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 calcium indicators (GECI). Calcium imaging can be used to optically probe intracellular calcium in living animals. This technique has allowed studies of Ca2+ signalling in a wide variety of cell types and neuronal activity in hundreds of neurons and glial cells within neuronal circuits .
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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 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 ubiqutous technique used on whole, isolated hearts such as mouse, rat, and rabbit species.
Genetically encoded calcium 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. Alternatively, variants of GFP are fused with the calcium binding protein troponin C (TnC), applying the mechanism of FRET (Förster Resonance Energy Transfer) for signal modulation.
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, GCaMP, TN-XXL and Twitch.
Usage
Regardless of the type of indicator used the imaging procedure is generally very similar. Cells loaded with an indicator or expressing it in the case of GECI, can be viewed using a fluorescence microscope and captured by a CCD camera. Confocal and two-photon microscopes provide improved sectioning ability so that calcium signals can be resolved in micro domains or (for example) synaptic boutons. Images are analyzed by measuring fluorescence intensity changes for a single wavelength or two wavelengths expressed as a ratio (ratiometric indicators). The derived fluorescence intensities and ratios are plotted against calibrated values for known Ca2+ levels to learn Ca2+ concentration.