Quantitative microscopy reveals 3D organization and kinetics of endocytosis in rat hepatocytes.

In order to demonstrate the power of quantitative microscopy, the endocytic apparatus of rat hepatocytes was reexamined using in situ liver and short term cultured hepatocyte couplets that were allowed to internalize endocytic markers for various time intervals. Correlative confocal light and electron microscopy demonstrate a tubulovesicular reticulum representing the endocytic apparatus. Volume and membrane area account for 2% of cell volume and 30% plasma membrane surface.

Colocalization analysis yields superior results after image restoration.

Colocalization analysis is a powerful tool for the demonstration of spatial and temporal overlap in the distribution patterns of fluorescent probes. In unprocessed images, background affects image quality by impairing resolution and obscuring image detail in the low-intensity range. Because confocal images suffer from background levels up to 30% maximum intensity, colocalization analysis, which is a typical segmentation process, is limited to high-intensity signal.