Diagnostic accuracy of two-photon fluorescence microscopy in the Mohs micrographic surgical margins of squamous cell carcinoma

Cutaneous squamous cell carcinoma (SCC) can be time-consuming to treat with Mohs micrographic surgery (MMS) due to the need for intraoperative frozen section (FS) preparation. Two-photon fluorescence microscopy (TPFM) can generate H&E-equivalent images from fresh tissue specimens in a fraction of this time. To determine the accuracy of TPFM for the evaluation of squamous cell carcinoma in MMS margins compared to conventional FS Mohs slide preparation. TPFM was used to image 144 first stage MMS margins from patients being treated for SCC. A Mohs surgeon reviewed 44 training images and then evaluated 100 margins. After a delay, the same surgeon evaluated the corresponding FS slides. Pairs of TPFM and FS slides were reviewed by an expert dermatopathologist to form a consensus diagnosis. Agreement with consensus diagnosis as assessed by an independent dermatopathologist. 3 margins (3%) unequivocally disagreed with the consensus on TPFM and 2 margins (2%) disagreed on FS. The sensitivity and specificity of TPFM were 95.1% and 98.2%, respectively. This study demonstrates that slide-free histology can be interpreted equivalently to conventional Mohs slide processing by both MMS surgeons and dermatopathologists with minimal training.