Fluorescein and Fluorescein Derivatives

Fluorescein and many of its derivatives exhibit multiple, pH-dependent ionic equilibria.ref 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.ref 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,ref as well as to study cell adhesion ref or, in combination with propidium iodide (P1304MPP3566P21493Nucleic Acid Stains—Section 8.1), to determine cell viability.ref 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 20.2.3 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.ref CFDA is moderately permeant to most cell membranes and, upon hydrolysis by intracellular nonspecific esterases, forms carboxyfluorescein (5(6)-FAM), 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 ref (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 (C1359C1360Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) 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.ref

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,ref the polar fluorescein derivative BCECF (B1151) and its membrane-permeant AM ester (B1150B1170) 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+.ref 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.ref 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.ref Alternative calibration methods have also been reported.ref

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) and specially packaged as a set of 20 vials that each contains 50 µg (B1170). 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:

The cell-impermeant BCECF acid (B1151) is useful for pH measurements in intercellular spaces of epithelial cell monolayers,ref interstitial spaces of normal and neoplastic tissue ref and isolated cell fractions.ref 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.ref The free acid of BCECF can be loaded into cells by microinjection ref or electroporation or by using our Influx pinocytic cell-loading reagent (I14402Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8). It has also been loaded into bacterial cells by brief incubation at pH ~2.ref


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).


Figure 20.2.5 Structures of the AM esters of BCECF (B1150, B1170).

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 ref (SFDA). 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 ref (S359Polar Tracers—Section 14.3). Fluorescein-5-(and 6-)sulfonic acid is more commonly used to measure barrier permeability of membranes ref (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 (C2925C7025) incorporates 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.ref 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.ref


Carboxynaphthofluorescein 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.ref

SNARF pH Indicator

The seminaphthorhodafluors (SNARF dyes) are visible light–excitable fluorescent pH indicators.ref The SNARF indicators have both dual-emission and dual-excitation properties, making them particularly useful for confocal laser-scanning microscopy ref (Figure 20.2.6), flow cytometry ref and microplate reader–based measurements.ref The dual-emission properties of the SNARF indicators make them preferred probes for use in fiber-optic pH sensors.ref 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 (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 Am J Physiol (1998) 275:H1937.

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 (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.ref 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.ref As with other ion indicators, intracellular environments may cause significant changes to both the spectral properties and pKa of carboxy SNARF-1,ref 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 ref and indo-1 ref (Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2), as well as those of the fluo-3,ref 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+ (photo). Carboxy SNARF-1 has also been used in combination with the Na+ indicator SBFI (S1262S1263S1264Fluorescent Na+ and K+ Indicators—Section 21.1) to simultaneously detect pH and Na+ changes.ref The relatively long-wavelength excitation and emission characteristics of carboxy SNARF-1 facilitate studies in autofluorescent cells ref and permit experiments that employ ultraviolet light–photoactivated caged probes ref (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 ref (photo).


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.ref 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 ref 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 (S23920S23922) and as cell-permeant AM ester acetate derivatives (S23921S23923).



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.

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 (contact Custom Services for more information) contains a chloromethyl group that is mildly reactive with intracellular thiols, forming adducts that improve cellular retention of the SNARF fluorophore (photo). 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 (S22801pH Indicator Conjugates—Section 20.4) is useful as an intracellular pH indicator ref in addition to its more common application as a cell tracer.ref

8-Hydroxypyrene-1,3,6-Trisulfonic Acid (HPTS)

8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS, also known as pyranine) is an inexpensive, highly water-soluble, membrane-impermeant pH indicator with a pKa of ~7.3 in aqueous buffers.ref The pKa of HPTS is reported to rise to 7.5–7.8 in the cytosol of some cells.ref 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 ref or liposome-mediated delivery,ref through ATP-gated ion channels ref 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.ref Because the excited state of HPTS is much more acidic than the ground state,ref it is frequently used as a photoactivated source of H+ in mechanistic studies of bacteriorhodopsin and other proton pumps.ref


Figure 20.2.11 The pH-dependent absorption spectra of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS).


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+ref (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.ref

Data Table

For a detailed explanation of column headings, see Definitions of Data Table Contents

                Acidic Solution              
               Basic Solution              
Cat #MWStorageSolubleAbsECEmSolventAbsECEmSolventpKaProduct *Notes
~615F,DDMSO<300 none      B1151
BCECF acid
BCECF acid
520.45LpH >648235,000520pH 550390,000528pH 97.0 2, 3
~615F,DDMSO<300 none      B1151
BCECF acid
5(6)-carboxyfluorescein376.32LpH >6, DMF47528,000517pH 549275,000517pH 96.4 2, 3
460.40F,DDMSO<300 none      5(6)-carboxyfluorescein 
5(6)-carboxynaphthouorescein476.44LpH >6, DMF51211,000563pH 659849,000668pH 107.6 2, 3, 5
5(6)-CFDA, SE
557.47F,DDMF, DMSO<300 none      C1311
5(6)-FAM, SE
5(6)-carboxy SNARF-1
453.45LpH >654827,000587pH 657648,000635pH 107.5 2, 3, 6
5(6)-carboxy SNARF-1 AM acetate
567.55F,DDMSO<350 none      C1270
5(6)-Carboxy SNARF-1
532.46F,DDMSO<300 none      5-FAM 
5(6)-FAM376.32LpH >6, DMF47529,000517pH 549278,000517pH 96.4 2, 3, 7
CellTracker Green CMFDA
464.86F,DDMSO<300 none      see Notes8
5(6)-chloromethyl SNARF-1 acetate499.95F,DDMSO<350 none      see Notes9
CellTracker Green CMFDA
464.86F,DDMSO<300 none      see Notes8
5(6)-carboxynaphthofluorescein diacetate560.52F,DDMSO<300 none      5(6)-carboxynaphthofluorescein 
fluorescein-5(6)-sulfonic acid
478.32D,LH2O, DMF47631,000519pH 549576,000519pH 96.4 2, 3
fluorescein reference standard
332.31LpH >6, DMF47334,000514pH 549093,000514pH 96.4 2, 3
416.39F,DDMSO<300 none      F1300
fluorescein reference standard
HPTS (pyranine)524.37D,LH2O40320,000511pH 445424,000511pH 97.3 2, 3, 10
724.97F,DMeOH<300 none        
SFDA518.43F,DDMSO<300 none      F1130
fluorescein-5(6)-sulfonic acid
SNARF-4F 5(6)-carboxylic acid
471.44LpH >655227,000589pH 558148,000652pH 96.4 2, 3
SNARF-4F 5(6)-carboxylic acid AM acetate
585.54F,DDMSO<350 none      S23920
SNARF-4F 5(6)-carboxylic acid
SNARF-5F 5(6)-carboxylic acid
471.44LpH >655527,000590pH 557949,000630pH 97.2 2, 3
SNARF-5F 5(6)-carboxylic acid AM acetate
585.54F,DDMSO<350 none      S23922
SNARF-5F 5(6)-carboxylic acid AM acetate
* 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 5(6)-carboxynaphthouorescein spectra obtained in our laboratories. Additional relevant data are reported elsewhere.ref
  6. Values of pKa for these SNARF indicators are as reported in published references.ref
  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 5(6)-FAM.
  9. 5(6)-Chloromethyl SNARF-1, acetateis converted to fluorescent products with spectra similar to 5(6)-carboxy SNARF-1 (C1270) after acetate hydrolysis.
  10. The pKa for HPTS (pyranine) was determined in 0.066 M phosphate buffers at 22°C.ref

For Research Use Only. Not for use in diagnostic procedures.