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In vivo multimodal nonlinear optical imaging of mucosal tissue

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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

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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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