Figure 5

In vivo imaging of hepatobiliary transport. Fluorophores such as β-Bodipy C5 phosphocholine, shown here, enabled in vivo elucidation of the biliary system and quantitation of blood to bile transport. (A) Brightfield microscopy of STII medaka at 30 dpf. Green algae can be seen in transport through lumen of the gut. (A1) Widefield fluorescence microscopy of region of interest indicated by gray square in A, showing β-Bodipy C5 phosphocholine fluorescence in intrahepatic biliary passageways (IHBPs) of the liver (L) and gall bladder (GB). (B) Confocal DIC microscopy, STII medaka, 9 dpf: Clearly resolved were hepatocytes and their nuclei/nucleoli. In longitudinal section 2 rows of hepatocytes characterize parenchymal architecture. Stacked ovate structures are red blood cells in circulation through the sinusoids (S/r). Red cells appear flattened due to active circulation of cells through sinusoids, and resulting distortion during imaging. (B1) Same as B: Single frame from in vivo confocal image stack capturing β-Bodipy C5 phosphocholine (green fluorescence) in transport from blood to bile, through intrahepatic biliary passageways (IHBPs) of the liver (imaged in vivo 30 minutes post fluorophore administration). (B2) Composite of frames B and B1 localizing fluorophore transport to area between apical membranes of adjacent hepatocytes, suggesting concentrative transport of the fluorophore into IHBPs. (C and D) Frame C is a surface map of region of interest (white square) in frame B1, illustrating concentrative transport of the fluorophore from sinusoidal space (S) to bile space (IHBP). (D) Quantitative evaluation of the white rectangular region of interest in frame B1, spanning an 18.3 μm area from blood to bile (sinusoid to canaliculus), suggested β-Bodipy C5 phosphocholine concentration (fluorescence intensity) to be ~20 times greater in the canalicular (IHBP) vs. sinusoidal spaces (S). Also evident is an increase in cytosolic concentration of the fluorophore, while no fluorescence was detected in the nucleus. These types of studies demonstrated concentrative transport of fluorescent probes from blood to bile can be imaged and quantitatively assessed in vivo. Confocal images captured with C-apochromatic objective, 1.2 NA w/correction, 488 nm excitation, Zeiss LSM 510. Field size: 76.8 × 76.8 μm.