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First published online October 21, 2005
Journal of Experimental Biology 208, 4137-4149 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01878

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Determination of pH by microfluorometry: intracellular and interstitial pH regulation in developing early-stage fish embryos (Danio rerio)

Andreas Mölich^* and Norbert Heisler

Department of Animal Physiology, Humboldt-Universität zu Berlin, D-10115, Germany

View larger version (29K): [in a new window]   Fig. 1. Principle of experimental chamber utilized in all series of experimentation. Embryos were fixed over small funnel-like chamber exits by slight medium suction. Stratification on the basis of diffusion limitation is prevented by the encircling flow of medium, provided by a peristaltic pump. The air space of the chamber was flushed with the same gas as used for equilibration of the medium.

View larger version (17K): [in a new window]   Fig. 2. Fluorescence intensities and intensity ratios of aqueous phosphate-buffered standards upon excitation of SNARF-1 (A) at 488 nm (dual-emission detection at 590-610 nm and >630 nm) and of SNAFL-2 (B) at 488 nm and 543 nm (emission detection at >570 nm). The extensive long-term laser instability is compensated for by ratiometric analysis (±2%) with the dual emission dye SNARF-1 (A), but is additively transmitted into the intensity ratio of the dual excitation dye SNAFL-2 (B). Referencing SNAFL-2 signals to quasi-simultaneous measurements of a fluorescent uranium glass standard is suitable to reduce fluctuations to ±6%. Abbreviations: exc., excitation wavelength; em, emission wavelength.

View larger version (38K): [in a new window]   Fig. 3. Determination of intracellular pH (pHi) with intracellular microelectrodes and by application of microfluorometry of SNARF-1-dextran, calibrated with standards containing 100 mmol l-1 phosphate, BSA and 150 mmol l-1 KCl. Embryonic cells were equilibrated with PCO2s alternating between 2.4 and 24.6 mmHg (A). Differences of optical measurements as compared with microelectrode readings indicate the best approximation to intracellular conditions to be achieved by addition of 10% BSA + 150 mmol l-1 KCl (B).

View larger version (17K): [in a new window]   Fig. 4. Hypercapnia in Danio rerio. pH, [HCO3-] and HCO3- equilibrium potentials (Eeq; according to the Nernst equation) of fluid compartments of Danio embryos in response to 10-fold changes in PCO2 from 0.73 to 7.4 mmHg. Indices denote: amb, ambient; int, interstitial; i, intracellular. Each point represents the mean of 7 (i) or 6 (int) measurements ± S.D.

View larger version (18K): [in a new window]   Fig. 5. Hypercapnia in Danio rerio. pH, [HCO3-] and HCO3- equilibrium potentials (Eeq; according to the Nernst equation) of fluid compartments of Danio embryos in response to 10-fold changes in PCO2 from 2.4 to 24.6 mmHg. Indices denote: amb, ambient; int, interstitial; i, intracellular. Each point represents the mean of 14 (i) or 5 (int) measurements ± S.D.

View larger version (16K): [in a new window]   Fig. 6. Post-hypercapnia in Danio rerio. pH, [HCO3-] and HCO3- equilibrium potentials (Eeq; according to the Nernst equation) of fluid compartments of Danio embryos in response to 10-fold changes in PCO2 from 7.4 to 0.73 mmHg. Indices denote: amb, ambient; int, interstitial; i, intracellular. Each point represents the mean of 7 (i) or 6 (int) measurements ± S.D.

View larger version (17K): [in a new window]   Fig. 7. Post-hypercapnia in Danio rerio. pH, [HCO3-] and HCO3- equilibrium potentials (Eeq; according to the Nernst equation) of fluid compartments of Danio embryos in response to 10-fold changes in PCO2 from 24.6 to 2.4 mmHg. Indices denote: amb, ambient; int, interstitial; i, intracellular. Each point represents the mean of 14 (i) or 5 (int) measurements ± S.D.