Non-ratiometric methods

On binding to calcium, indicators used for non-ratiometric measurements show a shift in their fluorescence intensity (the free indicator has usually a very weak fluorescence). In this case, cytosolic free calcium concentration is related with fluorescence intensity as follow:

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

Where:

Advantages of non-ratiometric indicators: an increase in fluorescence signal can be related directly to an increase in calcium concentration.

Drawbacks of non-ratiometric indicators: fluorescence intensity depends on many factors not related to calcium concentration. Some of these are acquisition conditions, probe concentration and optical path length.

There are many non-ratiometric calcium indicators, some of them are listed below (data from Molecular Probes). The most suitable for each experiment depends on the range of calcium concentration that has to be evaluated, instrumentation, loading requirements, etc (see our tips for choosing a dye).

Indicator
Kd (nM)
λexcitation (nm)
λemission (nm)
Comments
Quin2
60
332
498
High intracellular buffering. UV excitation required.
Fluo-3
390
506
526
 
Fluo-4
345
494
516 
More sensitive than Fluo-3 due to its λexc closer to 488 nm excitation laser
Calcium Green-2
550
503
536
 
Calcium Orange
185
549
575
 
Fluo-4FF
9700
494
516
Derivative of Fluo-4 suitable for [Ca2+] between 1 µM and 1 mM