from cellular components and collagen. Although relatively small amounts of elastic fibers are also found in the submucosa [29

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

], several studies have reported the absence of SHG from elastin using 800 nm excitation [21

21. 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]

,27

27. 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]

].

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. (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.

Quantitative analysis of MPM images enabled measurement of epithelial thickness and cellular nucleus diameter (Table 1). As an example, the y-z cross-section image in Fig. 3(a) has been used to determine the epithelial thickness by measuring the thickness of the darker layer located between the two brighter layers in the sandwich structure. The measurement was repeated at 20 positions evenly apart along the y direction, resulting in an average thickness of 30.0±4.8 µm. The epithelial thickness was also measured with the histology image shown in Fig. 3(d), resulting in an average value of 40.3±7.2 µm. For measuring nuclear diameter, we took into account that the nuclei in histological sections are in a plane (y-z) perpendicular to the tissue surface rather than in a plane parallel to the tissue surface as in individual x-y scan taken with multiphoton fluorescence. A fair comparison between histology and multiphoton imaging involved measuring cells along the same y direction (parallel to tissue surface in y direction). The superficial epithelium was defined as the first cell layer below the keratin layer and the deep epithelium as the cell layer above the basement membrane for both histology and MPM. In MPM measurements, images were first thresholded following histogram analysis to reveal nuclei of individual cells. The results of the above quantitative analysis on MPM images are listed in Table 1, showing good agreement with histology. Some discrepancy in epithelial thickness was noted between the two methods. This error is attributed to artifacts such as delamination of the keratinized layer during histological processing.

Figure 4 demonstrates the emission properties of normal buccal pouch upon excitation of three distinct wavelengths (730, 780, and 800 nm). In the case shown, the sample was imaged at a single depth of 35 µm where both epithelial cells and collagen are present near the basement membrane. Figure 4(a)–(d) are taken upon illumination with 730 nm radiation, whereas Fig. 4(e)–(h) were acquired using 780 nm, and Fig. 4(i)–(m) using 800 nm. Emission band pass ranges are listed in the left column in Fig. 4. There are a number of observations that can be made based on assessment of these images. First, cellular structures are most

Table 1. Quantitative measurement of morphologic features

table-icon
View This Table

evident using 730 nm excitation (Fig. 4(a)–(c)). Although individual nuclei can be identified using 800 nm excitation (Fig. 4(i)), the cell boundaries are not as distinct as in the case of 730 nm using the same incident power. Autofluorescence originating from epithelial cells due to 730 nm excitation is primarily observed in the 400–500 nm region (Fig. 4(b) and (c)). Recent studies on mucosal tissues using fluorescence spectroscopy have identified the major endogenous fluorophores in epithelial tissues [7

7. 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]

,10

10. A. Gillenwater, R. Jacob, R. Ganeshappa, B. Kemp, A.K. El-Naggar, J.L. Palmer, G. Clayman, M.M. Follen, and 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]

,15

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

19

19. 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]

]. These include reduced nicotinamide adenine dinucleotide (NAD(P)H), flavin adenine dinucleotide (FAD), and tryptophan in the epithelial cells, and collagen in the underlying submucosa. In these studies, NAD(P)H fluorescence was found to be strongest in the 400–500 nm region, corresponding to the two emission ranges shown in Fig. 4(b) and (c), while FAD autofluorescence peak falls within 500–550 nm corresponding to Fig. 4(d) [30

30. 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]

].

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.

In the image series using 780 nm illumination, fluorescence from cells is lower (Fig. 4(e)–(h)). However one observes collagen autofluorescence in the 450–550 nm range (collagen appears on right side of the field), which was not observed using 730 nm excitation. This collagenous region appears bright and diffuse (Fig. 4(g) and (h)). At 800 nm excitation, however, the same region appears highly structured in the 400–450 nm range (Fig. 4(j)), but diffuse at emission wavelengths >450 nm. The corresponding SHGM image is shown in Fig. 4(m), demonstrating a highly structured fibrillar pattern for collagen similar to the pattern of Fig. 4(j). These results indicate that at 800 nm, the main sources of signal are autofluorescence and SHG from collagen with a weaker optical signal originating from cellular structures. The cellular microstructure and the extracellular matrix can be differentiated by multimodal MPM and SHGM imaging to discriminate SHG signals of collagen from the background autofluorescence signal as shown in Fig. 4(m). At excitation wavelengths greater than 800 nm we observed a strong signal from collagen (both autofluorescence and SHG), but virtually no signal from cellular structures. The above results suggest that 800 nm excitation is an optimal wavelength for simultaneous visualization of cellular microstructures and extracellular matrix in the oral mucosa and submucosa. Shorter excitation wavelengths are optimal for imaging of epithelial cells alone.

Our above preliminary results on in vivo imaging of hamster cheek pouch demonstrate that a high-resolution multimodal imaging modality combining multiphoton fluorescence with second harmonic generation microscopy can clearly elucidate the microstructure of both the epithelium and deeper submucosa in mucosal tissues. Cellular and epithelial morphology measurements were in excellent agreement with histology, demonstrating the potential of this technique for the study of disease of epithelial tissues. In particular, we demonstrated the ability to visualize microstructure and measure morphological parameters that are known to change with disease progression. For example, during neoplastic transformation cellular morphology is altered, epithelial thickness increases, and collagen found in the submucosa undergoes architectural changes [1

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

2

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

]. Submucous fibrosis is marked by the accumulation of subepithelial (submucosal) collagen [4

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

] and could in particular benefit from SHGM for direct study of collagen. Diabetes mellitus is also associated with cellular morphological changes in the epithelium and alterations in epithelial thickness [5

5. S. Alberti, C.T. Spadella, T.R.C.G. Francischone, G.F. Assis, T.M. Cestari, and 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]

]. Finally, it has been suggested the susceptibility of vaginal mucosal tissue to HIV transmission may be correlated to epithelial thickness and may be reduced by estrogen treatment [3

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

]. The method presented in this paper will not only allow for these pathological changes to be studied in vivo, but also will allow for the assessment of drug interactions with mucosal tissue. Although in this study MPM was used to measure microstructure autofluorescence, there is the possibility to further study tissue function by MPM using functional biomarkers or fluorescent probes. Finally, this technique shows potential to be developed into a diagnostic tool. While the expense of ultrafast lasers could be a hindrance to translation into the clinic, such sources will likely become more user-friendly, less expensive, and more available in the future.

4. Conclusions

In this study, we have demonstrated the ability of MPM/SHGM for high resolution imaging of the oral mucosa and submucosa in vivo. The technique allows the identification of specific microstructures that may alter in morphology and function with disease progression. These include cellular/nuclear morphology, thickness of the keratinized stratum corneum, thickness of the epithelium, and direct identification of collagen in the submucosa. Imaging results of normal oral epithelial tissues are in excellent agreement with histology. An illumination wavelength of 800 nm was found to be optimal for imaging of both cellular morphology and submucosal collagen using the combined MPM/SHGM approach, however 730 nm is preferred for imaging only cellular structures.

Acknowledgments

Financial support from the John Sealy Memorial Endowment Fund for Faculty Recruitment (#6074-03), National Institutes of Health (# R21-CA89266), and NASA (# NAS2-0205) are gratefully acknowledged.

References and links

1.

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

2.

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

3.

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

4.

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

5.

S. Alberti, C.T. Spadella, T.R.C.G. Francischone, G.F. Assis, T.M. Cestari, and 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]

6.

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]

7.

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]

8.

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

9.

C. Pitris, C. Jesser, S.A. Boppart, D. Stamper, M.E. Brezinski, and 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]

10.

A. Gillenwater, R. Jacob, R. Ganeshappa, B. Kemp, A.K. El-Naggar, J.L. Palmer, G. Clayman, M.M. Follen, and 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]

11.

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]

12.

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]

13.

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]

14.

K. Sokolov, R. Drezek, K. Gossage, and R.R. Richards-Kortum, “Reflectance spectroscopy with polarized light: is it sensitive to cellular and nuclear morphology,” Opt. Express 5, 302–317 (1999), http://www.opticsexpress.org/abstract.cfm?URI=OPEX-5-13-302. [CrossRef] [PubMed]

15.

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

16.

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

17.

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]

18.

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]

19.

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]

20.

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]

21.

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]

22.

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]

23.

A.T. Yeh, N. Nassif, A. Zoumi, and B.J. Tromberg, “Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence,” Opt. Lett. 27, 2082–2084 (2002). [CrossRef]

24.

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]

25.

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]

26.

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

27.

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]

28.

L. Coghlan, U. Utzinger, R.A. Drezek, D. Heintzelmann, A.F. Zuluaga, C. Brookner, R.R. Richards-Kortum, I. Gimenez-Conti, and M. Follen, “Optimal fluorescence excitation wavelengths for detection of squamous intra-epithelial neoplasia: results from an animal model,” Opt. Express 7, 436–446 (2000), http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-12-436. [CrossRef] [PubMed]

29.

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

30.

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]

OCIS Codes
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(170.5810) Medical optics and biotechnology : Scanning microscopy
(190.4180) Nonlinear optics : Multiphoton processes

ToC Category:
Research Papers

History
Original Manuscript: April 27, 2004
Revised Manuscript: May 21, 2004
Published: May 30, 2004

Citation
Ju Sun, Tuya Shilagard, Brent Bell, Massoud Motamedi, and Gracie Vargas, "In vivo multimodal nonlinear optical imaging of mucosal tissue," Opt. Express 12, 2478-2486 (2004)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-12-11-2478


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References

  1. William O. Dobbins, �??Diagnostic Pathology of the Intestinal Mucosa: an Atlas and Review of Biopsy Interpretation,�?? (Springer-Verlag, New York, NY, 1990).
  2. Histological Typing of Cancer and Precancer of the Oral Mucosa,�?? J.J. Pindborg, ed. (Springer-Verlag, New York, NY, 1997).
  3. 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]
  4. S.M. Sirsat and J.J. Pindborg, �??Subepithelial changes in oral submucous fibrosis,�?? Acta Pathol. Microbiol. Scand. 70, 161-173 (1967). [CrossRef] [PubMed]
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  6. 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]
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