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Figure S1 shows fluorescence that correlates with the presence of benign lesions

Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes ex vivo immediately following excision. “Puzzle-fit” analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%–78% and specificities of 92%–97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment.Significance:A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed.

Figure S3A-C shows histology of false positive (FP) foreign body granuloma, benign inclusion cyst, and seborrheic keratosis skin samples.

Figure S2 shows histology of all false negative (FN) skin cancer samples.

Supplementary Methods include description of: 1) "Puzzle"-fit" analysis, 2) Intensity-based objective sensitivity and specificity analysis, 3) Blinded physician "Reader Study" analysis, and 4) Evaluation of reader responses regarding intensity.

Figure S3D shows histology of false positive (FP) skin samples with presence of healing area and inflammation.

Table S1 shows cancer type and histologic detection status following excision; Table S2 characterizes patients and specimens