Itoh Toshiya, Yamada Satoshi, Ohta Isao, Meguro Shiori, Kosugi Isao, Iwashita Toshihide, Itoh Hiroaki, Kanayama Naohiro, Okudela Koji, Sugimura Haruhiko, Misawa Kiyoshi, Hariyama Takahiko, Kawasaki Hideya
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Japan; Institute for NanoSuit Research, Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Institute for NanoSuit Research, Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan; Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Lab Invest. 2023 Jan;103(1):100020. doi: 10.1016/j.labinv.2022.100020.
Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus particles in FFPE sections using light microscopy is difficult because of the light diffraction resolution limitations of an optical microscope. In this study, light microscopy and field emission scanning electron microscopy were performed to observe 3-dimensional virus particles in FFPE sections in a nondestructive manner using NanoSuit or osmium conductive treatment methods. The virus particles in FFPE sections were immunostained with specific antibodies against the surface antigens of the viral particles and stained with 3,3'-diaminobenzidine. A metal solution (0.2% gold chloride or 2% osmium tetroxide) was applied to enhance the 3,3'-diaminobenzidine-stained area. This procedure is nondestructive for FFPE sections and is a simpler method than transmission electron microscopy. To validate the applicability of this technique, we performed 3-dimensional imaging of the virus particles of different sizes, such as human papillomavirus, cytomegalovirus, and varicella-zoster virus. Furthermore, ultrathin sections from the FFPE sections that were observed to harbor viral particles using field emission scanning electron microscopy were prepared and assessed using transmission electron microscopy. In the correlative areas, transmission electron microscopy confirmed the presence of large numbers of virus particles. These results indicated that the combination of marking viral particles with 3,3'-diaminobenzidine/metal staining and conductive treatment can identify active progeny virus particles in FFPE sections using scanning electron microscopy. This easy correlative imaging of field emission scanning electron microscopy of the identical area of FFPE in light microscopy may help elucidate new pathological mechanisms of virus-related diseases.
福尔马林固定石蜡包埋（FFPE）组织块的免疫组织化学分析常用于识别病毒感染细胞。然而，由于光学显微镜的光衍射分辨率限制，在FFPE切片中使用光学显微镜检测病毒颗粒很困难。在本研究中，采用光学显微镜和场发射扫描电子显微镜，使用纳米套装或锇导电处理方法以无损方式观察FFPE切片中的三维病毒颗粒。FFPE切片中的病毒颗粒用针对病毒颗粒表面抗原的特异性抗体进行免疫染色，并用3,3'-二氨基联苯胺染色。应用金属溶液（0.2%氯化金或2%四氧化锇）来增强3,3'-二氨基联苯胺染色区域。该程序对FFPE切片无损，且比透射电子显微镜方法更简单。为验证该技术的适用性，我们对不同大小的病毒颗粒进行了三维成像，如人乳头瘤病毒、巨细胞病毒和水痘-带状疱疹病毒。此外，制备了用场发射扫描电子显微镜观察到含有病毒颗粒的FFPE切片的超薄切片，并使用透射电子显微镜进行评估。在相关区域，透射电子显微镜证实存在大量病毒颗粒。这些结果表明，3,3'-二氨基联苯胺/金属染色标记病毒颗粒与导电处理相结合，可利用扫描电子显微镜识别FFPE切片中的活性子代病毒颗粒。这种在光学显微镜下对FFPE相同区域进行场发射扫描电子显微镜的简易相关成像，可能有助于阐明病毒相关疾病的新病理机制。
