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Fig. 2. Simultaneous identification of acinar lumens and agonist-evoked intracellular Ca²⁺ changes show near-synchronous global Ca²⁺ signals in submandibular acinar cells. Extracellular SRB fluorescence and intracellular Fura-2 fluorescence were recorded with two-photon microscopy to identify the acinar lumen and intracellular Ca²⁺ changes, respectively, in pancreatic fragments (10 µM ACh; A,B) and submandibular fragments (10 µM ACh C,D; 300 nM ACh; E,F). 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 pancreatic acinar cells (image shows approx. seven 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 taken at the time points (i, ii, iii) shown on the graphs in B, which plot the average Fura-2 self-ratio changes over time in the ROIs. (C) In the top left panel, SRB outlines submandibular acinar cells (image shows approx. five cells on the edge of a tissue fragment). Acinar lumens are apparently elongated structures. 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 10 µM ACh-induced Fura-2 self-ratio changes 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. (E) In the top left panel, SRB outlines submandibular acinar cells (image shows approx. six cells on the edge of a tissue fragment). The pseudocolour images show 300 nM ACh-induced Fura-2 self-ratio changes taken at the time points (i, ii, iii) shown on the graphs in F, which plot the average Fura-2 self-ratio changes over time in the ROIs. Scale bars: 10 µm.

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