We then examined the ability of Mito-pH2 to sense mitochondrial pH changes in live cells. We perturbed mitochondrial pH by known chemicals. We used extracellular addition of ammonium chloride and sodium acetate (Poburko, Santo-Domingo, and Demaurex 2011; Cano ) to potentially increase or decrease the mitochondrial pH respectively and examined Mito-pH2 fluorescence in cells with confocal imaging. Extracellular treatment with NH4Cl resulted in an increase in dye fluorescence (Figure ). This is in line with a reported increase in mitochondria pH when cells are treated with NH4Cl. On the contrary, fluorescence images showed that a treatment of cells with sodium acetate caused a decrease in Mito-pH2 fluorescence. This is in line with a putative decrease in mitochondrial pH with sodium acetate treatment (Figure ). These results suggested that Mito-pH2 is reporting pH changes at the mitochondria. Notably, even with sodium acetate treatment used here, no emission was seen from the protonated species of Mito-pH2 (Figure ). This suggested that the treatment used was unable to reduce the mitochondrial pH below the dye pKa. This also suggested that the dye pKa may have to be tweaked to obtain a dual-fluorescence response in the mitochondria of living cells. To quantify the confocal micrographs, we also performed image analyses to quantify the Mito-pH2 fluorescence intensity from individual mitochondria from each single cell in the data set. For this, we adapted a previously reported methodology ) wherein we attempted to segment each individual mitochondrion and quantify the fluorescence (refer methods section of Image analysis of confocal micrograph). Figure and F show a clear and significant change in dye fluorescence upon extracellular treatments. Ammonium chloride causes an increase in Mito-pH2 fluorescence in cells while sodium acetate causes a decrease.