Imaging of Mitochondrial pH Using SNARF-1

Laser scanning confocal microscopy provides the ability to image submicron sections in living cells and tissues. In conjunction with pH-indicating fluorescent probes, confocal microscopy can be used to visualize the distribution of pH inside living cells. Here we describe a confocal microscopic technique to image intracellular pH in living cells using carboxyseminaphthorhodafluor-1 (SNARF-1), a ratiometric pH-indicating fluorescent probe. SNARF-1 is ester-loaded into the cytosol and mitochondria of adult cardiac myocytes or other cell type. Using 568-nm excitation, emitted fluorescence longer and shorter than 595-nm is imaged and then ratioed after background subtraction. Ratio values for each pixel are converted to values of pH using a standard curve (lookup table). Images of the intracellular distribution of pH show cytosolic and nuclear areas to have a pH of ~7.1, but in regions corresponding to mitochondria, pH is 8.0, giving a mitochondrial ΔpH of 0.9. During hypoxia, mitochondrial pH decreases to cytosolic values, signifying the collapse of ΔpH. These results illustrate the ability of laser scanning confocal microscopy to image the intracellular distribution of pH in living cells and to determine mitochondrial ΔpH.