We have developed hemicyanine based fluorescent Mito-pH2 probe using a simple, one-step microwave based synthesis as described above (see methods section and Scheme-1). This facile synthesis is a hallmark of donor-acceptor hemicyanine dyes that we have recently developed First, to check the pH response of the synthesised Mito-pH2 in solution, we prepared Britton Robinson (BR) buffers of varying pH from pH 3 to pH 9, keeping the ionic strength the same. The absorbance spectra in Figure show that at low pH, one peak at 465 nm dominates, and with an increase in pH, a second peak at 585 nm gradually arises and finally dominates at high pH. A well-defined isosbestic point at 500 nm clearly indicates coexistence of two absorbing species in the ground state. The observed pH-dependent large bathochromic shift in absorbance spectra suggests the possibility of Intramolecular Charge Transfer (ICT). Clear color change was visible with change in pH (Figure ). The absorbance data was used to calculate pKa of Mito-pH2 using Henderson-Hasselbach equation (see Figure ). This analysis yielded a pKa of 6.6. Next, we recorded fluorescence spectra of Mito-pH2 with increasing pH from pH-3.0 to pH-9.0. The intensity of 465 nm excitation-specific emission peak at 560 nm reduces with increasing pH, while the opposite was observed for 585 nm excitation-specific fluorescence emission at 610 nm (Figure ). The observed red-shift in absorbance and fluorescence for the longer wavelength band likely suggests a pH induced increase in intramolecular conjugation for the deprotonated species formed at higher pH. This is likely achieved via pHinduced deprotonation of phenolic group in the donor moiety.