Manipulations of extracellular pH reveal distinct fractions of pHluorin-tagged GluR-A receptors visualized in TIRF mode. Right panel shows zoomed images of the transfected neuron presented in the left panel. A, pHluorin-GluR-A clusters under control conditions (pHo = 7.4). Scale bar: 5 μm. B, image obtained 30 seconds into the application of acidic solution (pHo = 5.4) shows strong but incomplete quenching of the pHluorin-GluR-A fluorescence. C, calculated image obtained by subtracting B from A. D, plot of changes in fluorescence intensity (averaged across the whole imaging field). Time points referring to each of the images shown in A and B are indicated by arrows with corresponding letters.