Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous epithelial tissues

Abstract

This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows high-resolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a two-photon excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multiphoton fluorescence endoscopy in a clinical setting.

Figure 1

MPLSM images of tissue biopsies (A) incubated in ethidium homodimer-1 and (B) soaked in methanol for 30 minutes first, and then incubated in ethidium homodimer-1. Both samples were imaged approximately 7 hours after biopsy under identical experimental conditions. Sample A was subject to the full imaging protocol before labeling. Bar, 30 µm

Figure 2

Representative MPLSM images at a two-photon excitation wavelength of 780 nm of tissues diagnosed as normal (A), moderate dysplasia (B), CIS (C), nonpapillary SCC (D), and papillary SCC (E). The images in the figure were corrected for the two-photon excitation peak power at the sample; thus, the fluorescence intensities in A to E can be compared (fluorescence intensity was pseudocolored, see calibration bar). The top image in the montage is at the surface of the tissue and each subsequent image in the montage represents a 10-µm z step, with the final image in the montage at a depth of 40 µm below the tissue surface. Bar, 30 µm