Fluorescent Calcium Indicators

Calcium acts as a universal second messenger in a variety of cells. Numerous functions of all types of cells are regulated by Ca²⁺, thus calcium measurement is critical for various biological investiga-tions. Since the 1920s, scientists have attempted to measure Ca²⁺, but few were successful due to the limited availability of Ca²⁺ probes. The first reliable measurement of Ca²⁺ was performed by Ridgway and Ashley by injecting the photoprotein aequorin into the giant muscle fiber of the barnacle. Subsequently, in the 1980s, Tsien and colleagues produced a variety of fluorescent indicators. Among them Indo-1, Fura-2, Fluo-3 and Rhod-2 have been the most valuable dyes for measuring Ca²⁺ with a fluorescence instrument. In recent years, AAT Bioquest has introduced the most robust calcium probes: Fluo-8^® and Cal-520™, both of which enable the high throughput screening of GPCR and calcium channel drug discovery targets through the convenient calcium detection. FLIPR^® and FlexStation^® instruments of Molecular Devices, FDSS^®/µCELL of Hamamatsu and NOVOstar of BMG Technologies have further accelerated the high throughput measurement of calcium for GPCR and ion channel research.

Fluorescent probes that show spectral responses upon binding Ca²⁺ have enabled researchers to investigate changes in intracellular free Ca²⁺ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Most of these fluorescent indicators are derivatives of BAPTA chelators that incorporate a PET system responsive to calcium. There are quite a few factors that need be considered when selecting a fluorescent Ca²⁺ indicator. These include:

• Spectral Properties: For UV excitation, Indo-1 and Fura-2 are widely used. Fura-8™ is a newly developed excitation-ratioable calcium dye. Its AM is superior to Fura-2 AM with higher signal/background ratio in cells. Fluo-8^® and Cal-520™ are preferred for 488 nm excitation while Cal-590™, Cal-630™, Rhod-2 and Rhod-4™ are used for red emissions.
• Measurement Mode: Ion indicators that exhibit spectral shifts upon ion binding can be used for ratiometric measurements of Ca²⁺ concentration, which are essentially independent of uneven dye loading, cell thickness, photobleaching effects and dye leakage. Excitation and emission wavelength preferences depend on the type of instrumentation being used, as well as on sample autofluorescence and on the presence of other fluorescent or photoactivatable probes in the experiment. Indo-1, Fura-2 and our newly developed Fura-8™ are primary choices for ratiometric measurements while Fluo-3, Fluo-4, Fluo-8^®, Cal-520™, Cal-590™, Cal-630™, Rhod-2 and Rhod-4™ are predominantly used for single wavelength measurements.
• Permeability of Ca²⁺ Indicators (salt or AM ester): The salt forms are typically loaded into cells by microinjection, microprojectile bombardment or electroporation, or used for extracellular assays. In contrast, the cell-permeant acetoxymethyl (AM) esters can be passively loaded into cells, where they are cleaved to cell-impermeant products by intracellular esterases.
• Dissociation Constant (K_d): The desired indicators must have a proper K_d compatible with the Ca²⁺ concentration range of interest. The K_d values of Ca²⁺ indicators are dependent on many factors, including pH, temperature, ionic strength, viscosity, protein binding, the presence of Mg²⁺ and other ions. Consequently, K_d values for intracellular indicators are usually significantly higher than the corresponding values measured in cell-free solutions.

Among the visible light-excitable calcium indicators, Fluo-8^®, Fluo-4, Fluo-3, Rhod-2 and Rhod-4™ are most commonly used. Fluo-8^® indicators are widely used in flow cytometry and confocal laser-scanning microscopy. More recently, Fluo-8^® AM has been extensively used for high throughput screening GPCR targets. Fluo-8^® is essentially nonfluorescent unless bound to Ca²⁺ and exhibits a quantum yield of ~0.15 in the presence of saturating Ca²⁺ and a K_d of 390 nM for Ca²⁺. Cal-520™ is by far the best 488 nm-excitable green fluorescent calcium indicator with a significantly improved signal/background ratio and intracellular retention.

The long-wavelength Rhod-4™ is a valuable alternative Ca²⁺ indicator to the green fluorescent Fluo-8^®, Fluo-4 and Fluo-3 for experiments in cells and tissues that have high levels of autofluo-rescence. Rhod-5N has a lower binding affinity for Ca²⁺ than any other BAPTA-based indicator (K_d = ~320 µM) and is suitable for Ca²⁺ measurements from 10 µM to 1 mM. Like the parent Rhod-2 indicator, Rhod-5N is essentially nonfluorescent in the absence of divalent cations and exhibits strong fluorescence enhancement with no spectral shift upon binding Ca²⁺. Both Fluo and Rhod indicators are available as cell-impermeant potassium salts or as cell-permeant AM esters.