Live Cell Calcium Indicators

Fluorescent Dyes for Calcium Imaging Experiments

Calcium ions (Ca²⁺) play vital cellular physiology roles in signal transduction pathways, in neurotransmitter release, in contraction of all muscle cell types, as enzyme cofactors, and in fertilization. Extracellular calcium is also important for maintaining the potential difference across excitable cell membranes, as is well known as a requirement for proper bone formation. Analyzing calcium flux using live cell calcium imaging techniques is important to understanding cellular function and dysfunctions that may be components of disease.

Chemical calcium indicators and sensors are small molecules that bind calcium ions via chelation. Many are based on widely-used calcium chelators like EGTA, (ethylene glycol-bis(β-aminoethyl ether), or on chelators with enhanced calcium ion (Ca²⁺)specificity or pH stability, such as BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). These dyes are often modified with acetoxymethyl esters (AM), in order to render the molecule lipophilic and to allow easy entrance into the cell. Once inside the cell, cellular esterases will free the carboxyl groups,enabling Ca²⁺ by the indicator. Binding of a Ca²⁺ ion to a fluorescent indicator molecule leads either to  an increase in quantum yield of fluorescence or an emission/excitation wavelength shift.

Traditional and Novel Calcium Imaging Probes

Fura-2: A UV light-excitable, ratiometric Ca²⁺ indicator that is a common dye  for ratio-imaging microscopy. Upon binding Ca²⁺, Fura-2 exhibits an absorption shift that can be observed by scanning the excitation spectrum between 300 and 400 nm, while monitoring the emission at ~510 nm.

Indo-1: A UV light-excitable Ca²⁺ indicator that is the preferred calcium sensor for flow cytometry that uses a single laser for excitation. The emission maximum of Indo-1 shifts from ~475 nm in Ca2+-free medium to ~400 nm when the dye is saturated with Ca²⁺.

Quin-2: Displays high selectivity for calcium. This calcium indicator is not affected by sodium gradients, membrane potential range, or intracellular pH, and displays high affinity for Ca2+ ions .Quin-2 is sensitive enough to monitor low levels of calcium, such as those found in resting cells. Excitation and emission maxima are 339/492 nm, respectively.

Fluo-3: A fluorescence indicator of intracellular calcium (Ca²⁺) used in flow cytometry and confocal laser scanning microscopy. Upon binding of Ca²⁺, its fluorescence increases sharply with an emission maximum at 525nm and is suitable for detection using a fluorescein isothiocyanate (FITC) channel.

Near-Infrared Calcium Dyes: BioTracker NIR Ca²⁺ dyes (SCT021, SCT022, SCT023) are far-red fluorescent calcium indicators. These dyes changes fluorescent intensity greatly (1000-fold) when it binds to calcium in living cells. The advantage of these long wavelength dyes is greater tissue penetration, lower phototoxicity along with multicolor imaging using Hoechst, Fluorescein, Rhodamine, GFP, YFP and RFP etc.

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