Fluorescence lifetime characterization of novel low-pH probes

The structures and functions of the cellular acidic compartments are strong ly dependent on the pH gradients across vesicular membranes. Measurement an d imaging of the vesicular pH require fluorophores with appropriate pK(a) v alues. In this report, we characterized the pH-dependent lifetime responses of a family of acidotropic probes, LysoSensors, to evaluate their usefulne ss to low-pH lifetime imaging. LysoSensors are cell-permeable weak bases th at selectively accumulate in acidic vesicles after being protonated. They h ave higher quantum yields at lower pH ranges to allow visualization of the lysosomes. For LysoSensors DND-167, DND-189, and DND-153, raising the buffe r pH increased the quenching effects of their basic side chains and substan tially reduced their steady-state fluorescence and lifetimes. The apparent pK, values determined from their lifetime responses were shifted to near ne utral values because of the dominant intensity contribution from their prot onated species. One unique property of LysoSensor DND-189 is its nonmonoton ic lifetime responses of the maxima occurring between pH 4 and 5. LysoSenso r DND-192 did not show significant lifetime changes over a wide pH range. L ysoSensor DND-160, which was the only excitation and emission ratiometric p robe, showed significant pH-dependent lifetime changes as well as its spect ral shifts. Its apparent pK, values determined from the lifetime responses were comparable to the lysosomal pH because of its bright basic form. Becau se of the pH-dependent absorption spectra, the apparent pK, values could be manipulated between 3 and 5 by changing the excitation and/or emission wav elengths. These results indicate that LysoSensor DND-160 is a promising pro be for lifetime imaging to determine lysosomal pH. (C) 2001 Academic Press.