Assessment of cutaneous tissue using light-sheet microscopy: Evaluating feasibility of 3D-microscopy for mohs micrographic surgery

Summary: Mohs micrographic surgery(MMS) offers high cure rates for high-risk skin cancers but is time-consuming. Hybrid open-top light sheet microscopy(OTLSM) enables fast 3D imaging and holds promise for MMS with rapid staining protocols. The aims of this research were to 1) develop a fast-staining protocol for imaging of debulk MMS tissue using OTLSM, and 2) determine feasibility, challenges, strengths, and future applications of the use of the OTLSM for MMS. Patients undergoing MMS for squamous cell(SCC) or basal cell carcinoma(BCC) were eligible to participate. For each tumor, a shave debulk was taken as part of MMS. Two novel ultra-fast protocols, using either DRAQ5 or SYBR Gold with NHS ester, were developed for staining the tissue in pseudo-H&E, followed by clearing in 2,2’-Thiodiethanol. The surface of the tissue was then imaged using OTLS with a custom sample holder with an adjustable-pressure lid. The total imaging and processing time was less than 5 minutes. The study included 32 MMS debulks from 27 patients, mostly from head or neck tumors (n=31). Diagnoses included BCC (65.6%, n=5), SCC (18.8%, n=6), and SCC <i>in-situ</i> (15.6%, n=5). The ultra-fast staining protocol enabled quicker visualization than traditional methods, but interpreting live images was challenging due to the OTLSM 45-degree geometry. Additional challenges included ensuring tissue flatness, balancing stain penetration with timing, and maintaining proper tissue orientation during imaging. Additional optimization of our OTLSM imaging protocol has the potential to reduce tissue processing and imaging times as compared to frozen pathology. Furthermore, if challenges are overcome, the 3D-nature of the OTLSM technology will allow for a more complete analysis of surgical margins compared to traditional pathology. Jorge A. Rios-Duarte and Shikhar Dhingra contributed equally. Shikhar Dhingra works for Alpenglow Biosciences. Jorge A. Rios-Duarte¹, Shikhar Dhingra², Steven N. Hart³, Nneka Comfere^{1, 3}, Addison M. Demer¹, Nahid Y. Vidal¹ 1. Department of Dermatology, Mayo Clinic, Rochester, MN, United States. 2. Alpenglow Biosciences, Inc., Seattle, WA, United States. 3. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States. Bioinformatics, Computational Biology, and Imaging