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

✍ Scribed by You Zhou; Elizabeth M. Marcus; Richard P. Haugland; Michal Opas

• Publisher: John Wiley and Sons
• Year: 1995
• Tongue: English
• Weight: 785 KB
• Volume: 164
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041640103

✦ Synopsis

A new p H indicator, seminaphthofluorescein (SNAFL)-calcein acetoxymethyl ester, was used for intracellular p H (pH,) 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. SNAFLcalcein excitation maxima are -492/540 nm (acid/base) and emission maxima are -535/625 nm (acid/base) with a pK, value at -7.0. The absorption/emission spectra of SNAFL-calcein indicate that the ratio of emission intensities of its basic1acidic 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 (4881568 nm) and dual-emission (525-61 4 nrn12615 nm) wavelengths (As). The simultaneous dual-excitation/emission LSCM system allows for efficient recording of pH, 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 (22°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 (AEm = 625 nm) has a slightly higher bleaching rate than the acidic form (AErn = 535 nm) in standard culture medium (pH 7.3) at either 22°C or 37°C. The pH, 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-cakein appears to be between 6.2 and 7.8, as the dye is difficult to calibrate outside this pH range.