Calibration of fura-FF signals. (A) Fluorescence recordings of mitochondria in the presence or absence of fura-FF loading (340:510 nm ex:em and 380:510 nm FuraFF or control, respectively) along with [Ca²⁺]_out (CaGreen: 505:535 nm ex:em). The Ca²⁺ signals were calibrated with multiple additions of 5 µM Ca²⁺ in the presence of the ionophore, 2 µM 4-bromo-A23187, 5 µg/ml oligomycin, and 5 µM FCCP. (B) The excitation ratio for fura-FF was calculated with (red) or without (black) correction for autofluorescence. (C) [Ca²⁺]_mito was determined after fitting the calibration curve to the equation [Ca²⁺] = Kd′β (R − R_min)/(R_max − R). The values obtained were R_max = 3.2, R_min = 0.5, K_d′ = 10.7, β = 2.5 without autofluorescence correction (black); R_max = 3.7, R_min = 0.5, K_d′ = 12.7, β = 2.75 for corrected ratio(red). (D) [Ca²⁺]_mito in intact mitochondria after multiple additions of Ca²⁺ (15.3 µM first addition, 5.7 µM for subsequent additions) show that autofluorescence correction had little effect on the calculated [Ca²⁺]_mito.