Three emission wavelengths from two-photon excitation techniques were use to determinefree Ca2+ using a new analytical calculation. This was achieved by considering theinteraction of protein with free Ca2+ and indo-1 in protein contaminated sample. The emissions obtained from the excitation with the dissociation constants were used in the calculation. Agreement values of the Ca2+ from the calculation with the known Ca2+ buffer solution used for the measurement was obtained for various Ca2+ and protein concentration. The analytical analysis shows that dissociation constants for each binding’sdeviation errors are small to affect free Ca2+ determination. This would provide an accurate measurement of free Ca2+ in biological cells than the typical two emission wavelength processes. Furthermore, using two-photon excitation methods, deep inside cells observation of free Ca2+ are possible using the methods introduced.
Key words: Free Ca2+, indo-1, two-photon excitation methods.
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