Structured illumination microscopy virtual pathology for real-time large-specimen surgical pathology imaging using SYBR Gold and Eosin

Virtual pathology techniques have demonstrated the capability to overcome surgical pathology's critical disadvantage, time inefficiency, by imaging surgical samples ex vivo. Removing extensive tissue preparation steps allows virtual pathology to provide same-day or even within-procedure diagnosis. Structured illumination microscopy (SIM) is a wide-field fluorescent optical-sectioning microscopy technique that allows higher speed imaging as compared to similar modalities, making it a good candidate for real-time diagnostics. Although SIM image acquisition is fast, the staining procedure must also be fast to meet real-time use, considering a maximum time allotment of 20-30 minutes for staining and imaging during surgery. Thus, we require our staining procedure to take no more than 4 minutes for nuclear and extracellular matrix (ECM) staining. In this work, we evaluated multiple candidates for imaging of large surgical resection specimens based on staining time and specificity, image quality, and cost per sample. Based on phantom studies using bovine muscle, we found that SYBR Gold provides the best results for nuclear staining (compared to TO-Pro3 and DRAQ5) on samples with large volume and surface area. Also, we found an optimal ratio between concentration and staining time for Eosin as an ECM stain. We then applied these protocols to imaging of radical prostatectomy specimens immediately after surgical removal, and present here for the first-time SYBR Gold and Eosin (Sg&E) virtual pathology for whole prostate imaging. In ten cases (2 with positive margin), we were able to identify healthy and pathological structures using SG&E virtual pathology that corresponds to hematoxylin and eosin pathology.