[HIn] The above-mentioned Henderson-Hasselbalch (Eq.1) used to calculate the pKa of Mito-pH2 dye by measuring the absorbance intensities of the ratiometric dye at two different wavelengths. The Mito-pH2 dye exhibits absorbance at two distinct wavelengths, 465 nm (denoted by λ1, protonated form) and 585 nm (denoted by λ2, deprotonated form). The ratio of [In -]/[HIn] can be re-written below [In⁻]