Fluorescent Dyes:
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Quantifying calcium - Calibration & Tips

Fluorescent Dyes - Quantifying calcium concentration

Changes in [Ca2+] are related to changes in fluorescence intensity (non-ratiometric indicators) or fluorescence intensity ratio (ratiometric indicators) of a chosen dye. Relative values of calcium concentration (semi-quantitative values) can be obtained without too many cautions. Roughly, fluorescence intensity for each cell is measured during an experiment and values are normalized by their resting intensities. Relative values can only be compared with those obtained using the same experimental conditions (ideally in the same experiment set). These values depend not only on the indicator used but also on the loading protocol, microscope settings, cell line, temperature, etc.

Absolute calcium concentration, though being one of the most desired goals on calcium imaging, is very tricky. Equation 1 shows the relationship between fluorescence and absolute calcium concentration ([Ca2+]).


[Ca2+] = Kd (F - Fmin)/(Fmax-F)

Equation 1.

Fmin and Fmax are values obtained at minimum and maximum calcium concentration (i.e. 0 and saturating values). These parameters should ideally be measured in each cell to obtain [Ca2+] accurately, but often this approximation is not viable.

Calcium chelators are used to obtain an accurate Fmin, but this kind of compounds interferes in the value of F. Another problem is that Fmax value of a cell might be modified during the measurement of F. Acquisition of Fmax value requires the use of high intracellular calcium concentrations (values >>10·Kd), which disturbs intracellular environment.

Kd is the dissociation constant for each indicator and should be established for each experimental condition. Kd tabulated values usually correspond to the dye in water or buffer (not inside a cell). Differences in pH, protein concentration, ionic strength, lipid interaction, etc. have been reported to modify dramatically Kd value (Molecular Probes), so a calibration is needed for each experimental conditions.


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