Fig. 1. TMRE localises to mitochondria in response to the m, where the fluorescence is quenched. (A) TMRE signal co-localises with mitochondrial NADH autofluorescence. Confocal imaging of TMRE loaded astrocytes shows the co-localisation of TMRE staining (top, orange emission) and NADH autofluorescence (bottom, blue emission). Bar, 10 µm. A small section from an image is shown at higher magnification in B, emphasising the colocalisation of the two signals. Bar, 1 µm. (C) A rise in TMRE signal reflects a loss of m. Dissipation of m with the protonophore, FCCP (1 µM) induced a rapid rise in TMRE fluorescence (i,ii), as TMRE fluorescence is quenched by concentration of the probe into mitochondria. When the image sequence was divided by the first image, the values of each pixel were normalised to the `resting' level. The resultant images (iii,iv) reveal the proportional change in each pixel value since the first image. After application of FCCP, the mitochondria appear dark against a bright background of cytosol, consistent with fluorescence dequench as the dye moves from mitochondria to cytosol. The TMRE signal measured over the cytosol of an astrocytes increased both (D) in response to the uncoupler FCCP, and (E) following inhibition of mitochondrial respiration by rotenone (2 µM, in the presence 2.5 µg ml^-1 oligomycin).

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