Enabling high-resolution diagnostic oral confocal laser endomicroscopy in mice

Therapeutic prevention of oral squamous cell carcinoma (OSCC) will avoid significant morbidity and mortality. To observe and measure the in vivo efficacy of therapeutic challenges, microscopic-level diagnosis without animal sacrifice is required. This study introduces a refined diagnostic methodology for non-invasive cellular-level imaging for diagnosis of micro-lesions by utilizing high-resolution scanning-fibre confocal laser endomicroscopy (ViewnVivo) with topical fluorescence imaging agents. We detail the development and standardization of imaging protocols using a fluorescent, cell-permeable cancer-targeting agent (PARPi-FL) as a cancer-targeting agent and a pan-cytoarchitectural (acriflavine) agent in a pre-clinical murine 4-NQO induced OSCC model. We provide comprehensive methodology for the in vivo identification of the progressive stages of oral carcinogenesis from microscopic lesions, supported by an annotated signature guide correlating with conventional histopathology. Our findings demonstrate that in vivo CLE imaging with both PARPi-FL and acriflavine clearly distinguishes between histologically normal and pathological oral tissue. Tissues with histologic dysplasia and carcinoma demonstrated PARPi-FL positivity and an aberrant nuclear staining pattern with acriflavine, compared to the regularly spaced nuclear staining of normal nuclei. Crucially, this methodology detects microscopic changes not visible to the naked eye, but histologically abnormal. Our observation model of progressive oral carcinogenesis has the potential to accelerate standardised interrogation of early molecular diagnostic applications and novel therapeutic efficacy, whilst reducing the need for animal sacrifice. This will result in faster validated translation to human applications, advancing effective early oral cancer detection and prevention.