Discerning protein pools by selective staining with self-labeling tags.

Transmembrane-spanning cell surface proteins undergo maturation from the site of synthesis in the endoplasmic reticulum to the plasma membrane, and may be endocytosed back into the cell. Microscopy allows the visual separation of such populations at the cell surface and in intracellular compartments and to monitor trafficking and dissect the relative abundance of such pools. Protein tags, like SNAP-tag, HTP, and FAST, combined with their respective (im)permeable ligands fused to fluorescent dyes, open up new possibilities to distinguish between intra- and extracellular protein pools. Introducing cleavable groups in dye-ligand molecules allows interrogation of endocytosed pools by stripping the remaining surface pools. Latest developments, for instance, fluorescence lifetime imaging microscopy and brightness demixing, give further opportunities in microscopy.