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Proceedings Paper

Fiber optic measurement of intracellular pH in intact rat liver using pH-sensitive dyes
Author(s): Franz Felberbauer; Juerg Graf
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Paper Abstract

The pH-sensitive fluorescent dye 1,3-dihydroxy-pyrene-6,8-disulfonic acid (DHPDS) was used to measure intracellular pH (pHi) from the surface fluorescence of the isolated perfused rate liver. 1 micrometers of the diacetyl ester of DHPDS was added to the perfusate. The dye readily accumulated in hepatocytes where it was hydrolyzed to form the fluorescent pH indicator. Surface fluorescence was excited by focusing monochromatic light from a spectrofluorometer onto the end of a fiber light guide that illuminated a part of the liver surface. Two excitation wavelengths were used, one where fluorescence intensity was pH sensitive, the other at the isosbestic point. Emitted light was collected by a second fiber optic and returned to the emission side of the spectrometer. A ratio was computed between fluorescence intensities at the pH-sensitive excitation peak wavelength and the isosbestic wavelength, thus yielding measurement insensitive to changes in dye concentration and in excitation light intensity. Intracellular calibration of dye fluorescence was done by clamping pHi to perfusate pH with the use of the cell membrane H+/K+ -ionophore Nigericin. The method was used to monitor changes in pHi caused by addition and removal of NH4Cl. Data obtained in the perfused liver were in good agreement with those reported for isolated liver cells. The technique appears adequate to determine hepatocellular pH in the liver and will allow study of regulatory mechanisms of hepatocyte pHi in the intact organ.

Paper Details

Date Published: 1 September 1991
PDF: 9 pages
Proc. SPIE 1510, Chemical and Medical Sensors, (1 September 1991); doi: 10.1117/12.47125
Show Author Affiliations
Franz Felberbauer, Univ. of Vienna School of Medicine (Austria)
Juerg Graf, Univ. of Vienna School of Medicine (Austria)

Published in SPIE Proceedings Vol. 1510:
Chemical and Medical Sensors
Otto S. Wolfbeis, Editor(s)

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