Widefield Calcium Imaging with Calcium Indicator Fura2

The Ca2+ unbound form of Fura2 gets excited at 380 nm and the Ca2+ bound form at 340 nm. The emitted light is measured at around 510 nm. The fluorescence intensity increases at 340 nm with increasing Ca2+ concentration and decreases at 387 nm for the unbound form. There are some techniques to introduce the fluorescent Ca2+ indicator into cells, like ester loading, microinjection, chemical loading techniques or diffusion from patch-clamp pipettes.

For further information on different Ca2+ indicators, handling of the dye, labeling of the cells and cell loading, please refer to the Molecular Probes Handbook and Takahashi et al. (see References).

Benefit of ratiometric measurement
Artifacts due to uneven dye distribution, leakage of dye, photo bleaching and unequal cell thickness may be avoided by ratioing methods. In a typical experiment where you detect Ca2+ changes over time a calibration must be done. Calibration is possible in vitro or in vivo. For in vivo calibration the ratio of maximum and minimum Ca2+ concentrations is measured. These values may be obtained within the same experiment. For the maximum amount of bound Ca2+ indicator you may add an ionophore like Ionomycin or Bromo-A2318 to equilibrate the Ca2+ concentration. To get the minimum Ca2+ concentration you may apply an EGTA buffered solution to chelate all Ca2+.

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