Fluorescence lifetime characterization of novel low-pH probes

Abstract

The structures and functions of the cellular acidic compartments are strongly dependent on the pH gradients across vesicular membranes. Measurement and imaging of the vesicular pH require fluorophores with appropriate pK(a) values. In this report, we characterized the pH-dependent lifetime responses of a family of acidotropic probes, LysoSensors, to evaluate their usefulness to low-pH lifetime imaging. LysoSensors are cell-permeable weak bases that selectively accumulate in acidic vesicles after being protonated. They have higher quantum yields at lower pH ranges to allow visualization of the lysosomes. For LysoSensors DND-167, DND-189, and DND-153, raising the buffer pH increased the quenching effects of their basic side chains and substantially reduced their steady-state fluorescence and lifetimes. The apparent pK(a) values determined from their lifetime responses were shifted to near neutral values because of the dominant intensity contribution from their protonated species. One unique property of LysoSensor DND-189 is its nonmonotonic lifetime responses of the maxima occurring between pH 4 and 5. LysoSensor DND-192 did not show significant lifetime changes over a wide pH range. LysoSensor DND-160, which was the only excitation and emission ratiometric probe, showed significant pH-dependent lifetime changes as well as its spectral shifts. Its apparent pK(a) values determined from the lifetime responses were comparable to the lysosomal pH because of its bright basic form. Because of the pH-dependent absorption spectra, the apparent pK(a) values could be manipulated between 3 and 5 by changing the excitation and/or emission wavelengths. These results indicate that LysoSensor DND-160 is a promising probe for lifetime imaging to determine lysosomal pH.

FIG. 1.

Chemical structures of the LysoSensor pH indicators, DND-167, DND-192, DND-153, DND-189, and DND-160.

FIG. 2.

Excitation and emission spectra of LysoSensor probes in different pH buffers. DND-160 can be used as a dual-excitation and dual-emission pH indicator since both of its excitation and emission maxima show blue shift as pH increases.

FIG. 3.

Absorption spectra of DND-189 at pH 2.87, 4.26, and 7.14. The red-shift phenomena were observed at pH 2.87 and 7.14, which were the same as those observed in the excitation spectra of DND-189.

FIG. 4.

Phase and modulation responses of LysoSensor probes at different pH values with frequency domain measurements. The observed emission depends on the emission filters.

FIG. 5.

The pH dependence of the steady-state fluorescence and mean lifetimes of DND-189. The excitation wavelength was 442 nm for both measurements, and the emission observation wavelengths were 500 and 470 ± 10 nm, respectively. Both profiles showed their maxima between pH 4 and 5.

FIG. 6.

Excitation wavelength dependence of the apparent pK_a of LysoSensor yellow/blue DND-160. When excitation wavelength was adjusted from 380 and 365 to 350 nm, the apparent pK_a dropped from4.5 and 3.8 to 3.1. The modulation frequency was fixed at 194.3 MHz.