Thus, to better understand the functional status of mitochondria, it is valuable to get a measure of the mitochondrial pH. Fluorescence imaging using molecular probes and biosensors has gained popularity in biological applications due to its many benefits, which include excellent sensitivity, quick response time, non-invasiveness, high signal-to-noise ratio and ability to provide new information on specific biochemical species and/or chemical microenvironment. Furthermore, because red and near-infrared (NIR) fluorescent dyes cause less harm to biological samples and are less affected by background auto-fluorescence in cells and tissues, they are desirable in the field of biological imaging. In this context, various probes have been developed to monitor mitochondrial pH Additionally, a few genetically encoded mitochondria pH sensors derived from fluorescent protein variants also exist. Those are MitoSypHer(Poburko, Santo-Domingo, and Demaurex 2011), MitoAlpHi (Cano , pHRed(Tantama, Hung, and Yellen 2011) and EYFPmito . However, fluorescent protein based pH sensors lead to spectral crowding in the already limited visible bandwidth required to monitor critical cellular processes. To further add to the complexity, many cells and tissues of physiological relevance including primary cells are difficult to transfect or suffer from transfection-mediated stress. Additionally, protein-based sensors are bulky, and may perturb mitochondrial function. Thus, the objective is to design and synthesise small molecule based red-emitting fluorescent probes to track mitochondrial pH in live cell in real space and time. Here, it is important to remember that often a single pH probe may not be effective in all physiological contexts including diverse cell and tissue types, therefore it is important to build diverse chemical probes to help develop a toolkit for efficacious and meaningful mitochondrial probing, including pH probing. To address this, we have developed a hemicynaine based fluorescent probe, Mito-pH2 with a pKa of 6.6, which therefore may be suitable for reporting change in mitochondrial pH. The Phenol hydroxyl group (-OH) in Mito-pH2 has the potential to act as a proton donor under alkaline conditions. In solution this probe works as a ratiometric pH probe and also shows pH induced reversible colorimetric changes visible through the naked eye. Importantly, this probe is capable of sensing dynamic changes in pH at the mitochondrial membrane, with spatial and temporal resolution. This low-cytotoxicity red-emitting probe reveals spatial heterogeneity in the mitochondrial network in living cells, with interesting inter-cellular and intra-cellular heterogeneity in pH seen across the mitochondrial network in single cells.