by Joseph Meacham

With so many possibilities, selecting the appropriate calcium indicator for a given Ca²⁺ flux assay can be difficult and tedious. To streamline the process, here is what we consider to be the eight best single-wavelength, green fluorescent Ca²⁺ indicators available.

Calcium is a ubiquitous ion involved in many physiological processes. For example, calcium can act as a second messenger in physiological pathways triggering the release of neurotransmitters from neurons. It plays an integral part as an enzyme cofactor regulating enzymatic activity and is essential for the contraction of all muscle cells. Because the physiological regulation of Ca²⁺ concentration is necessary to initiate and sustain such vital cellular processes, many qualitative and quantitative tools have been developed for this purpose.

A common signaling pathway regulating cytoplasmic Ca²⁺ concentration is the phospholipase C pathway. This pathway is initiated by activating G protein-coupled receptor (GPCR) targets. To monitor GPCR activity, we recommend robust Ca²⁺ flux assays that employ highly-sensitive indicators capable of detecting Ca²⁺ signals. Detection of intracellular Ca²⁺ is widely used to characterize GPCRs agonists and antagonists in drug discovery and in monitoring synaptic activities in neuroscience research.

Fluo-3 is one of the most commonly utilized green calcium indicators available. It was first developed by Tsien and colleagues in the late 1980s (Grynkiewicz et al. 1985). Since then, Fluo-3 and its analogs have been utilized in many Ca²⁺ imaging applications making considerable contributions to understanding the spatial dynamics of processes associated with Ca²⁺ signaling pathways.

Fluo-3 is a visible light-excitable, single-wavelength Ca²⁺ indicator with spectral characteristics similar to fluorescein. Fluo-3 is well excited by the 488 nm argon laser line and can be visualized using the FITC emission filter sets. It has a maximum absorption and emission wavelength at 506 and 526 nm, respectively. Compared to other Ca²⁺ indicators, Fluo-3 has a relatively lower affinity for Ca²⁺. It exhibits a calcium dissociation constant (K_d) of ~390 nM, which can alleviate drawbacks associated with cytosolic buffering at resting Ca²⁺ levels of ~100 nM. In the Ca²⁺ free form, Fluo-3 is essentially non-fluorescent. Upon binding Ca²⁺, the fluorescence intensity of Fluo-3 increases ~100 fold, and at saturating Ca²⁺ levels, exhibits a quantum yield of ~0.14.

In past comparative studies, cells loaded with Fluo-3 have displayed favorably large dynamic ranges sensitive to detecting elementary and global Ca²⁺ fluxes. However, Fluo-3 requires incubation at 37 °C resulting in significant indicator loss by increased activity of anion transporters sensitive to the same temperature. To improve cellular retention, probenecid, an organic anion transporter inhibitor, is used during cell loading. Unfortunately, probenecid can be toxic to cells, and while it inhibits specific anion transporters, it may activate others. In addition to being sensitive to temperature changes, Fluo-3 is also pH-sensitive and sensitive to protein binding.

Fluo-4 is a visible light-excitable calcium indicator derived from its predecessor, Fluo-3. Like Fluo-3, Fluo-4 is a non-fluorescent indicator whose intensity increases ~100-fold upon binding Ca²⁺. Additionally, Fluo-4 is an analog of Fluo-3 exhibiting minor structural modifications attributing to a brighter and more photostable indicator. By replacing two chlorine substituents with fluorines, Fluo-4 displays a wavelength shift of ~12 nm towards the blue spectrum. This establishes a new maximum absorption wavelength at 494 nm making Fluo-4 more efficiently excitable by the 488 nm argon laser line. This attributes to a brighter fluorescence signal at lower dye concentrations making Fluo-4 less phototoxic than Fluo-3. Fluo-4 has a slightly higher Ca²⁺ binding affinity than Fluo-3, with a K_d value of 345 nM. For these reasons, Fluo-4 is a better alternative for use with confocal microscopy than Fluo-3.

Although Fluo-4 shows a brighter signal and an improved signal-to-background ratio in analyzing Ca²⁺ fluxes of adherent cells, it is still only moderately fluorescent in live samples. Like its predecessor, Fluo-3, Fluo-4 also employs probenecid to improve dye retention, which is known to be toxic to cells.

Fluo-8® is a novel green calcium indicator incorporating the same fluorescein core utilized in Fluo-3 and Fluo-4 to monitor Ca²⁺ concentration and flux in cells. This enables Fluo-8® to retain spectral properties identical to Fluo-4 while improving the limitations plaguing Fluo-3 and Fluo-4. Minor structural modifications attributing to the inception of Fluo-8® have led to several enhancements associated with its employment, one being Fluo-8®'s improved loading conditions. Compared to Fluo-3 and Fluo-4, which require indicator loading at 37 °C, Fluo-8® can be loaded successfully at room temperature in just 20 minutes.

Like the other Fluo-dyes, Fluo-8® is non-fluorescent in the Ca²⁺ free form. Upon binding to Ca²⁺, Fluo-8® displays a more considerable fluorescence intensity increase of ~200 fold. This is significantly two times brighter than Fluo-4 and four times brighter than Fluo-3. Fluo-8® is less temperature dependent than other probes, allowing for more consistent and reproducible results at room temperature or 37 °C. Fluo-8® and its analogs are available in four distinct forms, each expressing different calcium binding affinities. Fluo-8® has a K_d of ~ 389 nM, Fluo-8H™ has a K_d of ~ 232 nM, Fluo-8L™ has a K_d of ~1.86 µM, and Fluo-8FF™ has a K_d of ~10 µM. Fluo-8L™ and Fluo-8FF™ have significantly lower Ca²⁺ binding affinities, making them better suited for detecting intracellular Ca²⁺ levels in the micromolar range.

Cal-520® is a novel fluorogenic calcium indicator with several noteworthy improvements and advantages compared to older generations of Ca²⁺ indicators such as Fluo-3 and Fluo-4. One improvement in Cal-520® is a shift in its optimum absorption and emission wavelengths ~12 nm shorter than that of Fluo-3, resulting in a maximum absorption wavelength of 494 nm. This considerably enhances the excitation efficiency of Cal-520® to 94% of its peak maxima when excited by the 488 nm argon laser line, which is significantly more efficient than Fluo-3's 40%. With a maximum emission at 514 nm, Cal-520® is an excellent indicator for multiplexing applications using an indicator with red fluorescence. Additionally, Cal-520®'s improved excitation efficiency permits its usage at less toxic or lower dye concentrations. This is advantageous for experiments designed to investigate intracellular Ca²⁺ measurements via confocal microscopy by reducing the effects of fluorescence saturation on imaging.

Compared to Fluo-3 and Fluo-4, Cal-520® has a slightly higher affinity for Ca²⁺ with a K_d value of 320 nM. Additionally, Cal-520® is optimized to localize in the cytosol. This reduces indicator compartmentalization in organelles such as the mitochondria and ensures that the signal detected accurately reflects the changes in cytosolic free Ca²⁺. Cal-520® is a robust probe for fluorescence-based assay detection of intracellular calcium mobilization. It is highly sensitive for evaluating GPCR and calcium channel targets and screening their agonist and antagonists.

Cal-520® is available in AM ester, salt, dextran conjugate, and bioconjugate forms to accommodate any experimental design. A significant advantage of using Cal-520® AM is the elimination of probenecid which can be toxic to live cells. Cal-520® bioconjugates, such as Cal-520® biocytin and biotin, can bind to avidin and streptavidin without altering their sensitivity to detect Ca²⁺ responses. Cal-520® dextran conjugates can be microinjected or taken up by cells via endocytosis. Ca1-520 dextran conjugates are available conjugated to either 3,000 or 10,000 molecular weight Dextran molecules depending on the tissue type being investigated.

Calbryte™ 520 is a next-generation Ca²⁺ indicator recently introduced as a superior replacement for traditional green calcium indicators such as Fluo-3 and Fluo-4. With fluorescence excitation and emission maxima of 492 nm and 514 nm, respectively, Calbryte™ 520 is the best indicator on this list suited for efficient excitation at the 488 nm argon laser line. Calbryte™ 520's spectral properties closely match that of Fluo-4, Calcium Green-1, and Oregon Green 488 BAPTA-1, allowing for a seamless transition between Calbryte 520 and any of these three indicators. Calbryte 520 is available in either AM ester form or as a potassium salt derivative. Calbryte 520, AM's cell-permeability, makes it well-suited for assaying Ca²⁺ concentrations in live cells, whereas, Calbryte 520, potassium salt's cell-impermeability, makes it suited for the calibration of Ca²⁺ indicators.

During Calbryte 520's development, improvements were centered on significantly enhancing its signal-to-background ratio. To reduce background interference, Calbryte 520, AM remains inactive and non-fluorescent showing minimal response to trace Ca²⁺ present in extracellular solution. AM esters facilitate Calbryte 520's passive diffusion across the cell membrane, where nonspecific intracellular esterases cleave off the AM ester functional groups activating Calbryte™ 520's fluorescence and responsiveness to Ca²⁺. A significant advantage of Calbryte™ 520's usage is the elimination of probenecid to improve dye retention (other than Cal-520®, all other Ca²⁺ indicators mentioned in this list require probenecid). Activated Calbryte™ 520 exhibits excellent cellular retention and localization within the cytosol. This ensures that the signal detected accurately assesses intracellular Ca²⁺ levels.

Like all calcium indicators, Calbryte™ 520 undergoes an increase in fluorescence intensity upon chelation to free Ca²⁺. The ~300-fold increase in fluorescence intensity exhibited by Calbryte™ 520 is the largest, followed second by Fluo-8®'s ~200-fold increase, and then a ~100-fold increase shown by Fluo-3, Fluo-4, Cal-520®, and Oregon Green 488 BAPTA-2. Calbryte™ 520's quantum yield is three times greater than that of Fluo-3 or Fluo-4, and it exhibits a K_d value of 1200 nM. Such characteristics make Calbryte™ 520 a highly sensitive indicator for calcium flux assays and high-throughput screening of GPCR agonists and antagonists. Calbryte™ 520 is optimized for use with confocal laser microscopy, fluorescence microplate readers, and flow cytometry.

Calcium Green – 1 is a visible light-excitable indicator derived from the fluorescent compound fluorescein. Like its predecessors, Fluo-3 and Fluo-4, Calcium Green-1 increases its fluorescence emission intensity upon binding to free Ca²⁺. With a maximum absorption and emission wavelength of 506 nm and 531 nm, respectively, Calcium Green-1 is suitable for excitation by the argon-laser line and visualized using a FITC emission filter set.

Compared to Fluo-3 and Fluo-4's ~100-fold intensity increase, Calcium Green-1's is substantially lower at ~14-fold. However, it boasts a stronger affinity for Ca²⁺, exhibiting a K_d value of 190 nM and a significantly larger quantum yield of 0.75 at saturating Ca²⁺ concentrations. At lower Ca²⁺ concentrations, Calcium Green-1 is more fluorescent than Fluo-3, which improves the visibility of resting cells, making it easier to establish baseline fluorescence and Ca²⁺ levels. Such characteristics also reduce Calcium Green-1's phototoxic effects. Because Calcium Green-1 is intrinsically more fluorescent, it requires lower illumination intensities and dye concentrations than Fluo-3. Unfortunately, like Fluo-3 and Fluo-4, Calcium Green-1 requires probenecid for improved cellular retention, which can be toxic to cells.

Calcium Green-1 is available as a cell-permeable AM ester or as cell-impermeable dextran conjugates and salt derivatives suitable for various calcium signaling investigations, including Ca²⁺ flux assays and multiphoton excitation imaging of Ca²⁺ in living tissues. Calcium Green-1 is adaptable to various fluorescence platforms such as fluorescence microscopy and flow cytometry.  

Calcium Green-5N is a relatively low-affinity Ca²⁺ indicator with spectrally identical properties to Calcium Green-1. Of all the Ca²⁺ indicators mentioned in this list, Calcium Green-5N has a significantly weaker affinity for Ca²⁺ with a larger dissociation constant at ~14 µM. Compared to indicators with K_d values < 1 µM, Calcium Green-5N's larger K_d makes it more suitable for tracking the kinetics of rapid calcium dynamics. In past studies, Calcium Green-5N has been shown to be an appropriate method for measuring Ca²⁺ concentrations up to at least 50 µM. When employed in conjunction with high-affinity Ca²⁺ indicators, Calcium Green-5N can provide an indication of the absolute magnitude of Ca²⁺ spikes inside cells.

Upon binding to free Ca²⁺, Calcium Green-5N experiences an increase in fluorescence intensity approximately ~ 38 fold which is twice that of Calcium Green-1's ~ 14-fold increase. However, even with a better emission intensity increase, Calcium Green-5N exhibits relatively little fluorescence except in cells experiencing a high-amplitude Ca²⁺ flux. Because of such characteristics and its low affinity for Ca²⁺, Calcium Green-5N may prove insensitive to detecting modest or transient Ca²⁺ changes.

Oregon Green 488 BAPTA-1 (OG488 BAPTA-1) is a group of bright fluorogenic calcium indicators with similar spectral characteristics to Calcium Green-1. The significant distinction between the two is OG488 BAPTA-1's shift in fluorescence absorption and excitation maxima ~10 nm shorter, resulting in a maximum absorption wavelength of 494 nm. This significantly improves OG488 BAPTA-1 excitation efficiency at the 488 nm argon laser line. In Ca²⁺ free solutions, OG488 BAPTA-1 is moderately fluorescent. Upon binding to free Ca²⁺, OG488 BAPTA-1 experiences a fluorescence emission intensity increase of ~14-fold. Like Calcium Green-1, OG488 BAPTA-1 possesses a quantum yield of approximately 0.7 while maintaining a small dissociation constant of ~140 nM. Such spectral properties of OG488 BAPTA-1 permit its use at lower dye concentrations, making it appropriate for investigating intracellular Ca²⁺ concentrations using a confocal laser-scanning microscope. In addition, studies have been known to implement a dual calcium indicator system utilizing OG488 BAPTA-1. For example, combinations of OG488 BAPTA-1 and Fluo-4 have been used to investigate responses that combine a finite basal signal level with a sizeable stimulus-dependent increase.