Background/Objectives:Accurate intraoperative margin assessment is essential for ensuring complete tumour excision in breast-conserving surgery, minimising local recurrence, and avoiding reoperations. Ex vivo fusion confocal microscopy (EVFCM) provides real-time, high-resolution imaging of fresh, unfixed tissues that closely resembles conventional histological imaging. This study aimed to validate the diagnostic performance and clinical feasibility of EVFCM for real-time intraoperative margin assessment during breast cancer surgery.Methods:A prospective observational diagnostic validation study was conducted using 144 breast tissue specimens. The samples were stained with acridine orange and fast green and scanned using a VivaScope 2500M-G4 system. Two breast pathologists independently evaluated the EVFCM images, blinded to the conventional histology results, which served as the reference standard. The diagnostic accuracy, sensitivity, specificity, and interobserver agreement were calculated using Cohen’s κ.Results:Interobserver agreement was almost perfect for neoplasia detection (97.3%, κ = 0.942) and tumour type classification (93.8%, κ = 0.883). The EVFCM achieved 93.7% sensitivity and specificity, with 94.0% accuracy for tumour detection (κ = 0.929,p< 0.001); 95.8% accuracy for tumour type classification (κ = 0.925,p< 0.001); and 95.1% accuracy for invasive subtype identification (κ = 0.907,p< 0.001). For margin assessment, EVFCM achieved 80% sensitivity, 100% specificity, and 99.3% accuracy (κ = 0.857,p< 0.001), whereas margin distance evaluation (<2 mm vs. ≥2 mm) yielded 75% sensitivity, 100% specificity, and 98.6% accuracy (κ = 0.854,p< 0.001).Conclusions:EVFCM enables rapid, high-resolution imaging of fresh breast tissue, facilitating real-time intraoperative margin evaluation with excellent diagnostic concordance and workflow efficiency. Its integration into surgical practice could reduce re-excisions, enhance oncological safety, and improve patient outcomes in breast-conserving surgeries.
**背景/目的：** 术中切缘的准确评估对于确保保乳手术中肿瘤的完整切除、最大限度减少局部复发及避免再次手术至关重要。离体融合共聚焦显微镜技术能够对新鲜、未固定的组织进行实时、高分辨率成像，其图像与传统组织学成像高度相似。本研究旨在验证EVFCM在乳腺癌手术中进行实时术中切缘评估的诊断性能与临床可行性。 **方法：** 本研究为一项前瞻性观察性诊断验证研究，共纳入144份乳腺组织标本。样本经吖啶橙和固绿染色后，使用VivaScope 2500M-G4系统进行扫描。两位乳腺病理学家在不知晓作为金标准的常规组织学结果的情况下，独立评估EVFCM图像。采用Cohen's κ系数计算诊断准确性、敏感性、特异性及观察者间一致性。 **结果：** 观察者间在肿瘤检测（97.3%，κ = 0.942）和肿瘤类型分类（93.8%，κ = 0.883）方面的一致性几乎完美。EVFCM在肿瘤检测方面达到93.7%的敏感性和特异性，准确性为94.0%（κ = 0.929，p < 0.001）；在肿瘤类型分类方面准确性为95.8%（κ = 0.925，p < 0.001）；在浸润性亚型识别方面准确性为95.1%（κ = 0.907，p < 0.001）。在切缘评估方面，EVFCM达到80%的敏感性、100%的特异性和99.3%的准确性（κ = 0.857，p < 0.001）；而在切缘距离评估（<2 mm 对比 ≥2 mm）方面，达到75%的敏感性、100%的特异性和98.6%的准确性（κ = 0.854，p < 0.001）。 **结论：** EVFCM能够对新鲜乳腺组织进行快速、高分辨率成像，有助于实现实时术中切缘评估，并具有优异的诊断一致性和工作流程效率。将其整合到外科实践中，可减少保乳手术中的再次切除，提高肿瘤学安全性并改善患者预后。
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