The pH-dependence fluorescence monitoring probes are of great significance and play many critical roles in studying of cells, enzymes, and tissue activities, including proliferation, apoptosis, multidrug resistance, ion transportation, endocytosis, and muscle contraction etc. Here a carbocyanine type anti-photobleaching pH fluorescence probe was synthesized in which four chlorine atoms were substituted. The probe had two fluorescent peaks at the wavelengths of 530 nm and 596 nm respectively

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... r the irradiation at the excited wavelength of 500 nm. The fluorescent emission at 596 nm indicated a large stokes shift of 96 nm and the strongest value at the pH value of 8.5, and the fluorescent emission was very weak at the pH values of <8.1 or >9.8. Then three configurational structures as A, B and C were proposed accordingly, which were further confirmed through calculation by the density functional theory in the Gaussian 03 soft package. The coplanarity of the structure B was found which was contributed by the intramolecular hydrogen bond between two sulfonic acid groups. The coplanarity thus led to the higher conjugation system in the molecule structure and the stronger fluorescence emission. The fluorescence quantum yield was measured as 0.35 by the relative measuring method and the transient fluorescence spectra were determined as well. In the photobleaching test, the probe showed a good anti-photobleaching performance with the stable time of more than 70 min, which was much better than the traditional organic probes. The anti-photobleaching performance was good enough to ensure the wide application in biology imaging. The fluorescence images of PANC-1 cells stained by the probe (1 μmol/L) incubated in PBS buffer solution were obtained by the inverted fluorescence microscope. The probe was found aggregated onto the cell-membrane during cell differentiation and proliferation processes.