Converting fluorescence data into Ca2+ concentration

In many situations, fluorescent Ca(2+) reporters are used to simply indicate that a change of Ca(2+) concentration has occurred. Monitoring the emission from a Ca(2+)-sensitive indicator can be sufficient to tell whether a signal has arisen, and what its kinetic/spatial parameters were. The emission from an indicator does not have a linear relationship to the Ca(2+) concentration within a cell; rather, the relationship between fluorescence emission and Ca(2+) concentration is described by a logistic function. Simply recording fluorescence emission, therefore, provides a relative indication of the magnitude of a Ca(2+) signal that should not be used for generating mean amplitude data. However, with a little consideration and effort, the fluorescence output can be calibrated to yield actual Ca(2+) concentration.