In-Vivo Nonlinear Optical Microscopy (NLOM) of Epithelial-Connective Tissue Interface (ECTI) Reveals Quantitative Measures of Neoplasia in Hamster Oral Mucosa

Abstract

The epithelial-connective tissue interface (ECTI) plays an integral role in epithelial neoplasia, including oral squamous cell carcinoma (OSCC). This interface undergoes significant alterations due to hyperproliferating epithelium that supports the transformation of normal epithelium to precancers and cancer. We present a method based on nonlinear optical microscopy to directly assess the ECTI and quantify dysplastic alterations using a hamster model for oral carcinogenesis. Neoplastic and non-neoplastic normal mucosa were imaged in-vivo by both multiphoton autofluorescence microscopy (MPAM) and second harmonic generation microscopy (SHGM) to obtain cross-sectional reconstructions of the oral epithelium and lamina propria. Imaged sites were biopsied and processed for histopathological grading and measurement of ECTI parameters. An ECTI shape parameter was calculated based on deviation from the linear geometry (ΔLinearity) seen in normal mucosa was measured using MPAM-SHGM and histology. The ECTI was readily visible in MPAM-SHGM and quantitative shape analysis showed ECTI deformation in dysplasia but not in normal mucosa. ΔLinearity was significantly (p < 0.01) higher in dysplasia (0.41±0.24) than normal (0.11±0.04) as measured in MPAM-SHGM and results were confirmed in histology which showed similar trends in ΔLinearity. Increase in ΔLinearity was also statistically significant for different grades of dysplasia. In-vivo ΔLinearity measurement alone from microscopy discriminated dysplasia from normal tissue with 87.9% sensitivity and 97.6% specificity, while calculations from histology provided 96.4% sensitivity and 85.7% specificity. Among other quantifiable architectural changes, a progressive statistically significant increase in epithelial thickness was seen with increasing grade of dysplasia. MPAM-SHGM provides new noninvasive ways for direct characterization of ECTI which may be used in preclinical studies to investigate the role of this interface in early transformation. Further development of the method may also lead to new diagnostic approaches to differentiate non-neoplastic tissue from precancers and neoplasia, possibly with other cellular and layer based indicators of abnormality.

Remodeling of the Epithelial–Connective Tissue Interface in Oral Epithelial Dysplasia as Visualized by Noninvasive 3D Imaging

“…The MPAM-SHGM system used for this study has been described previously (29, 34). Illumination was provided using a titanium sapphire (Ti:sapphire) femtosecond (~100fs, 82MHz) laser (Tsunami, Spectra Physics), tunable within 750–1000nm.…”

“…In a previous study, we reported on the use of in vivo MPAM-SHGM to delineate 2D cross-sectional morphology of the ECTI (akin to histology) in a hamster model for OED (29). An ECTI line-shape parameter (ΔLinearity, a measure of ECTI curviness) was defined and shown to increase from normal to OED.…”

Recent advances and applications of nonlinear optical microscopy: perspectives in cancer imaging

“…premalignancy and oral malignancy imaging through multiphoton microscopy which was combined with OCT, optical Doppler tomography (ODT). By using autofluorescence phenomenon, multiphoton autofluorescence microscopy (MPAM) applied for hamster oral mucosal neoplasia [13]. This group was successfully demonstrated quantitative aspects of oral cancer in a hamster model.…”

“…This group was successfully demonstrated quantitative aspects of oral cancer in a hamster model. Also recently in 2016, there was a development of multiphoton microscopy (MPM) for oral epithelial dysplasia based on MPAM by the same group [13]. This method was non-invasive and very important, because this dysplasia could progress to cancer [14].…”

Development of a multimodal foveated endomicroscope for the detection of oral cancer

“…In an effort to overcome the limitations of COE and autofluorescence imaging, several modalities have been explored including nonlinear microscopy [16,17], Raman spectroscopy [18,19], fluorescence lifetime imaging (FLIM) [20,21], optical coherence tomography [22,23], spectroscopy with point spectrometers [24,25], multispectral imaging [26,27] and confocal microscopy [20,28]. While each of these methods delivers valuable information to aid in the diagnosis of oral cancer, studies have suggested that a combination of these techniques could improve performance [5,7,21,27].…”