Figure 3: Multiparameter imaging of H₂O₂, GSH/GSSG, Ca²⁺ and pH.

(a) Inhibition of SERCA Ca²⁺ pump by TG induces temporal H₂O₂ increase in the mitochondrial matrix, whereas GSH/GSSG ratio remains unchanged. HEK-293 cells were co-transfected with HyPerRed-mito (upper row) and roGFP-mito (lower row). Numbers indicate timing in minutes. Scale bar, 20 μm. Lookup tables indicate HyPerRed brightness (3,700 to 25,000, 16 bit scale) and roGFP-mito ratio (0.5 to 4). (b) H₂O₂ does not spread from the mitochondria to the cytoplasm of the cells. HEK-293 cells were co-transfected with HyPerRed-mito (upper row) and HyPer3-cyto (lower row). Numbers indicate timing in minutes. Scale bar, 30 μm. Lookup tables indicate HyPerRed brightness (3,700 to 25,000, 16 bit scale) and HyPer-3 ratio (0.5 to 6). (c) Mitochondrial pH remains stable upon SERCA Ca²⁺ pump inhibition and matrix H₂O₂ release. HEK-293 cells were co-transfected with HyPerRed-mito (upper row) and SypHer-mito (lower row). Numbers indicate timing in minutes. Scale bar, 40 μm. Lookup tables indicate HyPerRed brightness (3,700 to 25,000, 16 bit scale) and SypHer ratio (0.5 to 4). (d) TG induces temporal H₂O₂ increase in the mitochondrial matrix (measured using HyPerRed-mito), while cytosolic H₂O₂ (HyPer3-cyto), mitochondrial GSH/GSSG (roGFP-mito) and mitochondrial pH (SypHer-mito) remain stable. In each experiment, HyPerRed-mito was co-expressed with either HyPer3-cyto or roGFP-mito or SypHer-mito. The panel shows values±s.d. for more than 100 cells in two or more experiments for each sensor combination. The agonist was added at a 1.7-min time point. For sample images see panels (a–c). (e) Similar to HyPerRed-mito, dmito-HyPer shows TG-induced matrix H₂O₂ production, confirming specificity of HyPerRed signal. TG was added at a 2.5-min time point.