Molecular Probes

Section 19.2 — Fluorescent Ca²⁺ Indicators Excited with UV Light

Fura-2, Indo-1 and Related Derivatives

Fura-2 and Indo-1

Fura-2 and indo-1 (Fura and Indo Ratiometric Calcium Indicators) are UV light–excitable, ratiometric Ca²⁺ indicators that are generally considered to be interchangeable in most experiments. 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 19.3). 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 19.4). Modern two-photon excitation imaging techniques used with fura-2 and indo-1 avoid the deleterious effects of conventional ultraviolet illumination on living 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 K_d 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 K_d for Ca²⁺ of fura-2 is ~135 nM in Mg²⁺-free Ca²⁺ buffers and ~224 nM in the presence of 1 mM Mg²⁺. 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 Section 19.7.

The sodium and potassium salts of fura-2 (F6799, F1200; Figure 19.5; ) 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, 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 (Acetoxymethyl (AM) and Acetate Esters). Once inside the cell, these esters are cleaved by intracellular esterases to yield cell-impermeant fluorescent indicators (Technical Focus: Loading and Calibration of Intracellular Ion Indicators). Molecular Probes offers 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; FluoroPure Grade, F14185). The 10,000 MW dextran conjugate of fura is described in Section 19.4.

Fura-2 Calcium Imaging Calibration Kit

The Fura-2 Calcium Imaging Calibration Kit (F6774, Calcium Calibration Buffer Kits), which is designed to facilitate rapid calibration and standardization of digital imaging microscopes, contains the same 11 prediluted buffers as our Calcium Calibration Buffer Kit #2 (C3009, Section 19.8). However, in this kit the buffers also include 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. All of our Calcium Calibration Kits are described further in Section 19.8.

A product information sheet (Fura and Indo Ratiometric Calcium Indicators) and extensive bibliographies are available for fura-2 and indo-1 (Bibliography for F1200, Bibliography for F1201, Bibliography for I1202, Bibliography for I1203). Our bibliographic database currently contains over 4000 publications that cite the use of fura-2 AM for measuring intracellular free Ca²⁺ and over 1000 publications for indo-1 AM. For more information, contact our Technical Assistance Department.

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 K_d for Ca²⁺ of ~370 nM and ~525 nM in the absence and presence of 1 mM Mg²⁺, respectively. In other aspects, the quantum yield of bis-fura-2 and its spectral response to Ca²⁺ (Figure 19.11) are virtually identical to those of fura-2. Although the difference between the K_d 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 19.3); this difference can improve the dynamic range for Ca²⁺ measurements in cells. Other potential advantages of bis-fura-2 include:

• Higher fluorescence output per indicator, which may allow the use of lower dye concentrations
• Lower affinity for Ca²⁺, which should decrease the buffering of intracellular Ca²⁺ and produce a faster response to Ca²⁺ spikes
• An additional negative charge, which may facilitate dye retention

The hexapotassium salt of bis-fura-2 (B6810) is available for loading by microinjection, by diffusion from a patch-pipette or using our Influx pinocytic cell-loading reagent (I14402, Section 19.8); we do not currently offer a membrane-permeant AM ester of bis-fura-2.

Quin-2 and Quin-2 AM

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. Molecular Probes offers quin-2 as a high-purity, cell-impermeant free acid (Q23918).

Indicators with Intermediate Calcium-Binding Affinity

Fura-4F, Fura-5F 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 offer three additional ratiometric Ca²⁺ indicators — fura-4F (F14174, F14175), fura-5F (F14177) and fura-6F (F14178) — and their corresponding membrane-permeant AM ester derivatives. Attachment of a single electron-withdrawing fluorine substituent at different positions on the BAPTA chelator moiety of fura-2 results in an increase of the K_d value to ~770 nM, ~400 nM and 5.3 µM for fura-4F, fura-5F and fura-6F, respectively (Figure 19.5). Except for the change in the Ca²⁺ concentration response range (Figure 19.13), the Ca²⁺-dependent spectral shifts produced by fura-4F, fura-5F and fura-6F are essentially identical to those of fura-2 (Figure 19.14) and the probes use the same optical filter sets (Table 23.11).

Fura-FF

Fura-FF is a difluorinated derivative of fura-2 (Figure 19.5, Figure 19.15) with a K_d value of ~5.5 µM. Fura-FF has high selectivity for Ca²⁺, a wide dynamic range and low pH sensitivity, making it an optimal low-affinity Ca²⁺ indicator for most imaging applications. Although its spectroscopic characteristics are very similar to those of mag-fura-2, fura-FF has negligible Mg²⁺ sensitivity, making Ca²⁺ detection less susceptible to interference. 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 fura-2 and mag-fura-5 to compare the actions of Sr²⁺ and Ca²⁺ as mediators of synaptic transmission. Fura-FF is available in water-soluble potassium salt form (F14180) and as a membrane-permeant AM ester derivative (F14181).

Low-Affinity Calcium Indicators

BTC

The coumarin benzothiazole–based Ca²⁺ indicator BTC (B6790, ) and its cell-permeant derivative BTC AM (B6791, BTC Ion Indicators) were developed by Molecular Probes 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 19.17), 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⁺ (K_d ~7 µM) allows accurate quantitation of high intracellular Ca²⁺ levels that are underestimated by fura-2 measurements. Furthermore, because BTC is excited at longer wavelengths than the ratioable fura-2 and indo-1 indicators, cellular photodamage and autofluorescence may be less of a problem. 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. BTC may also be useful as an indicator for Zn²⁺. A report by Papazoglou and co-workers describes the unusual use of the tetrapotassium salt of BTC to localize atherosclerotic plaque.

Mag-Fura-2 and Mag-Indo-1

Mag-fura-2 (also called furaptra, ) and mag-indo-1 (Fluorescent Magnesium Indicators) were originally designed to report intracellular Mg²⁺ levels (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 19.18). 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. Imaging of mag-fura-2 using a single excitation wavelength (420 nm) is reported to improve the detection of high-level Ca²⁺ transients in various cells, including Purkinje neurons and frog muscle. 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. Measurements of Ca²⁺ currents in presynaptic boutons and granule cell parallel fibers with our mag-fura-5 and Magnesium Green indicators were shown to be superior to those made using fura-2. Mag-fura-2 (M1290, M1291, M1292) and mag-indo-1 (M1295) are available as cell-impermeant potassium salts or as cell-permeant AM esters and are accompanied by a detailed protocol (Fluorescent Magnesium Indicators).