A random rayburst sampling (RRBS) framework was developed to detect the nucleus and cell membrane boundaries in three-dimensional (3-D) space. Raw images were acquired through a full-field optical coherence tomography system with submicron resolution—i.e., 0.8 μm in lateral and 0.9 μm in axial directions. The near-isometric resolution enables 3-D segmentation of a nucleus and cell membrane for determining the volumetric nuclear-to-cytoplasmic (N/C) ratio of a single cell. The RRBS framework was insensitive to the selection of seeds and image pixel noise. The robustness of the RRBS framework was verified through the convergence of the N/C ratio searching algorithm. The relative standard deviation of the N/C ratio between different randomly selected seed sets was only 2%. This technique is useful for various in vitro assays on single-cell analyses.
In vivo harmonic generation microscopy (HGM) has been applied successfully in healthy human skin and can achieve a submicron resolution, similar to histopathologic examination, even at a penetration depth up to 270 μm. This study aims to investigate the clinical applicability of HGM imaging for differential diagnosis of nonmelanoma pigmented skin lesions. A total of 42 pigmented skin tumors, including pigmented basal cell carcinoma, melanocytic nevus, and seborrheic keratosis were evaluated by HGM ex vivo or in vivo. Based on the standard histopathologic characteristics, we established the corresponding HGM imaging criteria for each pigmented tumor. Diagnostic performance of HGM for differentiating nonmelanoma pigmented skin tumors was evaluated through the observers’ direct general assessment (overall evaluation) or the presence of two imaging criteria with the highest sensitivity and specificity (major criteria evaluation). Our results show that, based on the direct general assessment, the sensitivity is 92% [95% confidence interval (CI): 67 to 97%] and the specificity is 96% (95% CI: 83 to 99%); by major criteria evaluation, 94% sensitivity (95% CI: 70 to 99%) and 100% specificity (95% CI: 87 to 100%) are achieved. Our study indicates that HGM serves as a promising histopathological examination tool for noninvasive differential diagnostics of nonmelanoma pigmented skin tumors.
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