Non-invasive Visualization and Characterization of Bile Canaliculus Formation Using Refractive Index Tomography

Area of Science:

• Hepatology and cell biology
• Biophysics and imaging techniques

Background:

• Bile canaliculus (BC) morphology is crucial for liver function.
• Traditional methods like electron microscopy are invasive and unsuitable for living specimens.
• A non-invasive method is needed to study BC formation and structure in real-time.

Purpose of the Study:

• To develop and validate a non-invasive method for characterizing bile canaliculus (BC) formation and structure using refractive index (RI) tomography.
• To investigate BC morphology in both 2D and 3D cell culture models.

Main Methods:

• Utilized refractive index (RI) tomography for non-invasive, label-free visualization of BCs.
• Investigated BCs in 2D cultured HepG2 cells and 3D HepG2 spheroids.
• Confirmed BC identification using morphology, functionality, and a fluorescence-labeled bile acid analog.

Main Results:

• Identified three common RI distribution features of BCs: low RI lumen, high RI surrounding membrane, and high RI microvilli.
• Successfully characterized BC structures in 3D spheroids, demonstrating RI features.
• Comparative analysis showed BC lumina in spheroids had higher circularity and lower RI standard deviation than in 2D cultures.
• Distinguished BC lumina from intracellular structures in spheroids based on RI and perimeter.

Conclusions:

• Refractive index tomography provides non-destructive, label-free visualization and quantitative characterization of living bile canaliculus structures.
• This technique is applicable to both 2D and 3D cell culture models, including complex spheroids.
• The findings lay the groundwork for advanced hepatological and pharmaceutical research and applications.