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J Oral Pathol Med (2010) 39: 318–327 Background: A rigid confocal endoscope has been developed to assess the oral squamous epithelium of mice and to determine sensitivity, specificity, and accuracy of this new technology. Methods: This endoscope is connected to the commercially available Heidelberg Retina Tomograph (HRT). HRT is a device with a 670-nm diode laser designed to acquire topographical measurements of the optic nerve head. Real-time rigid confocal endoscopy is demonstrated by imaging the epithelial lesions of a mice model. Six-week-old male C57Bl/6 mice were randomly divided into a non-treated group ( n = 10) and into a 4-nitroquinoline 1-oxide (4-NQO)-treated group ( n = 50). In the 4-NQO-treated group, the mice obtained 4-nitroquinoline 1-oxide in the drinking water (100 μg/ml) to induce tumourigenesis in the mouse tongue. The 4-NQO-solution was diluted in the drinking water for mice. After an 8–16-week carcinogen treatment with 4-NQO ( ad libitum), mouse tongues were dissected within 3 h after CO2 overdose. After confocal microscopy of all lesions of the tongue, conventional histopathological investigation was performed. Results: The inter-rater reliability for the two observers of the confocal microscopic findings was found to be Kappa = 0.59 ( P < 0.001). The penetration depth varied in the healthy tissue of the underside of the tongue throughout this study and was measured between 104 and 240 μm. In keratotic lesions, the penetration depths were diminished and varied between 80 and 140 μm. Strong keratinization inhibits the evaluation of the epithelium. For differentiation between low-grade and high-grade squamous intra-epithelial lesions, a sensitivity and specificity of 73% and 88% was reached. Conclusions: The animal experiment with this non-invasive new technology indicates that this imaging technology facilitates the detection of pre-cancerous lesions of the underside of the oropharynx. Human studies on oropharyngeal and laryngeal lesions are needed to prove the applicability of this method in the field of otorhinolaryngology. [ABSTRACT FROM AUTHOR]