FIGURE 4.

Live-cell PALM imaging revealed homogeneous spatial distribution and fast dynamics of PI(4,5)P₂ in INS-1 cells. a and b, TIRF and superresolution images of mEos3.2-labeled syntaxin1A in an INS-1 cell at 35 °C. c–e, enlarged images of the white box regions in b at 0 and 30 s. d, the intensity profile of the syntaxin1A cluster as indicated by the white line in c. e and f, the pseudocolor image of c (green) merged on e (red, f). Arrows indicate the generation, disappearance, and movement of syntaxin1A clusters during 30 s of imaging. g, TIRF image of PI(4,5)P₂ in a live INS-1 cell expressed with mEos3.1-PH_PLCδ1. The image was acquired rapidly using the green channel before PALM imaging (35 °C). h and i, sequential live-cell PALM images visualized at 10-s intervals. The insets show the intensity profiles of the local PI(4,5)P₂ density along the same straight line 1 position indicated at different times. Note the completely different intensity profile changes in 10 s. j, live PALM image of the same cell 30 s after 60 mm K⁺ stimulation. k, time course of the average intensity of PI(4,5)P₂ in the large area (box 2 in h, 3 × 3 μm) and small circles (circle 3, 4, and 5, 500-nm diameter) in h during 5 min of PALM imaging with an interval of 10 s. Note the rapid intensity fluctuations of local PI(4,5)P₂ probe changes (circle 3, 4, and 5) compared with very small changes in the relatively broad area (box 2). l, enlarged PALM images of the box 2 region in h at the indicated times. Arrows indicate PI(4,5)P₂-enriched membrane patches under physiological conditions. Scale bars = 3 μm (b and j) and 500 nm (f and l).