Simultaneous measurement of water volume and pH in single cells using BCECF and fluorescence imaging microscopy

¹ Wright State University, SOM
² Instituto Nacional de Psiquiatría
³ Wright State University

^* To whom correspondence should be addressed. E-mail: francisco.alvarez-leefmans{at}wright.edu.

Submitted on June 24, 2005
Revised on July 26, 2005
Accepted on 14 September 2005

	Abstract

Regulation and maintenance of cell water volume(CWV) 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 pHi and volume in single cells loaded with the fluorescent probe BCECF. CWV is measured at the excitation isosbestic wavelength, while pH_i is determined ratiometrically. The method has a time resolution of <1 second 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 fluorescent signals into changes in CWV and examples of applications are presented.

Key Words: BCECF, cell osmotic behavior, cell water volume, fluorescent dyes, intracellular pH, isosbestic wavelength

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