Next, to check if the pH-induced changes in Mito-pH2 fluorescence yield is reversible we monitored the I610/I565 while cycling between two different pH states (pH 5.0 and 8.0). Figure clearly shows that the fluorescence modulation due to deprotonation-protonation of Mito-pH2 process is reversible for at least three cycles. The I610/I565 value shows only a negligible decrease even after three cycles, demonstrating that Mito-pH2 to be photo-stable under the conditions tested. Then we checked if the observed changes in fluorescence is solely mediated by pH and not by other ions. For this, we examined the Mito-pH2 in the presence of various ions and measured the ratio (I610/I565). Figure shows little or no effect of examined ions on Mito-pH2 fluorescence.