Abstract：
The vital role of bile canaliculus (BC) in liver function is closely related to its morphology. Electron microscopy has contributed to understanding BC morphology; however, its invasiveness limits its use in living specimens. Here, we report non-invasive characterization of BC formation using refractive index (RI) tomography. First, we investigated and characterized the RI distribution of BCs in two-dimensional (2D) cultured HepG2 cells. BCs were identified based on their distinct morphology and functionality, as confirmed using a fluorescence-labeled bile acid analog. The RI distribution of BCs exhibited three common features: (1) luminal spaces with a low RI between adjacent hepatocytes; (2) luminal spaces surrounded by a membranous structure with a high RI; and (3) multiple microvillus structures with a high RI within the lumen. Second, we demonstrated the characterization of BC structures in a three-dimensional (3D) culture model, which is more relevant to the in vivo environment but more difficult to evaluate than 2D cultures. Various BC structures were identified inside HepG2 spheroids with the three features of RI distribution. Third, we conducted comparative analyses and found that the BC lumina of spheroids had higher circularity and lower RI standard deviation than 2D cultures. We also addressed comparison of BC and intracellular lumen-like structures within a HepG2 spheroid, and found that the BC lumina had higher RI and longer perimeter than intracellular lumen-like structures. Our demonstration of the non-destructive, label-free visualization and quantitative characterization of living BC structures will be a basis for various hepatological and pharmaceutical applications.
摘要：
胆小管（BC）在肝功能中的重要作用与其形态密切相关。电子显微镜有助于理解BC形态；然而，它的侵入性限制了它在活体标本中的使用。这里,我们使用折射率（RI）断层扫描报告了BC形成的非侵入性表征。首先,我们研究并表征了二维（2D）培养的HepG2细胞中BCs的RI分布。BCs根据其独特的形态和功能进行鉴定，使用荧光标记的胆汁酸类似物证实。BCs的RI分布表现出三个共同特征:(1)相邻肝细胞之间具有低RI的管腔空间;(2)由具有高RI的膜状结构包围的管腔空间;和(3)管腔内具有高RI的多个微绒毛结构。第二,我们在三维（3D）培养模型中展示了BC结构的表征，这与体内环境更相关，但比2D培养更难以评估。在HepG2球状体内鉴定出各种BC结构，具有RI分布的三个特征。第三，我们进行了比较分析，发现球体的BC腔比2D培养物具有更高的圆形度和更低的RI标准偏差。我们还讨论了HepG2球体中BC和细胞内腔样结构的比较，发现BC腔比胞内腔样结构具有更高的RI和更长的周长。我们展示了非破坏性的，活BC结构的无标签可视化和定量表征将是各种肝病和药物应用的基础。
