Whole mount liver images cleared with iDISCO+ were initially processed in ImageJ for maximum z-projection and segmentation. The images were filtered using the unsharp mask and integral image filter function. Images were next processed in Amira. In Amira images were filtered using the Gaussian filter and background detection correlation. Images were manually segmented. The manual segmentation was further traced using the autoskeleton function. The skeletons were further analyzed in Amira for length, volume and branching.

Images of ink injected liver were proceeds for filament tracing. Bile duct and portal vein filament tracing was performed using Amira. The images were filtered using the unsharp mask and mean filter. The signal was manually segmented to remove artificial signal. The manual segmentation was further traced using the autoskeleton function. The skeletons were analyzed in Amira for length, volume and branching. For double ink injection (Figure 1—figure supplement 1A) the background was changed for esthetic purposes using the lasso tool in Adobe Photoshop.

DUCT 2D slices were exported from MyVGL (Volumegraphics) and processed in ImageJ for maximum contrast and brightness.

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