Applying intravital fluorescence microscopy, we assessed sinusoidal delivery and biliary clearance of two different polymethine dyes. DY635, a benzopyrylium-based hemocyanine dye with shorter excitation wavelength than indocyanine green (ICG), was validated for assessment of hepatic excretory function. Decrease of DY635 and ICG reflecting transcellular transport was 83 ± 4% (DY635) and 14 ± 2% (ICG; p < 0.05) over 35 minutes, respectively. In cholestasis, hepatobiliary excretion of DY635 was markedly impaired (control 3176 ± 148 pmol vs. cholestatic 1929 ± 179 pmol; p < 0.05). DY635 even enabled an analysis at high resolution suggesting 1.) hepatocyte uncoupling and 2.) failure of primarily the canalicular pole, allowing in vivo insights into molecular mechanisms of this critical facet of hepatobiliary function.