Fig. 6. Single submandibular cells show a decreased Ca²⁺ wave velocity compared with cells within a tissue fragment. Intracellular Fura-2 fluorescence was recorded with two-photon microscopy, as before, but in single isolated submandibular cells (A,B). All Fura-2 self-ratio images have been overlaid with a binary mask (in white) obtained from the SRB image. (A) The pseudocolour images show ACh-induced Fura-2 self-ratio changes taken at the time points (i, ii, iii) shown on the graphs in B, which plots the average Fura-2 self-ratio changes over time in ROI 1 and 2. (C) Immunocytochemistry shows ZO-1 (red) and InsP₃R3 (green) are still colocalized in these single cells (overlay also shows the nuclear Hoechst 33258 stain in blue). The phase-contrast image of the single cell is also shown. Scale bars: 10 µm.