Fura-2 and Indo-1
Fura-2 and indo-1 are UV light–excitable, ratiometric Ca2+ indicators. Fura-2 has become the dye of choice for ratio-imaging microscopy (), in which it is more practical to change excitation wavelengths than emission wavelengths. 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 (Figure 19.2.1). In contrast, indo-1 is a preferred dye for flow cytometry, where it is more practical to use a single laser for excitation—usually the 351–364 nm spectral lines of the argon-ion laser—and monitor two emissions. The emission maximum of indo-1 shifts from ~475 nm in Ca2+-free medium to ~400 nm when the dye is saturated with Ca2+ (Figure 19.2.2).
Modern two-photon excitation imaging techniques used with fura-2 and indo-1 avoid the deleterious effects of conventional ultraviolet illumination on live specimens. Indo-1 may be less subject to compartmentalization than fura-2, whereas fura-2 is more resistant to photobleaching than indo-1. Both fura-2 and indo-1 exhibit Kd values that are close to typical basal Ca2+ levels in mammalian cells (~100 nM) and display high selectivity for Ca2+ binding relative to Mg2+. Nevertheless, Ca2+ binding is discernibly perturbed by physiological levels of Mg2+; the Kd for Ca2+ of fura-2 is ~135 nM in Mg2+-free Ca2+ buffers and ~224 nM in the presence of 1 mM Mg2+ (measured at 37°C in 100mM KCl, 10 mM MOPS, pH 7.0). Fura-2 and indo-1 also exhibit high affinities for other divalent cations such as Zn2+ and Mn2+, a property that is discussed further in Fluorescent Indicators for Zn2+ and Other Metal Ions—Section 19.7.
The sodium and potassium salts of fura-2 (F6799, F1200; Figure 19.2.3; ) and potassium salt of indo-1 (I1202, ) are cell-impermeant probes that can be delivered into cells by microinjection or using our Influx pinocytic cell-loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8). Free acids of fura-2 and indo-1 can also be loaded into some plant cells at pH 4–5. In addition, these salts are useful as standards for calibrating Ca2+ measurements.
Unlike the salt forms, the acetoxymethyl (AM) esters of fura-2 () and indo-1 () can passively diffuse across cell membranes, enabling researchers to avoid the use of invasive loading techniques. Once inside the cell, these esters are cleaved by intracellular esterases to yield cell-impermeant fluorescent indicators (Loading and Calibration of Intracellular Ion Indicators—Note 19.1). We offer fura-2 AM and indo-1 AM in 1 mg vials (F1201, I1203) or specially packaged in 20 vials of 50 µg each (F1221, I1223); the special packaging is recommended when small quantities of the dyes are to be used over a long period of time. We also provide stock solutions of fura-2 AM and indo-1 AM in anhydrous DMSO at 1 mg/mL (~1 mM; F1225, I1226). Our standard analytical specifications for fura-2 AM require ≥95% purity by HPLC. We also offer a special packaged high-purity grade of fura-2 AM that is specified to have ≥98% purity by HPLC (as a set of 20 vials, each containing 50 µg; F14185). The 10,000 MW dextran conjugate of fura is described in Fluorescent Ca2+ Indicator Conjugates—Section 19.4.
Figure 19.2.1 Fluorescence excitation spectra of fura-2 (F1200, F6799) in solutions containing 0–39.8 µM free Ca2+.
Figure 19.2.2 Fluorescence emission spectra of indo-1 (I1202) in solutions containing 0–39.8 µM free Ca2+.
Figure 19.2.3 Fura indicators with varying Ca2+ affinities.
Fura-2 Calcium Imaging Calibration Kit
The Fura-2 Calcium Imaging Calibration Kit (F6774) is designed to facilitate rapid calibration and standardization of digital imaging microscopes. This kit provides 11 CaEGTA:EGTA buffer solutions with free Ca2+ concentrations from zero (10 mM EGTA) to 39 µM. Each solution also includes 50 µM fura-2, as well as 15 µm unstained polystyrene microspheres to act both as spacers that ensure uniform separation between the slide and the coverslip and as focusing aids. We also provide a twelfth buffer—identical to the 10 mM CaEGTA standard but lacking fura-2—that serves as a control for background fluorescence. Our Calcium Calibration Kits are described further in Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8.
Bis-Fura-2: Brighter Signal with Lower Affinity for Ca2+
By linking two fura fluorophores with one BAPTA chelator (), we have produced bis-fura-2, a Ca2+ indicator that exhibits approximately twice the absorptivity of fura-2. Bis-fura-2 has a Kd for Ca2+ of ~370 nM and ~525 nM in the absence and presence of 1 mM Mg2+, respectively (measured at ~22°C using our Calcium Calibration Buffer Kits). In other aspects, the quantum yield of bis-fura-2 and its spectral response to Ca2+ (Figure 19.2.4) are virtually identical to those of fura-2. Although the difference between the Kd of fura-2 and bis-fura-2 for Ca2+ is small, the change in excitation ratio for bis-fura-2 in response to Ca2+ concentrations >500 nM is larger than that of fura-2 (Figure 19.2.1); this difference can improve the dynamic range for Ca2+ measurements in cells. Other potential advantages of bis-fura-2 include:
The hexapotassium salt of bis-fura-2 (B6810) is available for loading by microinjection or by infusion from a patch pipette. We do not currently offer a membrane-permeant AM ester of bis-fura-2.
Figure 19.2.4 Fluorescence excitation spectra of bis-fura-2 (B6810) in solutions containing 0–39.8 µM free Ca2+.
Quin-2 belongs to the first generation of Ca2+ indicators developed by Tsien (). Quin-2 has lower absorptivity and quantum yield values than the fura-2, indo-1, fluo-3, fluo-4 and Calcium Green indicators and thus requires higher loading concentrations. The resulting high intracellular concentration of the indicator may buffer intracellular Ca2+ transients. Quin-2 AM has been used to intentionally deplete cytosolic free Ca2+ and to ensure unidirectional Ca2+ influx. Measurement of cytosolic free Ca2+ with quin-2 has been thoroughly reviewed by Tsien and Pozzan. We offer quin-2 as a high-purity, cell-impermeant free acid (Q23918).
Fura-4F and Fura-6F
Calcium concentrations above 1 µM produce almost complete binding saturation of fura-2 but very low fractional saturation of the low-affinity fura analog mag-fura-2 (M1290, see below). To bridge this gap in the Ca2+ measurement range of fura-type indicators, we currently offer two additional ratiometric Ca2+ indicators—fura-4F (F14174) and fura-6F (F14178)—as well as the membrane-permeant fura-4F AM (F14175); fura-5F may be available upon request through Invitrogen Custom Services. Attachment of a single electron-withdrawing fluorine substituent at different positions on the BAPTA chelator moiety of fura-2 (Figure 19.2.3) results in an increase of the Kd value to ~770 nM, ~400 nM and 5.3 µM for fura-4F, fura-5F and fura-6F, respectively (measured at 22°C at 100 mM KCl, 10 mM MOPS, pH 7.2). Except for the change in the Ca2+ concentration response range (Figure 19.2.5), the Ca2+-dependent spectral shifts produced by fura-4F, fura-5F and fura-6F are essentially identical to those of fura-2 (Figure 19.2.6) and the probes use the same optical filter sets.
Figure 19.2.5 Fluorescence excitation ratio versus Ca2+ concentration curves for fura-2 (red), fura-5F (orange), fura-4F (green) and fura-6F (blue).
Figure 19.2.6 Ca2+-dependent fluorescence excitation spectra of fura-4F (F14174).
Fura-FF is a difluorinated derivative of fura-2 (Figure 19.2.3) with a Kd value of ~5.5 µM (measured at 22°C in 100 mM KCl, 10 mM MOPS, pH 7.2) and similar spectroscopic properties (Figure 19.2.7). Fura-FF has negligible Mg2+ sensitivity, making Ca2+ detection less susceptible to interference than with mag-fura-2. These properties have made fura-FF particularly useful for spatial and functional characterization of intracellular Ca2+ stores and for tracking Ca2+ oscillations driven by the inositol 1,4,5-triphosphate receptor. The low-affinity indicator fura-FF detected NMDA- and kainate-induced neuronal Ca2+ fluxes that were not detectable with the higher-affinity indicator fura-2. Fura-FF has also been used in combination with FluoZin-3 (Fluorescent Indicators for Zn2+ and Other Metal Ions—Section 19.7) for simultaneous detection of Ca2+ and Zn2+. Fura-FF is available in water-soluble potassium salt form (F14180) and as a membrane-permeant AM ester derivative (F14181).
Figure 19.2.7 Ca2+-dependent fluorescence excitation spectra of fura-FF (F14180).
The coumarin benzothiazole–based Ca2+ indicator BTC (B6790, ) and its cell-permeant derivative BTC AM (B6791) were developed in collaboration with Haralambos Katerinopoulos of the University of Crete. This Ca2+ indicator exhibits a shift in excitation maximum from about 480 nm to 400 nm upon binding Ca2+ (Figure 19.2.8), permitting ratiometric measurements that are essentially independent of uneven dye loading, cell thickness, photobleaching and dye leakage. Its high selectivity and moderate affinity for Ca2+ (Kd ~7 µM) allows accurate quantitation of high intracellular Ca2+ levels that are underestimated by fura-2 measurements. When loaded into neurons as its AM ester, BTC exhibits little compartmentalization; however, prolonged excitation appears to cause conversion of the indicator to a calcium-insensitive form.
BTC has been employed in investigations of Ca2+-dependent exocytosis in pancreatic β-cells, CHO fibroblasts and phaeochromocytoma cells. Neuronal Ca2+ transients detected by the low-affinity Ca2+ indicators BTC and mag-fura-2 are significantly more rapid than those reported by the higher-affinity indicators fura-2 and Calcium Green-2.
Figure 19.2.8 Fluorescence excitation spectra of BTC (B6790) in solutions containing 0–100 µM free Ca2+.
Mag-Fura-2 and Mag-Indo-1
Mag-fura-2 (also called furaptra, ) and mag-indo-1 were originally designed to report intracellular Mg2+ levels (Fluorescent Mg2+ Indicators—Section 19.6); however, these indicators actually have much higher affinity for Ca2+ than for Mg2+. Although Ca2+ binding by these indicators may complicate analysis when they are employed to measure intracellular Mg2+, their increased effective range and improved linearity for Ca2+ measurements has been exploited for measuring intracellular Ca2+ levels between 1 µM and 100 µM.
The spectral shifts of mag-fura-2 and mag-indo-1 are very similar to those of fura-2 and indo-1 but occur at higher Ca2+ concentrations. Because the off-rates for Ca2+ binding of these indicators are much faster than those of fura-2 and indo-1, these dyes have been used to monitor action potentials in skeletal muscle and nerve terminals with little or no kinetic delay (Figure 19.2.9). The spectral properties, kinetics and selectivity of several of our low-affinity Ca2+ indicators have been reviewed by Zhao, Hyrc and their co-workers.
The moderate Ca2+ affinity of mag-fura-2 and the tendency of its AM ester to accumulate in subcellular compartments have proven useful for in situ monitoring of inositol 1,4,5-triphosphate–sensitive Ca2+ stores. Mag-fura-2 has also been employed to follow Ca2+ transients in presynaptic nerve terminals, gastric epithelial cells and cultured myocytes. Mag-indo-1 has been used to detect gonadotropin-releasing hormone–induced Ca2+ oscillations in gonadotropes and to investigate the role of Ca2+/K+ exchange in intracellular Ca2+ storage and release processes. Mag-fura-2 (M1290, M1291, M1292) and mag-indo-1 (M1295) are available as cell-impermeant potassium salts or as cell-permeant AM esters.
Figure 19.2.9 Ca2+ transients evoked by trains of 1–4 action potentials in rat cerebellar granule cells detected by fura-2 (upper panel, F1200) and mag-fura-2 (lower panel, M1290). The stimulus pulses are 50 milliseconds apart (20 Hz); timing is indicated by the double-headed arrows. The amplitude of the transients detected by fura-2 decreases with each successive stimulus due to Ca2+ saturation. Mag-fura-2 avoids saturation due to its lower Ca2+ binding affinity (Kd for Ca2+ = 25 µM), recording transients of approximately equal amplitude from successive action potentials. Adapted with permission from Biophys J (1995) 68:2165.
|Low Ca2+||High Ca2+|
|B6790||844.03||F,D,L||pH >6||464||29,000||533||H2O||401||20,000||529||H2O/Ca2+||7.0 µM||1, 2, 3|
|B6810||1007.14||F,D,L||pH >6||366||56,000||511||H2O||338||68,000||504||H2O/Ca2+||370 nM||1, 2, 4, 5|
|F1200||832.00||F,D,L||pH >6||363||28,000||512||H2O||335||34,000||505||H2O/Ca2+||145 nM||1, 2, 4, 5|
|F6799||751.45||F,D,L||pH >6||363||28,000||512||H2O||335||34,000||505||H2O/Ca2+||145 nM||1, 2, 4, 5|
|F14174||835.96||F,D,L||pH >6||366||21,000||511||H2O||336||23,000||505||H2O/Ca2+||770 nM||1, 2, 4|
|F14178||835.96||F,D,L||pH >6||364||25,000||512||H2O||336||28,000||505||H2O/Ca2+||5.3 µM||1, 2, 3|
|F14180||853.95||F,D,L||pH >6||364||25,000||510||H2O||335||28,000||506||H2O/Ca2+||5.5 µM||1, 2, 3|
|I1202||840.06||F,D,L||pH >6||346||33,000||475||H2O||330||33,000||401||H2O/Ca2+||230 nM||1, 2, 4, 5|
|M1290||586.68||F,D,L||pH >6||369||22,000||511||H2O||329||26,000||508||H2O/Ca2+||25 µM||1, 2, 3|
|mag-indo-1||594.74||F,D,L||pH >6||349||38,000||480||H2O||328||35,000||390||H2O/Ca2+||35 µM||1, 2, 8, 9|
|Q23918||541.51||D,L||pH >6||353||4000||495||H2O||333||3900||495||H2O/Ca2+||60 nM||1, 2, 10|