Fig. 5. Agonist-evoked intracellular Ca²⁺ changes in submandibular acinar cells are dependent on InsP₃Rs. Extracellular SRB fluorescence and intracellular Fura-2 fluorescence were recorded with two photon microscopy, as before, in submandibular fragments stimulated with 10 µM ACh in the absence of extracellular Ca²⁺ (A,C) and in the presence of ryanodine (B,D). All Fura-2 self-ratio images have been overlaid with a binary mask (in white) obtained from the SRB image. (A) In the top left panel, SRB outlines submandibular acinar cells (image shows approx. four cells on the edge of a tissue fragment) and fills the acinar lumens allowing placement of ROIs on the apical (ROI 1) and basal (ROI 2) pole of the cell. The pseudocolour images show ACh-induced Fura-2 self-ratio changes, in the absence of extracellular Ca²⁺, taken at the time points (i, ii, iii) shown on the graphs in C which plot the average Fura-2 self-ratio changes over time in the ROIs. (B) In the top left panel, SRB outlines submandibular acinar cells (image shows three cells on the edge of a tissue fragment). ROIs were placed in the narrow, apparent apical pole of the cell (ROI 1) and wider, apparent basal (ROI 2) pole of the cell. The pseudocolour images show ACh-induced Fura-2 self-ratio changes, obtained in the presence of ryanodine, taken at the time points (i, ii, iii) shown on the graphs in D, which plot the average Fura-2 self-ratio changes over time in the ROIs. Scale bars: 10 µm.