Measurement of individual intracellular pH and membrane potential values in living cells

Jan Slavik; Edvard Lanz; Petr Cimprich

doi:10.1117/12.351014

2 July 1999 Measurement of individual intracellular pH and membrane potential values in living cells

Jan Slavik, Edvard Lanz, Petr Cimprich

Proceedings Volume 3600, Biomedical Imaging: Reporters, Dyes, and Instrumentation; (1999) https://doi.org/10.1117/12.351014
Event: BiOS '99 International Biomedical Optics Symposium, 1999, San Jose, CA, United States

Abstract

It was assumed that each cell is a homogeneous suspension may have a slightly different pH and membrane potential. A wide range of pH-sensitive fluorescent dyes BCECF, SNARF, FITC, carboxyfluorescein, fluorescein and pyranine have been carefully tested for the accuracy and reliability of their pH-response inside living cells. The intracellular milieu was simulated by a series of mineral buffers with addition of proteins. The pH values have been determined from the excitation ratios 490/435 nm for BCECF, FITC, carboxyfluorescein and fluorescein, and 450/400 nm for pyranine, emission ratios 518/529 nm for BCECF and 635/590 nm for SNARF. The spectrally determined values were then compared with the pH values of buffers measured by a glass electrode. Using the data from the calibration procedure, we evaluated individual intracellular pH values of a large number of cells within one cell population. The confocal ratio fluorescence microscopy revealed pH maps from which both cytoplasmic and vacuolar pH values could be determine, flow cytometry gave enormous amount of average intracellular pH values of individual cells of a whole cell population. Each cell population exhibited significant differences in both cytoplasmic pH values among individual cells. The pH distribution of a typical cell population appeared to fit a Gaussian curve. In yeast it was a Gaussian curve with half- width values around 0.4 pH unit. The men pH values depended on the growth phase, H-ATPase activity and external pH values. The preliminary result with the new membrane potential dye tetramethylrhodamine methyl ester indicated that similarly to pH values, there is a heterogeneity in membrane potential values among cell sin one cell population. The data presented above suggest that each ell behaves as an individual with an individual set up of its metabolism. This 'fine tuning' of the metabolism result in slightly higher or lower pH or membrane potential values that can be detected by fluorescence techniques.

Citation Download Citation

Jan Slavik, Edvard Lanz, and Petr Cimprich "Measurement of individual intracellular pH and membrane potential values in living cells", Proc. SPIE 3600, Biomedical Imaging: Reporters, Dyes, and Instrumentation, (2 July 1999); https://doi.org/10.1117/12.351014

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available