Quantitative Autofluorescence Imaging of Oral Mucosa and Lesions: A Proof-of-Concept Study

<b>Background/Objectives:</b> This study aimed to quantitatively assess site-specific mean autofluorescence intensity across normal oral mucosal subsites and to evaluate the effectiveness of Autofluorescence Imaging (AFI) as an adjunct tool for distinguishing benign lesions, OPMDs, and oral cancers by comparing lesion intensity with anatomically matched healthy subsites. <b>Methods:</b> This observational study employed dual-mode imaging, comprising paired White Light Imaging (WLI) and AFI, captured from different oral cavity subsites using a smartphone-based point-of-care device. The Region of Interest (ROI) was annotated on WLI and automatically mapped to the corresponding AFI for both normal mucosa and lesions. WLI and AFI images were separated into their constituent red, green, and blue (RGB) channels, and AFI intensity was quantified via ImageJ. <b>Results:</b> A total of 1380 dual-mode images were acquired from 86 healthy participants. AFI intensities were comparable across most oral subsites, except for the lateral and ventral tongue. The lateral border showed the lowest fluorescence (Green channel-GC: 68.12 ± 28.27; Blue channel-BC: 25.29 ± 7.93), whereas the ventral tongue showed the highest (GC: 98.89 ± 42.22; BC: 37.08 ± 11.04; both <i>p</i> < 0.001). Among 611 lesions, predominantly from the buccal mucosa, AFI intensity declined progressively with increasing disease severity. Homogeneous leukoplakia (<i>n</i> = 149; GC: 38.62 ± 25.05; BC: 21.60 ± 9.50), non-homogeneous leukoplakia (<i>n</i> = 25; GC: 30.42 ± 18.66; BC: 18.25 ± 7.17) and oral cancer (<i>n</i> = 21; GC: 23.39 ± 15.53; BC: 15.82 ± 7.15; all <i>p</i> < 0.001) showed markedly reduced fluorescence, while benign lesions (<i>n</i>: 44; GC: 66.99 ± 30.88; BC: 32.01 ± 13.62) exhibited intermediate intensities, supporting AFI's discriminative potential. <b>Conclusions:</b> This phase-1, proof-of-concept study highlights subsite-specific variations in autofluorescence intensity within healthy oral mucosa, providing an essential baseline for objective interpretation of lesion-associated fluorescence changes. AFI has the potential to be used as a non-invasive adjunct for monitoring OPMDs. Further validation in larger and more diverse cohorts is required before clinical implementation.