Eukaryotic cells contain compartments with different degrees of acidity. For example, biomolecules brought into cells by receptor-mediated endocytosis or phagocytosis (Probes for Following Receptor Binding and Phagocytosis—Section 16.1) are initially processed through organelles of decreasing pH, and specialized organelles such as plant vacuoles and the acrosome of spermatozoa are intrinsically acidic. A low intracompartmental pH activates enzymes and other protein functions—such as iron release from transferrin—that would be too slow at neutral pH, thereby facilitating cellular metabolism. Abnormal lysosomal or endosomal acidification is associated with various pathological conditions. For example, lysosomes in some tumor cells have been reported to have a lower pH than normal lysosomes, whereas other tumor cells contain lysosomes with higher pH.
The fluorescent pH indicators used to detect acidic organelles and to follow trafficking through acidic organelles must have a lower pKa than those described in Probes Useful at Near-Neutral pH—Section 20.2. Also, unlike most pH indicators for cytosolic measurements, pH indicators for acidic organelles need not be intrinsically permeant to membranes. Often they are covalently attached to large biomolecules that are actively taken up and processed through acidic organelles by the cell's own endocytic mechanisms (Probes for Endocytosis, Receptors and Ion Channels—Chapter 16).
LysoSensor probes are weak bases that are selectively concentrated in acidic organelles as a result of protonation (Summary of the pH response of our LysoSensor probes—Table 20.2). This protonation also relieves the fluorescence quenching of the dye that results from photoinduced electron transfer (PET) by its weak-base side chain. Thus, unlike most other pH indicators in this chapter, the LysoSensor dyes become more fluorescent in acidic environments. Because accumulation of LysoSensor probes also appears to cause lysosomal alkalinization, pH measurements should be made rapidly using the lowest practicable concentration of dye.
LysoSensor Yellow/Blue Dye
LysoSensor Yellow/Blue DND-160 (L7545, ) undergoes a pH-dependent emission shift to longer wavelengths in acidic environments when illuminated near its excitation isosbestic point (~360 nm) (Figure 20.3.1). It also undergoes a pH-dependent excitation shift when detected near its emission isosbestic point (~490 nm) (Figure 20.3.1). These properties can be exploited for dual-emission ratio imaging of lysosomal pH () (emission ratio ~450/510 nm, excitation ~365 nm). Yellow-fluorescent staining by LysoSensor Yellow/Blue DND-160 has been used to identify lysosomes as the accumulation site for anthracyclines in a drug-resistant cell line. LysoSensor Yellow/Blue DND-160, frequently referred to by the acronym PDMPO, has been widely used as a tracer of silica deposition and transport in marine diatoms. We also offer a 10,000 MW dextran conjugate of the LysoSensor Yellow/Blue dye (L22460, pH Indicator Conjugates—Section 20.4).
Figure 20.3.1 The pH-dependent spectral response of LysoSensor Yellow/Blue DND-160 (L7545): A) fluorescence excitation spectra and B) fluorescence emission spectra.
LysoSensor Green and LysoSensor Blue Dyes
The green-fluorescent LysoSensor Green dyes are available with optimal pH sensitivity in either the acidic or neutral range (pKa ~5.2 or ~7.5). The blue-fluorescent LysoSensor Blue DND-167 has a pKa of ~5.1 (Figure 20.3.2). With their low pKa values, LysoSensor Green DND-189 (L7535, ) and LysoSensor Blue DND-167 (L7533) are almost nonfluorescent except when inside acidic compartments, whereas LysoSensor Green DND-153 (L7534, ) is brightly fluorescent, even at neutral pH. LysoSensor Green DND-189 has been used to monitor the dissipation of neurite vesicle transmembrane pH gradients by bafilomycin A1.
Figure 20.3.2 The pH titration curve of LysoSensor Blue DND-167 (L7533), which exhibits a pKa ~5.1.
pHrodo pH indicator is an aminorhodamine dye that exhibits increasing fluorescence as the pH of its surroundings becomes more acidic (Figure 20.3.3). Consequently, pHrodo fluorescence (excitation/emission ~560/585 nm) provides a positive indication of processes such as phagocytic ingestion and lysosomal sequestration, in contrast to the negative indication generated by fluorescein and Oregon Green dyes. Because the pHrodo dye has more than one protolytically ionizable substituent, it exhibits a complex pH titration profile that typically changes upon conjugation to proteins and other biomolecules. We currently offer an amine-reactive succinimidyl ester form of the pHrodo dye (P36600, pH Indicator Conjugates—Section 20.4) and various bioconjugates (pH Indicator Conjugates—Section 20.4, Probes for Following Receptor Binding and Phagocytosis—Section 16.1). In cases where the nonreactive carboxylic acid form of the dye is required, pHrodo carboxylic acid can be obtained by mild alkaline hydrolysis of the succinimidyl ester (e.g., 12-hour incubation in aqueous solution at pH 8 in the dark will yield >99.9% hydrolysis, higher pH will give a more rapid reaction).
Figure 20.3.3 The pH response profile of pHrodo dextran (P10361) monitored at excitation/emission wavelengths of 545/590 nm in a fluorescence microplate reader. Citrate, MOPS and borate buffers were used to span the pH range from 2.5 to 10.
Introduction of electron-withdrawing groups into fluorescein dyes lowers the pKa of the phenolic group to 5 or below, as exemplified by our Oregon Green dyes and their insensitivity to pH changes in the near-neutral pH range. However, these fluorinated fluorescein dyes are still pH sensitive in moderately acidic solutions, with pKa values of ~4.7 (Figure 20.3.4). With the exception of their lower pKa values, the pH-dependent spectral characteristics of the Oregon Green dyes closely parallel those of other fluorescein-based dyes, allowing dual-excitation ratiometric measurements with the same general configuration used for BCECF (Probes Useful at Near-Neutral pH—Section 20.2).
Oregon Green 514 carboxylic acid (O6138, ), Oregon Green 488 carboxylic acid (O6146, ) and 5-(and 6-)carboxy-2',7'-dichlorofluorescein (C368) do not readily enter cells but may be useful as fluid phase markers for endocytosis. These fluorinated and chlorinated fluorescein derivatives will likely be most useful in the form of conjugates that are endocytosed and processed through acidic organelles (pH Indicator Conjugates—Section 20.4); dextran conjugates of the Oregon Green 488 and Oregon Green 514 dyes are described in pH Indicator Conjugates—Section 20.4.
Cell-permeant diacetate derivatives of carboxydichlorofluorescein (carboxy-DCFDA, C369) and carboxydifluorofluorescein (carboxy-DFFDA, O6151) are also available. Carboxy-DCFDA has been used to measure the pH in acidic organelles, as well as in the cytosol and vacuoles of plants and yeast. Furthermore, the mechanism of vacuolar pH rectification following exposure to ammonia has been investigated in rice and maize root hair cells loaded with carboxy-DFFDA.
Figure 20.3.4 The pH-dependent spectra of Oregon Green 514 carboxylic acid (O6138): A) absorption spectra and B) fluorescence excitation spectra.
The nucleic acid stain 9-amino-6-chloro-2-methoxyacridine (ACMA, A1324; ) apparently binds to membranes in the energized state, and its fluorescence becomes quenched if a pH gradient forms. Mechanistically, this probe resembles the membrane potential–sensitive carbocyanines (Slow-Response Probes—Section 22.3) more than the other probes in this chapter. ACMA is primarily employed to detect the proton-translocating activity of Escherichia coli ATP synthase and yeast vacuolar H+-ATPase.
8-Hydroxypyrene-1,3,6-Trisulfonic Acid (HPTS)
Although 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, (H348; Probes Useful at Near-Neutral pH—Section 20.2) has a pKa ~7.3 and is primarily used as a pH indicator in the near-neutral range, it offers several advantages for monitoring intraorganelle pH in endosomal/lysosomal pathways:
- The highly polar character that results from its three sulfonic acid groups prevents leakage across intracellular membranes.
- Uptake by fluid-phase endocytosis is efficient and easily accomplished.
- Determination of excitation ratios allows pH measurements that are independent of vesicular size and indicator concentration.
- Precise calibration permits pH values as low as 4.4 to be accurately measured.
- HPTS is nontoxic and does not perturb normal physiological function.
HPTS can be introduced into cells by microinjection, electroporation or liposome-mediated delivery or through ATP-gated ion channels. HPTS has also been loaded into cells using a patch pipette. HPTS has been reported to be less phototoxic than BCECF.
| Acidic Solution || Neutral/Basic Solution |
|A1324||258.71||L||DMF, DMSO||412||8200||471||MeOH||see Notes||see Notes||8.6||1, 2|
|C368||445.21||L||pH >6, DMF||495||38,000||529||pH 4||504||107,000||529||pH 8||4.8||3, 4|
|L7533||376.50||F,D,L||DMSO||373||11,000||425||pH 3||373||11,000||425||pH 7||5.1||3, 4, 6, 7|
|L7534||356.43||F,D,L||DMSO||442||17,000||505||pH 5||442||17,000||505||pH 9||7.5||3, 4, 6, 7|
|L7535||398.46||F,D,L||DMSO||443||16,000||505||pH 3||443||16,000||505||pH 7||5.2||3, 4, 6, 7|
|L7545||366.42||F,D,L||DMSO||384||21,000||540||pH 3||329||23,000||440||pH 7||4.2||3, 4, 6, 8|
|O6138||512.36||L||pH >6, DMF||489||26,000||526||pH 3||506||86,000||526||pH 9||4.7||3, 4, 9|
|O6146||412.30||L||pH >6, DMF||478||27,000||518||pH 3||492||85,000||518||pH 9||4.7||3, 4, 10|
|1. Absorption and fluorescence spectra of the protonated and deprotonated forms of A1324 are quite similar. This observation, and the listed pKa value, are reported in published references. Accumulation of the probe in the presence of transmembrane pH gradients results in fluorescence quenching.|
|2. Spectra of this compound are in methanol acidified with a trace of HCl.|
|3. 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.|
|4. Spectra are in aqueous buffers adjusted to >1 pH unit above and >1 pH unit below the pKa.|
|5. C369 is converted to a fluorescent product (C368) after acetate hydrolysis.|
|6. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."|
|7. This LysoSensor dye exhibits increasing fluorescence as pH decreases with no spectral shift. L7533 has additional absorption and fluorescence emission peaks at Abs = 394 nm and Em = 401 nm.|
|8. The pKa value for this product is determined from the pH-dependent variation of the absorption spectrum.|
|9. The fluorescence lifetime (τ) of the Oregon Green 514 dye in pH 9.0 buffer at 20°C is 4.2 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.|
|10. The fluorescence lifetime (τ) of the Oregon Green 488 dye (O6146) in pH 9.0 buffer at 20°C is 4.1 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.|
|11. Acetate hydrolysis of this compound yields a fluorescent product with similar spectral characteristics to O6146 .|