TIRF imaging highlights the perimembrane fraction of pHluorin-tagged GluR-A receptors. A, schematic representation of TIRF imaging of pHluorin-tagged molecules located either in the endoplasmic reticulum (ER) and vesicles or inserted in the PM. The pHluorin-tagged molecules that are close enough to the PM to be excited by evanescent field are marked with numbers 2 (ER-residing molecules) and 3 (membrane-inserted molecules and those residing in the perimembrane vesicles). The more distant pHluorin-tagged molecules that are not visualized in TIRF imaging mode are marked with number 1. B, example of TIRF image of a neuron transfected with pHluorin-GluR-A revealing individual clusters (magnified field is shown in C). D, schematic representation of epifluorescence imaging of pHluorin-tagged receptors located either in the endoplsasmic reticulum (ER) and vesicles or inserted in the PM. Note that all pHluorin-tagged receptors irrespective of their subcellular localization are visualized in epifluorescence mode. E, epifluorescence image of the same neuron that appears in B (magnified field of the epifluorescence image is shown in F). Note that the spatial resolution of individual clusters in epifluorescence is inferior compared to TIRF imaging mode (B and C). Scale bars: 10 μm.