A novel far-visible and near-infrared pH probe for monitoring near-neutral physiological pH changes: imaging in live cells

A novel vis-NIR neutral pH probe ESCY was facilely prepared by the ethylene bridging of N-alkylated indolium and 4-(diphenylamino)benzaldehyde. The pH titration indicated that ESCY displayed a remarkable emission enhancement in the vis-NIR fluorescence region (λ_em = 650 nm) when the pH is decreased from 10.00 to 5.00, and the pK_a value was 7.38 near the physiological pH, which is valuable for near-neutral cytosolic pH research. The probe exhibited strong pH-dependent behavior and responded linearly and rapidly to minor pH fluctuations within the range of 6.50-8.20. In addition, ESCY displayed a notably large Stokes shift of 120 nm, high reversibility and could effectively avoid the influence of autofluorescence in biological systems. The confocal fluorescent microscopic images of subcellular distribution and the detection of pH in SW480 cells were achieved successfully, suggesting that ESCY has excellent cell membrane permeability and could image near-neutral cytosolic pH fluctuations in live cells with negligible autofluorescence.