Yojet, that is a very broad set of questions. I will oversimplify and I hope chemists reading this will not cringe too much and correct me if I end up missing any important detail.

Nearly all calcium indicators consist of two components: a fluorophore and a ionophore (the actual calcium binding unit). These two are linked in a way that binding of calcium by the ionophore influences the fluorescence of the fluorophore. All modern synthetic calcium indicators use BAPTA as ionophore. BAPTA complexes calcium ions using four carboxylic acid groups. This complexation ('chelation') process leads to a shift of electron density within the BAPTA molecule. The binding kinetics and affinity can be modified by changing substituents on the BAPTA molecule, changing the availability of electrons for complexation (oversimplification!).

But how does binding of calcium to BAPTA change the fluorescence? There are two main mechanisms for this. In the case of Fura-type indicators, calcium binding leads to a shift of electrons from the fluorophore to to ionophore. This change ('photo-induced charge transfer' or PCT) shifts the energy levels of the ground state and excited state of the fluorophore. This change is reflected in a change in the fluorescence spectrum. Due to this color shift, it is possible to measure fluorescence at two different excitation wavelengths (one preferentially exciting the calcium-free indicator molecules and the other preferentially exciting the calcium-bound ones). The ratio of the resulting intensities is proportional to the calcium concentration. The ability to do this is what 'ratiometric' means. Careful calibration of such indicators will allow you to determine absolute calcium concentrations.

The second mechanism is found in indicators such as Oregon-Green BAPTA 1, Fluo-3 or XRhod-2. Here, the calcium-free ionophore is capable of quenching the fluorescence of the fluorophore, leading to very low fluorescence. This process is known as photo-induced electron transfer (PET). Once the ionophore binds calcium, it is no longer able to quench the fluorophore, so the fluorescence increases. This process does not change the energy levels of the fluorophore and thus the fluorescence spectra do not change. The result is that these indicators cannot be used rationmetrically. Instead, changes in fluorescence are reported only as changes in fluorescence intensity. These probes are sometimes referred to as intensiometric probes. Because the change in fluorescence is proportional to the change in calcium concentration, it will be impossible to determine absolute calcium concentration using these indicators without additional tricks.

Genetically-encoded indicators are another topic that I won't go into here. If that is relevant, do feel free to ask.

Regarding the situation of having multiple different indicators in the same cell, these indicators will of course be in competition for free calcium ions. It is important to remember that this is the case already, as cells contain many calcium binding proteins (often at high concentrations), that are also competing for free calcium. Any detailed quantification will need to take these effects into consideration. As for physiology the dynamics of calcium are relevant, this will often require a model that takes binding kinetics into consideration, to simulate the changes of calcium load of the different binding molecules. 

I hope this is at least somewhat helpful. I realize that I cut corners to reduce the length. I just hope I didn't cut the wrong ones.

The dyes work because the fluorescence of the dye changes as it binds calcium.

There are 2 types of dyes . One type (such as Fluo-4) changes fluorescence intensity directly based on the calcium concentration. Ratiometric dyes, like FURA, show a change in excitation or emission spectra depending on whether the molecule is bound or not to calcium. There are also protein based dyes such as Cameleon- this particular one is also ratiometric. They dyes are all cheleting agents with a preference for calcium.

I'm not sure why one would use 2 different dyes in the same area of a cell, but yes, one dye could affect the other by depleting calcium.

Yojet, that is a very broad set of questions. I will oversimplify and I hope chemists reading this will not cringe too much and correct me if I end up missing any important detail.

Nearly all calcium indicators consist of two components: a fluorophore and a ionophore (the actual calcium binding unit). These two are linked in a way that binding of calcium by the ionophore influences the fluorescence of the fluorophore. All modern synthetic calcium indicators use BAPTA as ionophore. BAPTA complexes calcium ions using four carboxylic acid groups. This complexation ('chelation') process leads to a shift of electron density within the BAPTA molecule. The binding kinetics and affinity can be modified by changing substituents on the BAPTA molecule, changing the availability of electrons for complexation (oversimplification!).

But how does binding of calcium to BAPTA change the fluorescence? There are two main mechanisms for this. In the case of Fura-type indicators, calcium binding leads to a shift of electrons from the fluorophore to to ionophore. This change ('photo-induced charge transfer' or PCT) shifts the energy levels of the ground state and excited state of the fluorophore. This change is reflected in a change in the fluorescence spectrum. Due to this color shift, it is possible to measure fluorescence at two different excitation wavelengths (one preferentially exciting the calcium-free indicator molecules and the other preferentially exciting the calcium-bound ones). The ratio of the resulting intensities is proportional to the calcium concentration. The ability to do this is what 'ratiometric' means. Careful calibration of such indicators will allow you to determine absolute calcium concentrations.

The second mechanism is found in indicators such as Oregon-Green BAPTA 1, Fluo-3 or XRhod-2. Here, the calcium-free ionophore is capable of quenching the fluorescence of the fluorophore, leading to very low fluorescence. This process is known as photo-induced electron transfer (PET). Once the ionophore binds calcium, it is no longer able to quench the fluorophore, so the fluorescence increases. This process does not change the energy levels of the fluorophore and thus the fluorescence spectra do not change. The result is that these indicators cannot be used rationmetrically. Instead, changes in fluorescence are reported only as changes in fluorescence intensity. These probes are sometimes referred to as intensiometric probes. Because the change in fluorescence is proportional to the change in calcium concentration, it will be impossible to determine absolute calcium concentration using these indicators without additional tricks.

Genetically-encoded indicators are another topic that I won't go into here. If that is relevant, do feel free to ask.

Regarding the situation of having multiple different indicators in the same cell, these indicators will of course be in competition for free calcium ions. It is important to remember that this is the case already, as cells contain many calcium binding proteins (often at high concentrations), that are also competing for free calcium. Any detailed quantification will need to take these effects into consideration. As for physiology the dynamics of calcium are relevant, this will often require a model that takes binding kinetics into consideration, to simulate the changes of calcium load of the different binding molecules. 

I hope this is at least somewhat helpful. I realize that I cut corners to reduce the length. I just hope I didn't cut the wrong ones.

@Christian Wilms

I have written all this down, it was so HELPFUL! Thank you so much. I couldn't find this explicit chemistry the way you described it. If you can talk about genetically-encoded indicators that would be beneficial or if you could provide me a link for it, that would be great as well! 

I have two follow-up questions:

1) Do I directly go on and dye my cells with Ca2+ indicators after growing my cells in the dishes? Is there a middle step there, like calibrating buffers?

2) Don't calcium indicators change the intracellular calcium dynamics in any way, as they are acting as exogenous calcium buffers? If so, then how does one go about it? 

Do a search on the Cameleon dye I mentioned for an example of a "genetically encoded" indicator.

Although, in theory, it is possible to determine the calcium concentration from the ratiometric measurement, one should verify the ratio by a buffer calibration.

Depending on the dye used, you will add the indicator after growing the cells and allow the cells to incorporate the dye. FURA comes as the acetoxymethyl ester which allows the dye to enter the cell membrane. Esterases in the cell cleave the acetoxymethyl ester and allow the dye to bind to calcium.

Here is a link to the original FURA paper which may answer a lot of questions: http://www.jbc.org/content/260/6/3440.long

If you are using cultured cells, the easiest way to load your cells is to use the AM-ester method that Jack mentioned above. There are numerous protocols available on how best to do this in different cell types. Alternatively, if you are using genetically encoded indicators, you would transfect the cells with your construct and wait for a few days for the cells to express the indicator.

Calibrating the indicator. Yes and no. It depends on what you are trying to achieve. If you only want to detect events, then a calibration is probably not necessary. If you are aiming to do quantitative work, then yes, you will need to calibrate the indicator. The values specified by the manufacturer tend to be off and the affinity has been found to vary massively between production batches. As Jack writes, you will need a buffered calibration system to achieve this. Making these solutions yourself requires very careful titration, so I would recommend purchasing them ready made.

One thing to be very aware of is that if you load the cells using an AM-ester, you will also be loading the high [Ca2+] endoplasmatic reticulum. Even if using a ratiometric indicator such as Fura-2, what you will determine is not the absolute calcium concentration of the cytosol, but the average calcium concentration of all indicator-loaded compartments.

And finally, yes, you are correct: the calcium indicator you are using is an exogenous calcium buffer, interfering with exactly the signal you are trying to measure. Depending on what variables you are interested in you can optimize your indicator selection to minimize the perturbation, but it is unavoidable and should be taken into consideration in any quantitative analysis you perform. 

Regarding genetically-encoded calcium indicators, I think the best reference (despite being a few years old now) is:

Genetically Encoded Calcium Indicators

Marco Mank and Oliver Griesbeck

Chem. Rev., 2008, 108 (5), pp 1550–1564

DOI: 10.1021/cr078213v

It discusses the different design strategies in detail and is very good reading.

http://pubs.acs.org/doi/abs/10.1021/cr078213v

@Christian Wilms Thank you.