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.
Structured illumination microscopy provides a compelling solution to location inaccuracy a in core-needle breast biopsy procedures. Fluorescent H and E analog dyes and dual-channel SIM imaging were utilized to create an digital pathology image analogous to a standard histology within minutes for point of care pathology. This study validated SIM’s ability to generate an image for on-site pathology validation of sample quality which minimizes the risk of a repeat procedure. Diagnoses from the diagnostic-quality SIM images were also proven to align with diagnoses obtained by standard histological processing of the same sample.
Accurate detection and diagnosis of prostate cancer remains challenging even with MRI fusion ultrasound biopsy procedures. These challenges contribute to diagnostic errors that can lead to repeat procedures. This study explores the application of ex vivo structured illumination microscopy (SIM) to generate pathology images of fresh biopsies to inform diagnostic decisions. Samples from the lesion of interest and a benign area of each patient were obtained, stained with fluorescent H&E analog dyes, imaged on a custom SIM system and diagnosed by a pathologist. This procedure could be extrapolated to a “see-and-treat” paradigm for localized prostate cancer ablation in the future.
Staining procedures in virtual H&E ex vivo microscopy is non-standardized, and image quality can be tuned by systematic parameter optimization. We are using structured illumination microscopy and image processing for an objective assessment of staining procedure. Bovine muscle tiles of 2x2 cm are used as experimental tissue. In these experiments we differ staining procedure by varying stain and rinse solvent, dye concentration, and immersion time. Quantitative measures of staining quality were compared. Our findings confirm the profound impact of the confluence of multiple factors in staining quality, and suggest general strategies for successful protocol development.
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