Use of a new fluorescent probe, seminaphthofluorescein‐calcein, for determination of intracellular pH by simultaneous dual‐emission imaging laser scanning confocal microscopy

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Abstract
A new pH indicator, seminaphthofluorescein (SNAFL)-calcein acetoxymethyl ester, was used for intracellular pH (pHi) measurement in living MDCK cells with a laser scanning confocal microscope (LSCM) equipped with an Argon/Krypton laser and dual-excitation and dual-emission (FITC/Texas Red) filter set. SNAFL-calcein excitation maxima are ∼492/540 nm (acid/base) and emission maxima are ∼535/625 nm (acid/base) with a pKa value at ∼7.0. The absorption/emission spectra of SNAFL-calcein indicate that the ratio of emission intensities of its basic/acidic forms is pH dependent. With an Argon/Krypton LSCM, we were able to monitor the acidic and basic forms of this dye simultaneously using dualexcitation (488/568 nm) and dual-emission (525–614 nm/∼615 nm) wavelengths (λs). The simultaneous dual-excitation/emission LSCM system allows for efficient recording of pHi dynamics (time resolution ≈︁ 1 sec) in living cells. We have analyzed emission stability of the dye at different temperatures (22°C and 37°C) and constant pH, and at the same temperature (22D°C) but various pHs (6.6, 7.0, and 7.4). Bleaching rate is slightly higher at 37°C than that at 22°C. The basic form of the dye (λEm ≈︁ 625 nm) has a slightly higher bleaching rate than the acidic form (λEm ≈︁ 535 nm) in standard culture medium (pH 7.3) at either 22°C or 37°C. The pHi in MDCK cells calculated from ratio images (535 nm/625 nm) was 7.19 ± 0.03 (mean ± SEM, n = 20). Calibration experiments show that the useful pH range of SNAFL-calcein appears to be between 6.2 and 7.8, as the dye is difficult to calibrate outside this pH range.