(A and B) TIRF images of a ND (A) and T2D (B) human β cell expressing NPY-mCherry (see “gr,” granule) and loaded with the Ca²⁺ sensor Fluo5F (right), before and during (stim) stimulation with 75 mM K⁺. Circles indicate the location of exocytosis events. Scale bar: 5 μm (A and B). (C) Time course of Fluo5F-Ca²⁺ fluorescence (the whole-cell signal was normalized to prestimulation, F^cell/F^cell₀; black lines) and cumulative exocytosis events (orange line) in a cell periodically stimulated with K⁺ as indicated. The stimulation was carried out for 1 second, with an interval of 9 seconds. Imaging was performed from 2 seconds before until 2 seconds after each pulse. (D) Exocytosis events as a function of time relative to the most recent K⁺ pulse in 22 cells, as in A. (E) Average Fluo5F fluorescence from responders (blue) and failures (black), aligned to the onset of the K⁺ application and the center position of each granule. There were 102 granules each in 22 cells from 8 ND donors. P = 0.033, by Student’s t test, for the difference in peak amplitude. (F) Examples of an individual granule undergoing exocytosis (gr), the Fluo5F signal for the same granule (Fluo5F), the average Fluo5F signals for 102 responders in ND cells (Avg ND), and 104 responders in T2D cells (Avg T2D). Sequences were aligned to the onset of K⁺ application (red arrow). Note the different spatial scale for the example and the averages. (G) As in F, but for granules that failed to undergo exocytosis (failures). In F and G, the image frames are shown for every 0.1 second. Arrowheads indicate onset of stimulation. Scale bars: 1 μm (F and G). (H–J) As in C–E, but for 104 granules each in 26 cells from 7 T2D donors. Dashed lines in J indicate a subset of 8 ND cells with the highest cell-averaged Fluo5F/Ca²⁺ responses.