Fluorescein and many of its derivatives exhibit multiple, pH-dependent ionic equilibria. Both the phenol and carboxylic acid functional groups of fluorescein are almost totally ionized in aqueous solutions above pH 9 (Figure 20.2.1). Acidification of the fluorescein dianion first protonates the phenol (pKa ~6.4) to yield the fluorescein monoanion, then the carboxylic acid (pKa <5) to produce the neutral species of fluorescein. Further acidification generates a fluorescein cation (pKa ~2.1).
Only the monoanion and dianion of fluorescein are fluorescent, with quantum yields of 0.37 and 0.93, respectively, although excitation of either the neutral or cationic species is reported to produce emission from the anion with effective quantum yields of 0.31 and 0.18, respectively. A further equilibrium involves formation of a colorless, nonfluorescent lactone (Figure 20.2.1). The lactone is not formed in aqueous solution above pH 5 but may be the dominant form of neutral fluorescein in solvents such as acetone. The pH-dependent absorption spectra of fluorescein (Figure 20.2.2) clearly show the blue shift and decreased absorptivity indicative of the formation of protonated species. However, the fluorescence emission spectrum of most fluorescein derivatives, even in acidic solution, is dominated by the dianion, with only small contributions from the monoanion. Consequently, the wavelength and shape of the emission spectra resulting from excitation close to the dianion absorption peak at 490 nm are relatively independent of pH, but the fluorescence intensity is dramatically reduced at acidic pH (Figure 20.2.2).
We offer a broad variety of fluorescein-derived reagents and fluoresceinated probes that can serve as sensitive fluorescent pH indicators in a wide range of applications. Chemical substitutions of fluorescein may shift absorption and fluorescence maxima and change the pKa of the dye; however, the effects of acidification on the spectral characteristics illustrated in Figure 20.2.2 are generally maintained in all fluorescein derivatives.
Figure 20.2.1 Ionization equilibria of fluorescein.
Figure 20.2.2 The pH-dependent spectra of fluorescein (F1300): A) absorption spectra, B) emission spectra.
Fluorescein and Its Diacetate
The cell-permeant fluorescein diacetate (FDA, F1303) is still occasionally used to measure intracellular pH, as well as to study cell adhesion or, in combination with propidium iodide (P1304MP, P3566, P21493; Nucleic Acid Stains—Section 8.1), to determine cell viability. However, fluorescein (F1300), which is formed by intracellular hydrolysis of FDA, rapidly leaks from cells (Figure 20.2.3). Thus, other cell-permeant dyes such as the acetoxymethyl (AM) esters of BCECF and calcein are now preferred for intracellular pH measurements and cell viability assays (Viability and Cytotoxicity Assay Reagents—Section 15.2).
Figure 20.3.3 Loading and retention characteristics of intracellular marker dyes. Cells of a human lymphoid line (GePa) were loaded with the following cell-permeant acetoxymethyl ester (AM) or acetate derivatives of fluorescein: 1) calcein AM (C1430, C3099, C3100MP), 2) BCECF AM (B1150), 3) fluorescein diacetate (FDA, F1303), 4) carboxyfluorescein diacetate (CFDA, C1354) and 5) CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, C2925, C7025). Cells were incubated in 4 µM staining solutions in Dulbecco's modified eagle medium containing 10% fetal bovine serum (DMEM+) at 37°C. After incubation for 30 minutes, cell samples were immediately analyzed by flow cytometry to determine the average fluorescence per cell at time zero (0 hours). Retained cell samples were subsequently washed twice by centrifugation, resuspended in DMEM+, maintained at 37°C for 2 hours and then analyzed by flow cytometry. The decrease in the average fluorescence intensity per cell in these samples relative to the time zero samples indicates the extent of intracellular dye leakage during the 2-hour incubation period.
Carboxyfluorescein and Its Cell-Permeant Esters
Fluorescein's high leakage rate out of cells makes it very difficult to quantitate intracellular pH because the decrease in the cell's fluorescence due to dye leakage cannot be easily distinguished from that due to acidification. The use of carboxyfluorescein diacetate (CFDA, C195) for intracellular pH measurements partially addresses this problem. CFDA is moderately permeant to most cell membranes and, upon hydrolysis by intracellular nonspecific esterases, forms carboxyfluorescein (5(6)-FAM, C194, C1904), which has a pH-dependent spectral response very similar to that of fluorescein. As compared with fluorescein, carboxyfluorescein contains an extra negative charge and is therefore better retained in cells (Figure 20.2.3). The mixed-isomer preparation of CFDA (C195) is usually adequate for intracellular pH measurements because the single isomers of carboxyfluorescein exhibit essentially identical pH-dependent spectra with a pKa ~6.5. For experiments requiring a pure isomer, the single-isomer preparations of carboxyfluorescein (C1359, C1360; Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) and CFDA (C1361, C1362; Viability and Cytotoxicity Assay Reagents—Section 15.2) are available. In addition, we offer the AM ester of CFDA (5-CFDA, AM, C1354) , which is electrically neutral and facilitates cell loading. Upon hydrolysis by intracellular esterases, this AM ester also yields carboxyfluorescein.
BCECF and Its AM Ester
Although carboxyfluorescein is better retained in cells than is fluorescein, its pKa of ~6.5 is lower than the cytosolic pH of most cells (pH ~6.8–7.4). Consequently, its fluorescence change is less than optimal for detecting small pH changes above pH 7. Since its introduction by Roger Tsien in 1982, the polar fluorescein derivative BCECF (B1151) and its membrane-permeant AM ester (B1150, B1170, B3051) have become the most widely used fluorescent indicators for estimating intracellular pH. Also, a flow cytometric assay has been developed that uses BCECF to estimate the concentration of intracellular K+. BCECF's four to five negative charges at pH 7–8 improve its retention in cells (Figure 20.2.3), and its pKa of 6.98 is ideal for typical intracellular pH measurements.
As with fluorescein and carboxyfluorescein, absorption of the phenolate anion (basic) form of BCECF is red-shifted and has increased molar absorptivity relative to the protonated (acidic) form (Figure 20.2.4); there is little pH-dependent shift in the fluorescence emission spectrum of BCECF upon excitation at 505 nm. BCECF is typically used as a dual-excitation ratiometric pH indicator. Signal errors caused by variations in concentration, path length, leakage and photobleaching are greatly reduced with ratiometric methods (Loading and Calibration of Intracellular Ion Indicators—Note 19.1). Intracellular pH measurements with BCECF are made by determining the pH-dependent ratio of emission intensity (detected at 535 nm) when the dye is excited at ~490 nm versus the emission intensity when excited at its isosbestic point of ~440 nm (Figure 20.2.4, ). Because BCECF's absorption at 440 nm is quite weak, increasing the denominator wavelength to ~450 nm provides improved signal-to-noise characteristics for ratio imaging applications. As with other intracellular pH indicators, in situ calibration of BCECF's fluorescence response is usually accomplished using 10–50 µM nigericin (N1495, see below) in the presence of 100–150 mM K+ to equilibrate internal and external pH. Alternative calibration methods have also been reported.
Loading of live cells for measurement of intracellular pH is readily accomplished by incubating cell suspensions or adherent cells in a 1–10 µM solution of the AM ester of BCECF. At least three different molecular species can be obtained in synthetic preparations of the AM ester of BCECF; however, all three forms shown in Figure 20.2.5 appear to be converted to the same product—BCECF acid (B1151, )—by intracellular esterase hydrolysis. Although we can readily prepare the pure tri(acetoxymethyl) ester form (Form I in Figure 20.2.5), some researchers have found that cell loading with a mixture of the lactone Forms II and III is more efficient. Consequently, we produce BCECF AM predominantly as a mixture of Forms II and III with a typical percentage composition ratio of 45:55, as determined by HPLC, NMR and mass spectrometry. The AM ester of BCECF is available in a single 1 mg vial (B1150), specially packaged as a set of 20 vials that each contains 50 µg (B1170) and as a 1 mg/mL solution (~1.6 mM) in anhydrous dimethylsulfoxide (DMSO) (B3051). We highly recommend purchasing the set of 20 vials in order to reduce the potential for product deterioration caused by exposure to moisture.
Our bibliography for BCECF AM (Bibliography for B1150) lists more than 1200 journal citations, including references for the use of BCECF AM to investigate:
- Cl–/HCO3– exchange
- K+/H+ exchange
- Na+/H+ exchange
- Na+/Ca2+ exchange
- NH4+ transport
- Lactate transport and metabolism
- Apoptosis (Assays for Apoptosis—Section 15.5)
- Phagocytosis (Probes for Following Receptor Binding and Phagocytosis—Section 16.1)
- Regulation of pancreatic insulin secretion
- Voltage-activated H+ conductance in neurons
The cell-impermeant BCECF acid (B1151) is useful for pH measurements in intercellular spaces of epithelial cell monolayers, interstitial spaces of normal and neoplastic tissue and isolated cell fractions. BCECF has also been employed for two-photon fluorescence lifetime imaging of the skin stratum corneum to detect aqueous acid pockets within the lipid-rich extracellular matrix. The free acid of BCECF can be loaded into cells by microinjection or electroporation or by using our Influx pinocytic cell-loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8). It has also been loaded into bacterial cells by brief incubation at pH ~2. In addition to the cell-permeant BCECF AM and cell-impermeant BCECF acid, we offer dextran conjugates of BCECF (D1878, D1880; pH Indicator Conjugates—Section 20.4).
Figure 20.2.4 The pH-dependent spectra of BCECF (B1151): A) absorption spectra, B) emission spectra and C) excitation spectra. The fluorescence excitation spectra on the left in panel C have been enlarged 10X to reveal BCECF's 439 nm isosbestic point. Note that the isosbestic point of the excitation spectra of BCECF is different from that of the absorption spectra (compare panels A and C).
Fluorescein Sulfonic Acid and Its Diacetate
The fluorescein-5-(and 6-)sulfonic acid (F1130, ) is much more polar than carboxyfluorescein. Consequently, once inside cells or liposomes, it is relatively well retained. Some cells can be loaded directly with 5-sulfofluorescein diacetate (SFDA, S1129). Direct ratiometric measurement of the pH in the trans-Golgi of live human fibroblasts was achieved by simultaneously microinjecting liposomes loaded with both fluorescein sulfonic acid and sulforhodamine 101 (S359, Polar Tracers—Section 14.3). Fluorescein-5-(and 6-)sulfonic acid is more commonly used to measure barrier permeability of membranes (Polar Tracers—Section 14.3).
Chemically Reactive Fluorescein Diacetates
One means for overcoming the cell leakage problem common to the above pH indicators, including BCECF, is to trap the indicator inside the cell via conjugation to intracellular constituents. CellTracker Green CMFDA (C2925, C7025; ) and chloromethyl SNARF-1 (C6826, see below) incorporate a thiol-reactive chloromethyl moiety that reacts with intracellular thiols, including glutathione and proteins, to yield well-retained products (Figure 20.2.3). Cleavage of the acetate groups of the CMFDA conjugate by intracellular esterases yields a conjugate that retains the pH-dependent spectral properties of fluorescein. Because of its superior retention as compared with SNARF AM and BCECF AM, CellTracker Green CMFDA was employed to monitor the intracellular pH response to osmotic stress in CHO, HEK 293 and Caco-2 cells. Similarly, the amine-reactive succinimidyl ester of CFDA (CFSE, C1157) can be used for long-term pH studies of live cells, producing a conjugate with the pH-sensitive properties of carboxyfluorescein.
Carboxynaphthofluorescein (C652, ) has pH-dependent red fluorescence (excitation/emission maxima ~598/668 nm at pH >9) with a relatively high pKa of ~7.6. The long-wavelength pH-dependent spectra of carboxynaphthofluorescein have been exploited in the construction of fiber-optic pH sensors. This long-wavelength pH indicator is also available in membrane-permeant diacetate form (C13196) for passive intracellular loading and as an amine-reactive succinimidyl ester (C653, pH Indicator Conjugates—Section 20.4) for preparing pH-sensitive conjugates.
The seminaphthorhodafluors (SNARF dyes) are visible light–excitable fluorescent pH indicators. The SNARF indicators have both dual-emission and dual-excitation properties, making them particularly useful for confocal laser-scanning microscopy (Figure 20.2.6), flow cytometry and microplate reader–based measurements. The dual-emission properties of the SNARF indicators make them preferred probes for use in fiber-optic pH sensors. These pH indicators can be excited by the 488 or 514 nm spectral lines of the argon-ion laser and are sensitive to pH values within the physiological range. Dextran conjugates of the SNARF dyes are described in pH Indicator Conjugates—Section 20.4.
Figure 20.2.6 Confocal fluorescence images of rabbit papillary muscle loaded by perfusion with carboxy SNARF-1 AM acetate (C1271, C1272). The first two images were acquired through 585 ± 10 nm bandpass and >620 nm longpass emission filters, respectively. The 620 nm/585 nm fluorescence ratio image in the third image is more uniform than the component images A and B due to cancellation of intensity variations resulting from heterogeneous uptake of the fluorescent indicator. Images contributed by Barbara Muller-Borer and John Lemasters, University of North Carolina and reprinted with permission from .
Carboxy SNARF-1 Dye and Its Cell-Permeant Ester
The carboxy SNARF-1 dye (C1270, ), which is easily loaded into cells as its cell-permeant AM ester acetate (C1271, C1272), has a pKa of about 7.5 at room temperature and between 7.3 and 7.4 at 37°C. Thus, carboxy SNARF-1 is useful for measuring pH changes between pH 7 and pH 8. Like fluorescein and BCECF, the absorption spectrum of the carboxy SNARF-1 pH indicator undergoes a shift to longer wavelengths upon deprotonation of its phenolic substituent (Figure 20.2.7). In contrast to the fluorescein-based indicators, however, carboxy SNARF-1 also exhibits a significant pH-dependent emission shift from yellow-orange to deep-red fluorescence as conditions become more basic (Figure 20.2.8, ). This pH dependence allows the ratio of the fluorescence intensities from the dye at two emission wavelengths—typically 580 nm and 640 nm—to be used for quantitative determinations of pH (Loading and Calibration of Intracellular Ion Indicators—Note 19.1) (Figure 20.2.6). For practical purposes, it is often desirable to bias the detection of carboxy SNARF-1 fluorescence towards the less fluorescent acidic form by using an excitation wavelength between 488 nm and the excitation isosbestic point at ~530 nm, yielding balanced signals for the two emission ratio components (Figure 20.2.8, ). When excited at 488 nm, carboxy SNARF-1 exhibits an emission isosbestic point of ~610 nm and a lower fluorescent signal than obtained with 514 nm excitation. Alternatively, when excited by the 568 nm spectral line of the Ar-Kr laser found in some confocal laser-scanning microscopes, carboxy SNARF-1 exhibits a fluorescence increase at 640 nm as the pH increases and an emission isosbestic point at 585 nm. As with other ion indicators, intracellular environments may cause significant changes to both the spectral properties and pKa of carboxy SNARF-1, and the indicator should always be calibrated in the system under study.
The spectra of carboxy SNARF-1 are well resolved from those of fura-2 and indo-1 (Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2), as well as those of the fluo-3, fluo-4, Calcium Green and Oregon Green 488 BAPTA Ca2+ indicators (Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3), permitting simultaneous measurements of intracellular pH and Ca2+ (). Carboxy SNARF-1 has also been used in combination with the Na+ indicator SBFI (S1262, S1263, S1264; Fluorescent Na+ and K+ Indicators—Section 21.1) to simultaneously detect pH and Na+ changes. The relatively long-wavelength excitation and emission characteristics of carboxy SNARF-1 facilitate studies in autofluorescent cells and permit experiments that employ ultraviolet light–photoactivated caged probes (Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes—Section 5.3). Incubation of cells for several hours after loading with carboxy SNARF-1 AM ester acetate results in compartmentally selective retention of the dye, allowing in situ measurements of mitochondrial pH ().
Figure 20.2.7 The pH-dependent absorption spectra of carboxy SNARF-1 (C1270).
Figure 20.2.8 The pH-dependent emission spectra of carboxy SNARF-1 (C1270) when excited at A) 488 nm, B) 514 nm and C) 534 nm.
SNARF-4F and SNARF-5F Dyes and Their Cell-Permeant Esters
Although the carboxy SNARF-1 indicator possesses excellent spectral properties, its pKa of ~7.5 may be too high for measurements of intracellular pH in some cells. For quantitative measurements of pH changes in the typical cytosolic range (pH ~6.8–7.4), we now recommend SNARF-5F carboxylic acid (), which has a pKa value of ~7.2, as the indicator with the optimal spectral properties for estimating cytosolic pH (Figure 20.2.9). SNARF-4F carboxylic acid () has a somewhat more acidic pH sensitivity maximum (pKa ~6.4) but retains its dual-emission spectral properties (Figure 20.2.10). SNARF-4F has been used for pH imaging in kidney tissues using two-photon excitation (780 nm) microscopy; the pH-dependent emission shift response was observed to be essentially the same as seen with one-photon excitation. This study also reported nigericin calibrations that yielded different pKa values (6.8 versus 7.4) in the kidney cortex and kidney ileum, respectively, emphasizing the importance of performing in situ calibrations. Both SNARF-4F and SNARF-5F allow dual-excitation and dual-emission ratiometric pH measurements, making them compatible with the same instrument configurations used for carboxy SNARF-1 in ratio imaging and flow cytometry applications. SNARF-4F and SNARF-5F are available as free carboxylic acids (S23920, S23922) and as cell-permeant AM ester acetate derivatives (S23921, S23923).
Figure 20.2.9 Fluorescence emission spectra of SNARF-5F 5-(and 6-)carboxylic acid (S23922) as a function of pH.
Figure 20.2.10 Fluorescence emission spectra of SNARF-4F 5-(and 6-)carboxylic acid (S23920) showing the pH-dependent spectral shift that is characteristic of this and other SNARF pH indicators.
Amine- and Thiol-Reactive SNARF Dyes
Our 5-(and 6-)chloromethyl SNARF-1 acetate (C6826, ) contains a chloromethyl group that is mildly reactive with intracellular thiols, forming adducts that improve cellular retention of the SNARF fluorophore (). As with CellTracker Green CMFDA (see above), improved retention of this conjugate in cells may permit monitoring of intracellular pH over longer time periods than is possible with other intracellular pH indicators. Similarly, amine-reactive SNARF-1 succinimidyl ester (S22801, pH Indicator Conjugates—Section 20.4) is useful as an intracellular pH indicator in addition to its more common application as a cell tracer.
8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS, also known as pyranine; H348; ) is an inexpensive, highly water-soluble, membrane-impermeant pH indicator with a pKa of ~7.3 in aqueous buffers. The pKa of HPTS is reported to rise to 7.5–7.8 in the cytosol of some cells. Unlike indicators based on the SNARF and fluorescein dyes, there is no membrane-permeant form of HPTS available. Consequently, HPTS must be introduced into cells by microinjection, electroporation or liposome-mediated delivery, through ATP-gated ion channels or by other relatively invasive means (Choosing a Tracer—Section 14.1, Techniques for loading molecules into the cytoplasm—Table 14.1). HPTS exhibits a pH-dependent absorption shift (Figure 20.2.11), allowing ratiometric measurements using an excitation ratio of 450/405 nm. Because the excited state of HPTS is much more acidic than the ground state, it is frequently used as a photoactivated source of H+ in mechanistic studies of bacteriorhodopsin and other proton pumps.
Figure 20.2.11 The pH-dependent absorption spectra of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, H348).
Intracellular calibration of the fluorescence response of cytosolic pH indicators is typically performed using the K+/H+ ionophore nigericin (N1495), which causes equilibration of intracellular and extracellular pH in the presence of a depolarizing concentration of extracellular K+ (Loading and Calibration of Intracellular Ion Indicators—Note 19.1). Nett and Deitmer have compared this technique with calibrations performed by direct insertion of pH-sensitive microelectrodes in leech giant glial cells.
| Acidic Solution || Basic Solution |
|Cat #||Links||MW||Storage||Soluble||Abs||EC||Em||Solvent||Abs||EC||Em||Solvent||pKa||Product *||Notes|
|B1151||520.45||L||pH >6||482||35,000||520||pH 5||503||90,000||528||pH 9||7.0||2, 3|
|C194||376.32||L||pH >6, DMF||475||28,000||517||pH 5||492||75,000||517||pH 9||6.4||2, 3|
|C652||476.44||L||pH >6, DMF||512||11,000||563||pH 6||598||49,000||668||pH 10||7.6||2, 3, 5|
|C1270||453.45||L||pH >6||548||27,000||587||pH 6||576||48,000||635||pH 10||7.5||2, 3, 6|
|C1904||376.32||L||pH >6, DMF||475||29,000||517||pH 5||492||78,000||517||pH 9||6.4||2, 3, 7|
|F1130||478.32||D,L||H2O, DMF||476||31,000||519||pH 5||495||76,000||519||pH 9||6.4||2, 3|
|F1300||332.31||L||pH >6, DMF||473||34,000||514||pH 5||490||93,000||514||pH 9||6.4||2, 3|
|H348||524.37||D,L||H2O||403||20,000||511||pH 4||454||24,000||511||pH 9||7.3||2, 3, 10|
|S23920||471.44||L||pH >6||552||27,000||589||pH 5||581||48,000||652||pH 9||6.4||2, 3|
|S23922||471.44||L||pH >6||555||27,000||590||pH 5||579||49,000||630||pH 9||7.2||2, 3|
|* Cat # of product generated in situ in typical intracellular applications.|
|1. MW value is approximate. BCECF, AM is a mixture of molecular species.|
|2. pKa values may vary considerably depending on the temperature, ionic strength, viscosity, protein binding and other factors. Unless otherwise noted, values listed have been determined from pH-dependent fluorescence measurements at 22°C.|
|3. Spectra are in aqueous buffers adjusted to >1 pH unit above and >1 pH unit below the pKa.|
|4. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."|
|5. Data on pH dependence of C652 spectra obtained in our laboratories. Additional relevant data are reported elsewhere.|
|6. Values of pKa for these SNARF indicators are as reported in published references.|
|7. This product is specified to equal or exceed 98% analytical purity by HPLC.|
|8. Acetate hydrolysis of this compound yields a fluorescent product with similar pH-dependent spectral characteristics to C1904.|
|9. C6826 is converted to fluorescent products with spectra similar to C1270 after acetate hydrolysis.|
|10. The pKa for H348 was determined in 0.066 M phosphate buffers at 22°C.|