MULTIWAVELENGTHS FLUORESCENCE IMAGING - THE PREREQUISITES FOR THE INTRACELLULAR APPLICATIONS

Since the importance of the intracellular concentrations and transmemb rane fluxes of cations has been recognized, various techniques for the evaluation of intracellular concentrations of H+, Ca2+, Mg2+ and othe r cations have been developped. Microfluorometry is a powerful and non invasive technique for it makes possible kinetic studies or mapping of ionic concentrations in different cellular compartments when there ar e fluorescent probes specific to given cations. Some previously publis hed results seem sometimes controversial and, as reported by W.N. Ross on the use of fluorescent calcium probes, it appears that often we ob tain only qualitative answer to physiological questions. To minimize t hese problems, a resolution method of complex fluorescence data is ana lyzed then the following protocol is proposed: i) careful and exhausti ve determination of the fluorescent compounds in the sample (determina tion of the physicochemical properties of the probe(s)), ii) optimizat ion of the choice of optical filters by analysis of the components spe ctra and study of the final error, iii) control the validity of the re sults, specially the number and quality of the fluorescent compounds. An example of the application of this protocol to the determination of intracellular pH using the C-SNARF-1 probe is given.