FIGURE 6.

Calcein fluorescence inside and outside mitochondria. (A) Time-resolved TMRE (1, 3) and calcein (2, 4) fluorescence in single mitochondria. TMRE and calcein fluorescence was simultaneously monitored in the same mitochondrion. Typical behaviors observed for two different mitochondria (Mitochondrion 1 and 2) are shown. The vertical axes for TMRE fluorescence are the same as those of Fig. 1 C. The vertical axes for calcein fluorescence are shown in arbitrary unit. Malate was added at t = 0. Arrows show the time when the rapid depolarizations occurred. (B) Release of calcein under the various conditions. The vertical axis represents the calcein release from mitochondria to the medium. The detailed explanation of the vertical axis is described in the text. Values represent the mean ± SE (n > 3). *, P < 0.05 versus the sample without ADP, oligomycin, CsA, BA, Ca²⁺, and Mg²⁺. (C) Fluorescence images of calcein entrapped in individual mitochondria in the same microscopic field. Bar, 5 μm. (1) Before addition of malate; (2) after incubation with malate in the absence of ADP.