Park Won Yeong, Kim Bumju, Chun Ji Hyun, Hong Seung-Mo, Oh Byung Ho, Kim Ki Hean
Department of Mechanical Engineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Republic of Korea.
Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Gyeongbuk, Republic of Korea.
Lasers Surg Med. 2022 Nov;54(9):1226-1237. doi: 10.1002/lsm.23600. Epub 2022 Sep 10.
Precise determination of cancer margin during skin cancer surgery is crucial for complete resection and further clinical prognosis. Although reflection confocal microscopy (RCM) has been used for perioperative guiding, its reflection contrast has limitations in detecting cancer cells in the dermis. We previously developed combined reflection confocal (RC) and moxifloxacin-based two-photon (MB-TP) microscopy for sensitive cancer detection by using multiple contrast mechanisms. In this study, the performance of combined microscopy was characterized in various skin cancer specimens and compared with standard methods.
Seven human skin specimens in total including two normal ones, three basal cell carcinomas (BCCs), and two squamous cell carcinomas (SCCs) were collected and imaged in fresh condition. Moxifloxacin ophthalmic solution was topically instilled for cell labeling for 3-5 minutes, then mosaic imaging with the combined microscopy was conducted. The imaged specimens were imaged again after exogenous nuclear labeling for comparison and then processed for standard hematoxylin and eosin histology.
Combined RC and MB-TP microscopy visualized both cell and extracellular matrix structures of the skin specimens with multiple contrasts of reflection, moxifloxacin fluorescence, autofluorescence, and second harmonic generation. It distinguished normal cell structures in the skin dermis such as hair follicles, sebaceous and eccrine glands from BCC nests, and SCCs based on cell organization. Normal cell structures had organized cell arrangements for their functions, while cancer cell structures had dense and disorganized cell arrangements. Cellular features found by combined microscopy images were confirmed by both TP microscopy with nuclear labeling and histological examination.
The imaging results showed the potential of combined microscopy for sensitive cancer detection and in vivo guiding of skin cancer surgery.
在皮肤癌手术中精确确定癌切缘对于完整切除及后续临床预后至关重要。尽管反射共聚焦显微镜（RCM）已用于围手术期引导，但其反射对比度在检测真皮层癌细胞方面存在局限性。我们之前开发了结合反射共聚焦（RC）和基于莫西沙星的双光子（MB - TP）显微镜，通过多种对比机制实现对癌症的灵敏检测。在本研究中，对联合显微镜在各种皮肤癌标本中的性能进行了表征，并与标准方法进行了比较。
总共收集了7个人类皮肤标本，包括2个正常标本、3个基底细胞癌（BCC）和2个鳞状细胞癌（SCC），并在新鲜状态下进行成像。局部滴注莫西沙星滴眼液进行细胞标记3 - 5分钟，然后用联合显微镜进行拼接成像。对外源核标记后的成像标本再次成像以作比较，然后进行标准苏木精和伊红组织学处理。
联合RC和MB - TP显微镜通过反射、莫西沙星荧光、自发荧光和二次谐波产生的多种对比，可视化了皮肤标本的细胞和细胞外基质结构。它基于细胞组织结构，将皮肤真皮中的正常细胞结构（如毛囊、皮脂腺和汗腺）与BCC巢和SCC区分开来。正常细胞结构为其功能具有有序的细胞排列，而癌细胞结构具有密集且无序的细胞排列。联合显微镜图像中发现的细胞特征通过带核标记的双光子显微镜和组织学检查得到了证实。
成像结果显示联合显微镜在灵敏检测皮肤癌及皮肤癌手术的体内引导方面具有潜力。
