A Feasibility Study on 3-D Imaging of Intrahepatic Bile Ducts in Patients with Biliary Atresia Using Airy Beam Excited Two-Photon Microscopy.

Abstract

The purpose of this study was to utilize a two-photon microscope excitation Airy beam to achieve three-dimensional imaging of intrahepatic bile ducts in BDL mice and patients with biliary atresia (BA). Ten male Balb/c mice aged 6-8 weeks underwent extrahepatic bile duct ligation (BDL), and 10 underwent sham operation as control. After the operation, the mice resulted in symptoms such as jaundice, darkened urine, and weight loss. Taken liver tissues from BDL and control mice and trimmed to 5*5*3 mm³ after 10 days. Sixteen patients with BA were included in this study. Liver transplantation was performed in 12 cases of them; liver hilar and liver margin tissues were taken during the operation. Kasai portoenterostomy (KPE) was performed in 4 cases, and liver margin tissues were taken. Intraoperative liver tissue samples were trimmed to a size of 5*5*5 mm³. The specimens were subjected to tissue fixation, antigen retrieval, antibody incubation, and subsequent tissue hyalinization following the principles of immunofluorescence staining. Subsequently, light-sheet fluorescence microscopy (LSFM) was followed, and intrahepatic bile ducts of the specimen were imaged utilizing Airy beam which was excited with high imaging depth attenuation-compensated two-photon. Deconvolution was applied to image processing to construct a three-dimensional model of intrahepatic bile ducts. Three-dimensional imaging of liver tissue was conducted in both BDL mice and BA patients, and the distribution of intrahepatic bile ducts was visualized. BDL mice exhibited notable widening of intrahepatic bile ducts, accompanied by bile duct hyperplasia. There was no obvious hyperplasia of intrahepatic bile duct in the control group. Significant small bile duct hyperplasia was seen on imaging of the intrahepatic bile ducts in patients with BA. The intrahepatic bile duct was disorganized and hyperplasia especially in patients who performed liver transplantation. The technique of Airy beam three-dimensional reconstruction can effectively image the intrahepatic bile ducts in Balb/c mice and BA patients in three dimensions. This approach contributes to a better understanding of the distribution of intrahepatic bile ducts in BA patients. Moreover, it facilitates the exploration of models that more accurately simulate BA disease by elucidating the distribution of intrahepatic bile ducts in animal models. Understanding the distribution characteristics of intrahepatic bile duct will facilitate the formulation of hilar bile duct microstructure classification, which can guide the operation and evaluate the prognosis better.