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Fig. 1. Two-photon permeabilization of pancreatic acinar cells and the effects of Ca²⁺ releasing messengers. (A) A pancreatic acinar cell doublet loaded with Fluo-5N AM before permeabilization. Blue dot shows the position of two-photon light application. (B) Same cell doublet after permeabilization and perfusion with Texas Red dextran (M_r 3x10³). Only the lower cell has been permeabilized and is therefore bright due to diffusion of Texas Red dextran into the cytoplasm. (C) Same cell doublet after washing out of Texas Red dextran. Note reduced fluorescence of Fluo-5N in the lower permeabilized cell. (D) Transmitted light picture of the doublet (after permeabilization) shown in A-C. (E) IP₃ (10 µM), applied to the doublet shown in A-D, elicited a reduction in [Ca²⁺] in the intracellular stores in the lower (permeabilized) cell (red trace), whereas there was no response in the upper (intact) cell (blue trace). (F) Typical reduction of [Ca²⁺] in the intracellular stores induced by NAADP (100 nM) in permeabilized cell. (G) Typical cADPR (10 µM)-induced Ca²⁺ release in the permeabilized cell. (H) IP₃ (10 µM) induces additional Ca²⁺ release after the NAADP-elicited reduction in the intra-organellar [Ca²⁺]. (I) NAADP (100 nM) induces additional Ca²⁺ release after the IP₃-elicited response.

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