Simultaneous Measurement of Water Volume and pH in Single Cells Using BCECF and Fluorescence Imaging Microscopy

Authors

Francisco J. Alvarez-Leefmans, Wright State University - Main CampusFollow
José J. Herrera-Pérez, National Institute of Psychiatry, Mexico
Martín S. Márquez, National Institute of Psychiatry, Mexico
Victor M. Blanco, Wright State University - Main Campus

Document Type

Article

Publication Date

1-2006

Abstract

Regulation and maintenance of cell water volume and intracellular pH (pH_i) are vital functions that are interdependent; cell volume regulation affects, and is in turn affected by, changes in pH_i. Disruption of either function underlies various pathologies. To study the interaction and kinetics of these two mechanisms, we developed and validated a quantitative fluorescence imaging microscopy method to measure simultaneous changes in pH_i and volume in single cells loaded with the fluorescent probe BCECF. CWV is measured at the excitation isosbestic wavelength, whereas pH_iis determined ratiometrically. The method has a time resolution of <1 s and sensitivity to osmotic changes of ∼1%. It can be applied in real time to virtually any cell type attached to a coverslip, independently of cellular shape and geometry. Calibration procedures and algorithms developed to transform fluorescence signals into changes in cell water volume (CWV) and examples of applications are presented.

Comments

Copyright © 2006, Biophysical Society. Originally published in the Biophysical Journal 90.2. Available on publisher's site at http://www.sciencedirect.com/science/article/pii/S0006349506722391

Repository Citation

Alvarez-Leefmans, F. J., Herrera-Pérez, J. J., Márquez, M. S., & Blanco, V. M. (2006). Simultaneous Measurement of Water Volume and pH in Single Cells Using BCECF and Fluorescence Imaging Microscopy. Biophysical Journal, 90 (2), 608-618.
https://corescholar.libraries.wright.edu/ptox/14

DOI

10.1529/biophysj.105.069450