FIGURE 3.

PI(4,5)P₂ spatial organization in the fixed PM is sensitive to probe blinking and sample fixation conditions. a, TIRF and a corresponding superresolution image of the INS-1 cell membrane sheet immunostained with a monoclonal PI(4,5)P₂ antibody and a secondary antibody labeled with Alexa Fluor 647. The samples were fixed with 4% PFA + 0.2% glutaraldehyde at 4 °C. b, cropped image corresponding to the box in a. c, enlarged image of the boxed region in b. Note the cluster structure in b and c caused by photoblinking. d and e, superresolution images of Alexa Fluor 647 molecules on a blank coverslip as a control of Alexa Fluor 647 single-molecule photobehavior. Note the fluorescence cluster artifact caused by Alexa Fluor 647 photoblinking in e. f–h, similar to a–c but using INS-1 cells expressed with mEos3.1-PH_PLCδ1. i, superresolution image of mEos3.1 expressed at isolated single-molecule levels in a COS-7 cell using the deltaCMV promoter. Note the cluster structure of the mEos3 molecule caused by moderate photoblinking. k, TIRF image of an INS-1 membrane sheet fixed at room temperature and immunostained by a monoclonal PI(4,5)P₂ antibody and AF647-conjugated secondary antibody. Note the abundant dense puncta visible under TIRF that were rarely observed in live cells. l, dSTORM superresolution image of PI(4,5)P₂ in k. m, enlarged TIRF and dSTORM images in the boxed area in l. n–p, similar to k–m, but, with fixation at 37 °C, PI(4,5)P₂ dense cluster artifacts were observed similarly. q–s, similar to k–m but fixed at 4 °C with 0.2% glutaraldehyde in addition to 4% PFA. The display contrast in q was different compared with other TIRF images to avoid saturation. Note the relative even fluorescence image and the enhanced fluorescence intensity under TIRF in q–s. Scale bars = 3 μm (a, f, i, o, and r), 500 nm (b, d, g, i, m, p, and s), and 200 nm (c, e, h, and j).