Compatibility of fast tissue clearing with targeted fluorescence imaging and pathology analysis using clinical head and neck cancer biopsies

Complete tumor removal is crucial for successful surgical oncological outcomes. Targeted fluorescence imaging facilitates differentiation between healthy and cancerous tissue, enabling intraoperative margin assessment. However, its depth specificity is still limited. Three-dimensional fluorescence imaging could improve margin assessment, especially after tissue clearing. Conventional tissue clearing may take up to 24 h, limiting clinical applicability. This study developed and validated a fast tissue clearing protocol, achieving transparency within one hour. Ten patients from a fluorescence-guided surgery trial were included, from whom three tumor and three healthy tissue biopsies were acquired. These biopsies were used as control, cleared using a standard Benzyl Alcohol Benzyl Benzoate (BABB) clearing protocol, or cleared using the newly developed fast BABB clearing protocol. This protocol uses an orbital shaker, decreasing total process time to one hour. No significant difference in transparency was observed between normal and fast cleared biopsies. Fluorescence signals of all cleared biopsies increased directly after clearing and slowly decreased back to pre-cleared fluorescence signals over time. In non-cleared tumor biopsies, fluorescence intensity decreased substantially upon fixation, yet remained stable. Finally, clearing did not impact immunohistochemical staining. Therefore, fast tissue clearing is possible within one hour, enabling optical microscopy after tissue clearing for intraoperative pathology-assisted surgery.