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 highresolution, 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 twophoton 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. (Cancer Res 2005; 65(4): 1180-6)

In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

“…There were also no significant changes in the cellular coefficient of variation of the redox ratio or lifetime variables between the first vs. last plane of the epithelium in either normal or precancerous tissues (P Ͼ 0.05). The thickness of the normal epithelium was 25 Ϯ 3 m, indicating that thickness measurements were consistent between animals and consistent with our previous studies (19,25). The epithelial thickness in the low-grade (29 Ϯ 9 m) and high-grade (31 Ϯ 9 m) dysplastic samples were determined by using the same protocol.…”

“…Diagnosis was based on established criteria (40). The diagnosis assigned to each image stack was based on the most severe diagnosis of the entire corresponding biopsy sample, consistent with our previous multiphoton microscopy studies in this animal model (19,25).…”

“…For each hamster, the right cheek pouch was either treated chronically with 0.5% DMBA in mineral oil (DMBA-treated animals) or mineral oil only (control animals) for 16 weeks. The treatment procedures were established from previous studies (19,25,35). At 16-21 weeks after the commencement of treatment, the cheek pouch of each animal was imaged by using multiphoton microscopy.…”

Optical Detection of Cancers

“…These methods have been studied for cancer detection with Raman spectroscopy and multiphoton microscopy and for analysis of the skin dermis by the second-harmonic signals. [67][68][69] …”

546 Quantifying Human Eosinophils Using 3-Dimensional Volumetric Images Collected With Multi-Photon Fluorescence Microscopy

“…14 FAD is a coenzyme in the mitochondrial electron transport chain that has a maximum absorption at 445 nm, resulting in a peak fluorescence emission of 525 nm. 15 Multi-photon microscopy (MPM) is a powerful method for collecting fluorescence images from cells and tissues, 16 and has been used to perform in vivo imaging of FAD from squamous epithelium in animals. 17,18 MPM imaging has inherent 3D resolution, uses near-infrared excitation for superior tissue penetration, has lower photobleaching effects, and is capable of providing quantitative information.…”