Individual vesicle fusion events delivering GPCRs to the cell surface can be resolved by TIR-FM. (A) Schematic of change in SpH fluorescence during a fusion event in SpH-cargo expressing cells. The fluorescence intensity of SpH-cargo increases upon exposure to the neutral extracellular environment. (B) Montage of a SpH-MOR expressing cell showing a typical fusion event (black square) and an endocytic cluster event (gray square) after 5 min 12.5 µM DAMGO treatment at 37°C. Scale bar = 8 µm. (C and D) Enlarged montages (top panels) of a fusion event (C) and an endocytic cluster event (D) shown in B. The fusion event showed an instantaneous increase in fluorescence intensity followed by a rapid (<0.4 s) diffusion that was visualized as a puff of fluorescence. On the other hand, the endocytic cluster event showed sustained fluorescence (∼8 s) followed by an exponential decay. Surface plots of fluorescence are shown in the bottom panels. Scale bar = 2 µm. (E and F) Traces of changes in maximum fluorescence (Max fluo.) of the fusion event (E) and the endocytic cluster event (F) shown in B. Time 0 represents the onset of image acquisition. Horizontal scale bar = 5 s and vertical scale bar = 2,000 count of fluorescence intensity (arbitrary unit, a.u.).