Calcium imaging is a microscopy technique to optically measure the calcium (Ca²⁺) status of an isolated cell, tissue or medium. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca²⁺ ions by fluorescence properties. Two main classes of calcium indicators exist: chemical indicators and genetically encoded calcium indicators (GECI). This technique has allowed studies of calcium signalling in a wide variety of cell types, and can be used to measure electrical activity in hundreds of neurons in cell culture, or in living animals during ongoing behavior.

Chemical indicators are small molecules that can chelate calcium ions. All these molecules are based on an EGTA homologue called BAPTA, with high selectivity for calcium (Ca²⁺) ions versus magnesium (Mg²⁺) ions.

This group of indicators includes fura-2, indo-1, fluo-3, fluo-4, Calcium Green-1.

These dyes are often used with the chelator carboxyl groups masked as acetoxymethyl esters, in order to render the molecule lipophilic and to allow easy entrance into the cell. Once this form of the indicator is in the cell, cellular esterases will free the carboxyl groups and the indicator will be able to bind calcium. The free acid form of the dyes (i.e. without the acetoxymethyl ester modification) can also be directly injected into cells via a microelectrode or micropipette which removes uncertainties as to the cellular compartment holding the dye (the acetoxymethyl ester can also enter the endoplasmic reticulum and mitochondria). Binding of a Ca²⁺ ion to a fluorescent indicator molecule leads to either an increase in quantum yield of fluorescence or emission/excitation wavelength shift. Individual chemical Ca²⁺ fluorescent indicators are utilized for cytosolic calcium measurements in a wide variety of cellular preparations. The first real time (video rate) Ca²⁺ imaging was carried out in 1986 in cardiac cells using intensified video cameras. Later development of the technique using laser scanning confocal microscopes revealed sub-cellular Ca²⁺ signals in the form of Ca²⁺ sparks and Ca²⁺ blips. Relative responses from a combination of chemical Ca²⁺ fluorescent indicators were also used to quantify calcium transients in intracellular organelles such as mitochondria.

Calcium imaging, also referred to as calcium mapping, is also used to perform research on myocardial tissue. Calcium mapping is a ubiquitous technique used on whole, isolated hearts such as mouse, rat, and rabbit species.

Genetically encoded calcium indicators (GECIs) are powerful tools useful for in vivo imaging of cellular, developmental, and physiological processes. GECIs do not need to be acutely loaded into cells; instead the genes encoding for these proteins can be introduced into individual cells or cell lines by various transfection methods. It is also possible to create transgenic animals expressing the indicator in all cells or selectively in certain cellular subtypes. GECIs are used to study neurons, T-cells, cardiomyocytes, and other cell types. Some GECIs report calcium by direct emission of photons (luminescence), but most rely on fluorescent proteins as reporters, including the green fluorescent protein GFP and its variants (eGFP, YFP, CFP).

Of the fluorescent reporters, calcium indicator systems can be classified into single fluorescent protein (FP) systems, and paired fluorescent protein systems. Camgaroos were one of the first developed variants involving a single protein system. Camgaroos take advantage of calmodulin (CaM), a calcium binding protein. In these structures, CaM is inserted in the middle of yellow fluorescent protein (YFP) at Y145. Previous mutagenesis studies revealed that mutations at this position conferred pH stability while maintaining fluorescent properties, making Y145 an insertion point of interest. Additionally, the N and C termini of YFP are linked by a peptide linker (GGTGGS). When CaM binds to Ca2+, the effective pKa is lowered, allowing for chromophore deprotonation. This results in increased fluorescence upon calcium binding in an intensiometric fashion. Such detection is in contrast with ratiometric systems, in which there is a change in the absorbance/emission spectra as a result of Ca2+ binding. A later developed single-FP system, dubbed G-CaMP, also invokes circularly permuted GFP. One of the termini is fused with CaM, and the other termini is fused with M13 (the calmodulin binding domain of myosin light kinase) The protein is designed such that the termini are close in space, allowing for Ca2+ binding to cause conformational changes and chromophore modulation, allowing for increased fluorescence. G-CaMP and its refined variants have nanomolar binding affinities. A final single protein variant is the CatchER, which is generally considered to be a lower affinity indicator. Its calcium binding pocket is quite negative; binding of the cation helps to shield the large concentration of negative charge and allows for recovered fluorescence.

In contrast to these systems are paired fluorescent protein systems, which include the prototypical Cameleons. Cameleons consist of two different fluorescent proteins, CaM, M13, and a glycylglycine linker. In the absence of Ca2+, only the donor blue-shifted fluorescent protein will be fluorescent. However, a conformational change caused by calcium binding repositions the red-shifted fluorescent protein, allowing for FRET (Förster resonance energy transfer) to take place. Cameleon indicators produce a ratiometric signal (i.e. the measured FRET efficiency depends on the calcium concentration). Original variants of cameleons were originally more sensitive to Ca2+ and were acid quenched. Such shortcomings were abrogated by Q69K and V68L mutations. Both of these residues were close to the buried anionic chromophore and these mutations probably hinder protonation, conferring greater pH resistance.