Establishing conditions for local manipulation of [Ca²⁺]. (A) A bright field image of a parotid acinar cell is shown together with pseudocolored fluorescence image series of cells immediately before and after flash photolysis. The pipette solution contained 10 mM NP-EGTA and 5 mM Ca²⁺ (free Ca²⁺, ∼160 nM). The images were acquired every 45 ms, and the single image containing the flash artifact was removed. Release of Ca²⁺ after laser exposure (green arrow) resulted in a marked increase in fluorescence at the photolysis site in the basal pole (blue dot), which decayed rapidly but spread toward the apical pole (red dot). (B) The change in ΔF/F₀ ratio in either the basal (blue line) or apical (red line) region, demonstrating that the increase in the “untargeted” pole was >10% when compared with basal values. (C) A similar experiment is shown, except the pipette solution contained 10 mM NP-EGTA and 2 mM Ca²⁺ (free [Ca²⁺], ∼40 nM). The series of images demonstrates that the increase in [Ca²⁺] remains localized to the apical pole (red dot) under these conditions. (D) The change in ΔF/F₀ ratio values for this experiment, indicating that the marked increase in fluorescence after laser exposure in the apical domain (red line) results in little increase (<10% increase over basal) in fluorescence in the untargeted region (blue line). (E) A spatial analysis of the peak change in fluorescence after photolysis with buffering conditions as in C. The peak change in fluorescence shown in E (left) was compared with an average image generated from the previous five frames under basal conditions. The change in ΔF/F₀ ratio along the yellow line is shown in the right panel and demonstrates that the fluorescence decays exponentially from the photolysis site, with a length constant of 2.5 ± 0.2 µm. The fit for the decay is shown in the red line. (F) A similar analysis in which the individual experiments are normalized to the peak fluorescence in the individual cell. The red line shows the mean fit of the data.