非线性光学显微镜观察肝脏纤维化的实验研究

Non-linear Optical Microscopy as a Sensitive and Quantitative Technique for Liver Fibrosis Studies

目的:了解非线性光学显微镜在肝纤维化成像及定量分析研究中的地位。方法:分别用前向二次谐波(SGH)及背向双光子荧光(TPEF)对肝脏标本的成纤维胶原(Ⅰ/Ⅲ)和肝细胞浆进行成像,将结果与传统的Masson’s三染色法进行比较。随后用非线性光学显微镜对不同肝纤维化阶段的大鼠肝脏标本成像,并对图像进行统计分析。结果:(1)用二次谐波成像的肝内成纤维胶原分布图较传统的方法更清晰,易于进一步定量分析;(2)双光子荧光信号可以清晰的显示肝细胞形态;(3)非线性光学显微镜得到的肝纤维化图像易于用软件进行定量分析。结论:非线性光学显微镜是研究肝纤维化进程的灵敏、准确、快速、简单、客观的新方法。其对肝纤维化的定量分析具有重要临床意义。

Fibrillar collagen （ Collagen type Ⅰ and Ⅲ） in sinusoids of normal liver was successfully observed through second harmonic generation （SHG） microscopy. Two photon excited fluorescence （TPEF） image, recorded simultaneously with SHG, clearly revealed the hepatocytes morphology. By comparing with traditional staining as Masson＇ s trichrome, SHG/TPEF is proved to be a sensitive tool for collagen characterization in liver tissue. In monitoring the progression of fibrosis in rat liver, SHG signal presented the subtle changes of collagen quantitatively and TPEF images provided detailed information of cell morphology. The image of collagen fiber captured by nonlinear microscopy can be quantified easily by Image Pro Plus. In conclusion, this study indicates that nonlinear optical microscopy is a sensitive, accurate, and objective tool for quantitative studies of the liver fibrosis with no staining required.