Ratiometric methods

Ratiometric methods are based on the use of a ratio between two fluorescence intensities. This allows correction of arctifacts due to bleaching, changes in focus, variations in laser intensity, etc. but makes measurements and data processing more complicated.

Ratiometric indicators show a shift in their emission or excitation spectra when they bind to calcium, therefore they can be classified as dual emission or dual excitation indicators. Measurement of calcium with these compounds is achieved by using two excitation lasers (if they are dual excitation indicators) or two detection ranges (it they are dual emission indicators).

If a so-called ratiometric indicator is used, intensity ratio is calculated at wavelenghts were difference of fluorescence between bound and free indicator is maximum.

A ratiometric quantification can also be done using a mixture of an intensity shift indicator and a insensitive fluorescence compound (i.e. Fluo-3 and Fura-Red). In this case, it is important to adjust conditions to obtain a similar fluorescence intensity response from both compounds.

Cytosolic free calcium concentration is related with fluorescence as follows:

[Ca2+] = Kd (R - Rmin)/(Rmax- R)·(Fλ1max/Fλ1min)

Where:

Advantages of ratiometric methods: [Ca2+] estimated with ratiometric compounds is not affected by probe loading (loading conditions is critical when mixtures are used), bleaching, optical path length, illumination intensity, etc. Therefore using ratios avoids many of the problems related to absolute fluorescence values.

Drawbacks of ratiometric methods: acquisition and data manipulation is more complex due to the use of fluorescence ratios. Not all microscopes are suitable for these measurements (changing excitation/emission wavelength at suitable rates is required) and many ratiometric indicators require the use of UV excitation.

Ratiometric calcium indicators are among the most popular due to their higher robustness than non-ratiometric indicators.

Some ratiometric calcium indicators are listed in the table below (data from Molecular Probes). The most suitable for each experiment will depend 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
Free
Bond
Free
Bond
Fura Red
140
472
436
657
637
Low quantum yield. Dual excitation.
Fura-2
145
363
335
512
505
Dual excitation. It is more ressistant to bleaching than indo-1.
Mag-fura-2
25000
369
329
511
508
Mg2+ sensitive. Dual excitation.
Indo-1
230
346
330
475
401
Dual emission.
YC2.1
100 and 4300
430
430
480
535
Biphasic yellow cameleon
YC3.1
1500
430
430
480
535
Monophasic yellow cameleon