Taken together, the in vitro studies suggested Mito-pH2 to be a promising probe for living cells. Mito-pH2 cytotoxicity was evaluated using the WST-8 assay to assess its cellular applicability. Figure shows no significant cytotoxicity till a Mito-pH2 concentration of 5000 nM. We cautiously used a concentration 500nM for cell imaging, which was 1/10 th of the limit tested. Fluorescence micrographs of Mito-pH2 treated NIH-3T3 cells appeared to suggest a mitochondrial localisation of the dye. Co-localization assay with a known mitochondrial probe, MTG showed that Mito-pH2 was specifically localizing to mitochondria (Figure ). The overlapping merged image and line scan profile, and high PCC of 0.91 (Figure ) confirm the localization of our Mito-pH2 dye at the mitochondria. It is important to mention that Mito-pH2 in cells showed strong fluorescence in the 580-620 nm channel, when excited with 561 nm.