The transport and targeting of internalized molecules to distinct intracellular organelles/compartments can prove challenging to visualize clearly, which can contribute to some of the difficulties associated with these studies. By combining several approaches, we show how the trafficking and processing of photoreceptor outer segments in the phagosome and autophagy-lysosomal pathways of the retinal pigment epithelium (RPE) can easily be quantified and visualized as 3D-reconstructed images. This protocol takes advantage of new developments in microscopy and image-analysis software which has the potential to help better understand dynamic intracellular processes that underlie RPE dysfunction associated with irreversible blinding diseases such as age-related macular degeneration. The method described herein can also be used to study the trafficking and co-localization of different intracellular cargos in other cell types and tissues.
Keywords: 3D imaging; Autophagy; Co-localization; Confocal; Lysosomes; Microscopy; Phagosome; Retinal pigment epithelium (RPE).