Calcium imaging is a scientific technique usually carried out in research which is designed to show the calcium

Calcium

Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...

 (Ca²⁺) status of a tissue or medium.
Calcium imaging techniques take advantage of so called calcium indicators, molecules that can respond to the binding of Ca²⁺ ions by changing their spectral properties.
Two main classes of calcium indicators exist: chemical indicators and genetically encoded indicators (GECI).

Chemical indicators

Chemical indicators are small molecules that can chelate calcium ions. All these molecules are based on an EGTA

EGTA (chemical)

EGTA is a polyamino carboxylic acid, a chelating agent that is related to the better known EDTA, but with a much higher affinity for calcium than for magnesium ions...

 homologue called BAPTA

BAPTA

BAPTA is a calcium-specific polyamino carboxylic acid. The presence of four carboxylic acid functional groups makes possible the binding of two magnesium ions. The extensive flexibility of the carboxylate ligands is critical to the coordination of magnesium, or other metal ions...

, with high selectivity for calcium (Ca²⁺) ions versus magnesium

Magnesium

Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...

 (Mg²⁺) ions.

This group of indicators includes fura-2

Fura-2

Fura-2, a polyamino carboxylic acid, is a ratiometric fluorescent dye which binds to free intracellular calcium. It was the first widely-used dye for calcium imaging, and remains very popular. Fura-2 is excited at 340 nm and 380 nm of light, and the ratio of the emissions at those...

, indo-1

Indo-1

Indo-1 is a popular calcium indicator similar to Fura-2. In contrast to Fura-2, Indo-1 has a dual emissions peak. The main emission peak in calcium-free solution is 475 nm while in the presence of calcium the emission is shifted to 400 nm...

, fluo-3, fluo-4

Fluo-4

Fluo-4 is used to measure calcium concentrations inside living cells, and is often used for high-throughput screening of receptor ligands and calcium permeable ion channels....

, 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 esterase

Esterase

An esterase is a hydrolase enzyme that splits esters into an acid and an alcohol in a chemical reaction with water called hydrolysis.A wide range of different esterases exist that differ in their substrate specificity, their protein structure, and their biological function.- EC classification/list...

s 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

Green fluorescent protein

The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...

 (GFP) or its variants (e.g. circularly permuted GFP, YFP, CFP), fused with calmodulin

Calmodulin

Calmodulin is a calcium-binding protein expressed in all eukaryotic cells...

 (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, Cameleon

Cameleon

Cameleon is an engineered protein based on variant of green fluorescent protein used to visualize calcium levels in living cells. It is a genetically encoded calcium sensor created by Roger Y. Tsien and coworkers...

s and GCaMP

GCaMP

GCaMP is a genetically encoded calcium indicator, or GECI. GECIs are designed to show the calcium ion status of a tissue or medium and are either transfected into cell lines or via transgenic crosses...

.

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

Fluorescence

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

 only after binding to calcium, or its excitation

Excited state

Excitation is an elevation in energy level above an arbitrary baseline energy state. In physics there is a specific technical definition for energy level which is often associated with an atom being excited to an excited state....

 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.