Calcium imaging

Calcium imaging is a scientific technique usually carried out in research which is designed to show the calcium (Ca2+) status of a cell, tissue or medium. Calcium imaging techniques take advantage of so called calcium indicators, fluorescent molecules that can respond to the binding of Ca2+ ions by changing their fluorescence properties. Two main classes of calcium indicators exist: chemical indicators and genetically encoded indicators (GECI). Calcium imaging can be used to optically probe intracellular calcium in living animals. This technique has allowed studies of neuronal activity in hundreds of neurons and glial cells within neuronal circuits.

Chemical indicators
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 generally 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 the indicator is in the cell, cellular esterases will free the carboxyl and the indicator will be able to bind calcium.

Genetically encoded indicator
These indicators are fluorescent proteins derived from green fluorescent protein (GFP) or its variants (e.g. circularly permuted GFP, YFP, CFP), fused with calmodulin (CaM) and the M13 domain of the myosin light chain kinase, which is able to bind CaM.

Genetically encoded indicators do not need to be loaded onto cells, instead the genes encoding for these proteins can be easily transfected to cell lines. It is also possible to create transgenic animals expressing the dye in all cells or selectively in certain cellular subtypes.

Examples of genetically encoded indicators are Pericams, Cameleons and GCaMP.

Usage
Regardless of the type of indicator used the imaging procedure is generally very similar. The spectral properties of the dye change upon binding to calcium. For example the dye could be fluorescent only after binding to calcium, or its excitation and emission spectra could be modified by the binding. Cells loaded with the indicator or expressing it in case of GECI, can be viewed using a fluorescence microscope, captured by a CCD camera and analyzed according to intensity, ultimately reflecting the Ca2+ status.