Department of Gastrointestinal and Pediatric Surgery, Division of Reparative Medicine, Institute of Life Sciences, Mie University Graduate School of Medicine, Tsu, Mie, Japan.
J Gastroenterol. 2010 May;45(5):544-53. doi: 10.1007/s00535-009-0187-7. Epub 2010 Jan 9.
Two-photon laser-scanning microscopy (TPLSM) is a powerful diagnostic tool for real-time, high-resolution structural imaging. However, obtaining high-quality in vivo TPLSM images of intra-abdominal organs remains technically challenging.
An organ-stabilizing system was applied to high-quality TPLSM imaging. Real-time imaging of visceral organs, such as the liver, spleen, kidney and intestine, of transgenic green fluorescent protein (GFP) mice was performed in vivo using TPLSM. The bacterial translocation model using dextran sodium sulfate (DSS)-induced colitis was also investigated in prepared GFP mice following simple surgery. This allowed the capture of morphological real images using in vivo TPLSM. Immunohistochemical analysis of ZO-1 was performed to support the morphological findings of TPLSM.
We established an organ-stabilizing system to evaluate the real-time imaging of visceral organs in actin-GFP transgenic mice using in vivo TPLSM. DSS-induced colitis showed irregularity of crypt architecture, disappearance of crypts, inflammatory cell infiltration and increased rolling of white blood cells along the vasculature. In addition, the intercellular distance of mucosal cells in the crypt and vascular endothelial cells in the intestinal wall was increased in the intestinal mucosa during DSS colitis. In DSS colitis, there was remarkable loss of mucosal and vascular endothelial ZO-1 expression, as could be seen by a decrease in ZO-1 staining. In conclusion, our observations suggested the possibility that our TPLSM imaging system can be used to clarify the pathophysiological changes in various diseases using longitudinal studies of microscopic changes in the same animal over long periods of time.
双光子激光扫描显微镜（TPLSM）是一种强大的诊断工具，可用于实时、高分辨率的结构成像。然而，获得高质量的腹腔内器官的体内 TPLSM 图像在技术上仍然具有挑战性。
应用器官稳定系统进行高质量 TPLSM 成像。使用 TPLSM 在体内对转基因绿色荧光蛋白（GFP）小鼠的内脏器官，如肝脏、脾脏、肾脏和肠道进行实时成像。还在经过简单手术的准备好的 GFP 小鼠中研究了使用葡聚糖硫酸钠（DSS）诱导结肠炎的细菌易位模型。这允许使用体内 TPLSM 捕获形态学实时图像。进行 ZO-1 的免疫组织化学分析以支持 TPLSM 的形态学发现。
我们建立了一个器官稳定系统，以使用体内 TPLSM 评估在肌动蛋白-GFP 转基因小鼠中内脏器官的实时成像。DSS 诱导的结肠炎显示隐窝结构不规则、隐窝消失、炎性细胞浸润和沿血管滚动的白细胞增加。此外，在 DSS 结肠炎期间，隐窝中的黏膜细胞和肠壁中的血管内皮细胞的细胞间距离增加。在 DSS 结肠炎中，黏膜和血管内皮 ZO-1 表达明显丢失，ZO-1 染色减少可见。总之，我们的观察结果表明，我们的 TPLSM 成像系统有可能通过对同一动物的微观变化进行长时间的纵向研究，用于阐明各种疾病的病理生理变化。
