Optical measurements of pH using fluorescence lifetimes and phase-modulation fluorometry

Abstract

We measured the pH-dependent fluorescence decay times of the seminaphthofluoresceins (SNA-FL), seminaphthorhodafluors (SNARF), and BCE-CF using phase-modulation fluorometry. The phase and modulation values were found to be strongly pH-dependent in the physiological pH range, over the easily accessible range of light modulation frequencies from 10 to 300 MHz, making these probes useful as lifetime-based pH sensors. The phase and modulation values are dependent on excitation and emission wavelength as well as pH. This dependence allows the range of pH sensitivity to be chosen by selection of the wavelength(s) and enables increased precision of the pH measurements by use of phase and/or modulation measurements at several wavelengths. These probes can be excited using a a green He-Ne laser at 543 nm, which allows their use in low cost instrumentation. Phase and modulation measurements are especially suitable for sensing applications because they are insensitive to the changes in signal intensity that result from photobleaching, probe washout, and/or light losses.

Figure 1.

pH-dependent absorption and emission spectra of cerboxy SNARF-6. The dashed line (- -) in the lower panel shows the transmission profile of a Corning 2–73 cutoff filter used for some of the measurements.

Figure 2.

Frequency responses of four SNARF probes at low and high pH. The acid and base forms were excited at 563 nm and observed at 600 nm.

Figure 3.

Frequency response of SNAFL-1 and carboxy SNAFL-1 for 563 excitation and 600 nm emission.

Figure 4.

pH-dependent phase and modulation of four SNARF probes excited with a 543-nm He-Ne laser and 135MHz light modulation.

Figure 5.

pH and emission wavelength-dependent phase and modulation of carboxy SNARF-6 with 543-nm excitation.

Figure 6.

pH calibration curves of cerboxy SNARF-6 for excitation at 543 and 563 nm.

Figure 7.

Time stability of pH sensing by SNARF-6 at room temperature, which varied by several °C. Excitation was at 563 nm, and emission was at 580 nm using an interference filter.

Scheme I.. Structures of the SNAFL, SNARF, and BCECF Probes^a

^a For a complete listing of the full chemical names, see ref .