We report what we believe to be the first near-infrared pH-sensitive fluorescence lifetime molecular probe suitable for biological applications in physiological range. Specifically, we modified a known fluorophore skeleton, hexamethylindotricarbocyanine, with a tertiary amine functionality that was electronically coupled to the fluorophore, to generate a pH-sensitive probe. The pKa of the probe depended critically on the location of the amine. Peripheral substitution at the 5-position of the indole ring resulted in a compound with pKa ∼ 4.9 as determined by emission spectroscopy. In contrast, substitution at the meso-position shifted the pKa to 5.5. The resulting compound, LS482, demonstrated steady-state and fluorescence-lifetime pH-sensitivity. This sensitivity stemmed from distinct lifetimes for protonated (∼1.16 ns in acidic DMSO) and deprotonated (∼1.4 ns in basic DMSO) components. The suitability of the fluorescent dyes for biological applications was demonstrated with a fluorescencelifetime tomography system. The ability to interrogate cellular processes and subsequently translate the findings in living organisms further augments the potential of these lifetime-based pH probes. © 2011 by the Biophysical Society.
Berezin, M. Y., Guo, K., Akers, W., Northdurft, R. E., Culver, J. P., Teng, B., … Achilefu, S. (2011). Near-infrared fluorescence lifetime pH-sensitive probes. Biophysical Journal, 100(8), 2063–2072. https://doi.org/10.1016/j.bpj.2011.02.050