Abstract
The measurement of the intracellular concentrations of ions is one of the challenges of modern cell biology. For that purpose, different kinds of fluorescent probes are now available. For some of them, the complexation induces a shift in the position of the excitation and/or the emission fluorescence spectrum. In that case the popular two-wavelength ratio method allows the determination of the intracellular pH although some problems have been reported in the obtaining of intracellular calibration curves.
We have previously demonstrated that the fluorescence emission or excitation spectrum of most of these probes was pH sensitive in the range of biological pH. Moreover it has also been proven that they are able to bind with intracellular proteins. So we have proposed another data processing method that allows us to extract quantitative information on each of these interactions from the cell fluorescence spectrum. That method was used to monitor pHi variations with the fluorescent probe C-SNARF-1.
The scope of this contribution is to present a protocol of data processing allowing to use video-imaging techniques for monitoring the changes in intracellular ionic concentrations. For sake of clarity, only applications to the determination of the kinetics of pHi equilibration following some external stimuli will be considered.
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Viallet, P., Salmon, J.M., Yassine, M., Vigo, J. (1996). Videomicrofluorometry and Intracellular Ionic Concentration in Single Living Cells. In: Kohen, E., Hirschberg, J.G. (eds) Analytical Use of Fluorescent Probes in Oncology. NATO ASI Series, vol 286. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-5845-3_22
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DOI: https://doi.org/10.1007/978-1-4615-5845-3_22
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