Ratiometric calcium indicators are designed to monitor calcium mobilization with greater accuracy and sensitivity. When bound to free Ca2+, the peak wavelength of either the excitation curve or emission curve of the ratiometric indicator will undergo a shift. This allows for very precise measurements of Ca2+ concentrations by correcting for uneven dye loading, dye leakage, photobleaching and varying cell thicknesses.

Since their introduction in 1985 by Tsien and collaborators, ratiometric indicators have been cited in countless scientific papers and have made considerable contributions to understanding the role of calcium in cellular regulation (Grynkiewicz et al. 1985). Ratiometric calcium indicators have been successfully used to monitor calcium mobilization, image the spatial dynamics of calcium signaling, and in HTS cell-based pharmacological screening of agonist-stimulated and antagonist-inhibited signaling through GPCRs. Among ratiometric calcium indicators, Fura-2 and Indo-1 are the most widely used.

Fura-2 and its derivatives are UV-light excitable ratiometric indicators for measuring intracellular Ca2+ concentrations. It was first introduced in 1985, and since then has become the preferred indicator for ratio-imaging microscopy. When free of Ca2+, Fura-2 exhibits a peak excitation and emission maxima of ~363 nm and ~510 nm, respectively. Upon binding Ca2+, the peak excitation wavelength of Fura-2 undergoes an absorption shift towards the blue spectrum that is detectable at ~335 nm. Fura-2 and its derivatives have a wide range of Ca2+ binding affinities ranging from ~145 nM to 5.5 µM. For studies requiring an excitation-ratioable indicator capable of detecting higher levels of calcium, use the low affinity indicator BTC (K_d 7 µM).

Fura-2 is also a proven tool for drug discovery and high-throughput screening. AAT Bioquest^® Screen Quest™ Fura-2 No Wash Calcium Assays offers a convenient no-wash, homogeneous fluorescence-based assay for monitoring GPCRs and calcium channels, and for detecting intracellular calcium mobilization across a broad spectrum of biological targets. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and can be easily adapted for automation. Screen Quest™ Calcium Assays are optimized for HTS screening and have been validated for the following instruments: FDSS^®, FLIPR^®, ViewLux™, NOVOStar, ArrayScan™, FlexStation^® and IN Cell Analyzer.

Fura-8™ and Fura-10™ AM are novel ratiometric calcium indicators designed to address the low sensitivity and poor cellular retention that is associated with Fura-2. Both Fura-8™ and Fura-10™ generate significantly brighter signals than Fura-2 while maintaining a similar affinity range for Ca2+ (K_d ~260 nm to ~6 µM). Their red-shifted dual-excitation and emission wavelengths help improve assay sensitivity by minimizing background interference caused by auto-fluorescing materials, and also improves compatibility with common filter sets. Fura-10™ has been modified for optimal cellular retention. This reduction in dye leakage enables Fura-10™ to generate notably higher signal-to-background ratios than Fura-2 and Fura-8™.

Fura Red is a visible light-excitable analog of Fura-2 that can be used to ratiometrically measure Ca2+ in single cells. It's visible-light excitation (~488 nm) and long-wavelength emission maxima (~639 nm) minimizes background interference and enables multicolor analysis with green fluorescent probes. Chelation of Ca2+ by Fura Red results in a decrease of the fluorescence emission when excited at 488 nm. Fura Red is available as a membrane-permeant AM ester or a membrane-impermeant salt derivative.

Cal Red™ R525/650 is a novel ratiometric Ca2+ indicator with similar spectral properties to those of Fura Red. In response to Ca2+, Cal Red™ R525/650 generates a brighter, more photostable fluorescence yielding a S/N ratio ~5-fold greater than Fura Red. Cal red™ R525/650 is ideal for Ca2+ imaging and other Ca2+ flux assays requiring increased sensitivity and visible-light excitability.

In contrast to Fura-2 and its derivatives, which exhibits a shift in absorption upon Ca2+ binding, Indo-1 and its analogs are emission-ratioable indicators for measuring intracellular Ca2+ concentrations. When saturated with Ca2+, the emission maximum of Indo-1 shifts from ~475 nm (Ca2+-free medium) to ~400 nm (see Figure 20). Indo-1 is ideal for multicolor fluorescence applications and is the preferred ratiometric indicator for flow cytometry, where it is more practical to use a single laser for excitation (e.g. the 351-364 nm spectral lines) and then adjust the emission filters, accordingly. Indo-1 is available as a membrane-permeant AM ester or as a membrane-impermeant salt derivative.

Cells loaded with ratiometric calcium indicators have displayed favorably large dynamic ranges sensitive at detecting elementary and global Ca²⁺ fluxes. However, many calcium indicators are susceptible to dye extrusion by organic anion transporters sensitive to 37°C. To improve cellular retention reagents such as probenecid can be added to the dye working solution. Probenecid works be inhibiting organic anion transporters located in the cell membranes. Unfortunately, probenecid can be toxic to cells and while it inhibits certain anion transporters it may activate others, therefor it should be used with discretion. To improve the aqueous solubility of AM esters, also add Pluronic^® F-127 to the dye working solution. Pluronic^® F-127 is a nonionic surfactant that is 100% active and relatively non-toxic to cells. At low temperatures it is soluble, however at room temperature Pluronic^® F-127 will gel.