In vivo imaging of hepatobiliary toxicity: hydropic vacuolation. Acute exposures to 2 to 6 μM ANIT resulted in a marked "pebbling" phenotype. This terminology was adopted due to the morphological appearance of the liver (L), first observed in vivo with widefield fluorescence microscopy. (A) STII medaka control, 20 dpf, showing the normal smooth appearance of the hepatic parenchyma in vivo, as viewed with widefield fluorescence microscopy. (B) Widefield fluorescence microscopy, FITC image capture, STII medaka, 20 dpf. Shown is the "pebbled" appearance (black arrowhead) of the liver (L) in vivo; distinct at 2 μM to 6 μM aqueous ANIT. This phenotypic response was characterized (in vivo) by ovate structures within the cytosol of hepatocytes, which resulted in a pebbled appearance in the plane of focus in the liver. This phenotype was observed with the aid of autofluorescence alone, no fluorophores were necessary for visualizing this cellular response. (B1) ANIT exposed medaka exhibiting the pebbling phenotype were treated with the nuclear stain DAPI (aqueous bath) to label hepatocyte and biliary epithelial cell nuclei. After 1 hr of DAPI exposure the hepatobiliary systems of medaka were imaged in vivo via widefield fluorescence microscopy. These investigations found that intracellular ovate structures (black arrowhead) did not label with DAPI, and were distinguishable from hepatic and biliary epithelial nuclei (blue). (B2) Transmission electron micrograph showing cellular changes consistent with hydropic vacuolation, which was observed in both hepatocytes (black arrowhead) and bile preductular epithelia (not shown). Vacuoles ranged from 2 μm to 10 μm in diameter, and were found to be partially to completely filled with electron dense infiltrate. (B3) In vivo confocal image of YO-PRO-1, DIC and TRITC composite: nuclear labeling experiments performed with YO-PRO-1 revealed uptake of YO-PRO-1 into cells with putatively compromised cell membranes. In grayscale DIC image hydropic vacuoles (black arrowhead) are distinct. Associated with hydropic vacuolation was a slight increase in apoptosis (ovate green fluorescence, cell type not known). Of interest is the almost literal appearance of hydropic vacuoles in the confocal DIC fluorescence image (grayscale), where vacuoles appear as liquid droplets. Gill (Gl), Ventral Aorta (Va), Heart Atrium (Ha), Heart Ventricle (Hv), Liver (L), Gall Bladder (GB), Gut (Gt).