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RATIOWORKS pH 165, AM: A Dual Excitation/Emission Fluorescence Probe For Imaging Live Cells


A novel dual excitation/emission fluorescent probe has been developed for specific and sensitive determination of intracellular pH in cellular processes. The present study explores the effectiveness of RatioWorks™ pH 165, AM as a pH-sensitive fluorescent indicator by analyzing the spectral characteristics in HeLa cell lines with pH measurements using a fluorescence microplate reader. Standard buffers in the range of pH 4-10 were used in analyzing the excitation/emission spectrum of the dye. The pH measurements were determined using the ratio of the emission and excitation intensities of the dye at dual wavelengths (Ex/Em: 497nm/594nm and 578nm/654nm, respectively). Cell cytotoxicity was evaluated using CCK-8 assay at an incubation period of 120 min post WST-8 addition. The fluorescence response of RatioWorks™ pH 165, AM obtained with Cy3/TRITC and Cy5 filters at different pH regions provide evidence for accurate pH measurements of intracellular organelles as well as low cytotoxic nature of the probe. Furthermore, in contrast to other fluorescein derivatives, RatioWorks™ pH 165, AM exhibits response in the red and far red spectrum, making the probe an ideal dual excitation/emission fluorescence pH sensor for multiplex immunoassay technologies involving GFP. RatioWorks™ pH 165, AM proves to be a competitive pH indicator among other fluorescent-based technologies.

Keywords: pH indicator, fluorescence, dual excitation, dual emission, cytotoxicity, WST-8™, intracellular pH



Fluorescent pH indicators are of pivotal relevance in measuring changes to intracellular proton concentrations. These are selected based on their pKa values and similarity to the pH of the investigated system. Under physiological conditions, the pH of intracellular fluid ranges between 6.8 and 7.4, in which case a fluorescent probe with a pKa around 7 is required to make quantitative pH measurements. However, this neutral pH range is not characteristic of all cellular components, as some organelles like lysosomes function optimally only under acidic conditions. A variation in pH will affect the different resonance forms of the dye, altering the stability of the excited state and consequently, the associated spectrum shifts of the species.

Fluorescent probes can be sophisticated sensors, but the diffusion properties and specificity of each probe vary profoundly with the structural characteristics of the associated fluorescent molecules. Several techniques have been developed for pH measurements in cells, including partitioning of weak acids and bases, pH selective microelectrodes, nuclear magnetic resonance, etc. Nevertheless, pH sensitive fluorescent probes continue to remain a powerful technique with their increased sensitivity and greater sampling capability when compared to microelectrodes and other fluorescence-based assays for quantifying intracellular pH in cellular processes. The most frequently used indicators for near neutral pH conditions are fluorescein and fluorescein derivatives, like BCECF AM, BCFL AM, and SNARF, SE. Despite the advantages of these pH indicators, there still exist limitations in their emission spectra that result in compromised image resolutions, rendering them ineffective for certain multiplex immunoassay techniques with GFP. In an attempt to overcome these shortcomings, AAT Bioquest has developed a novel pH sensitive fluorescent probe, RatioWorks™ pH 165, AM which not only reflects all the advantages of the fluorescein based pH indicators listed above, having essentially identical basic structural characteristics, but also addresses the issue of compromised image resolution by providing well-resolved dual emissions at two discrete wavelengths.

RatioWorks™ pH 165, AM is an indispensable tool for intracellular pH measurements in living organisms. The emission spectrum of the probe undergoes a pH-dependent wavelength shift, making it a long wavelength fluorescent probe. This allows for the ratio of the fluorescence intensities from the dye at two emission wavelengths to be used for quantitative pH measurements in near-neutral regions. The indicator operates by exciting the dye at two regions (497 nm and 578 nm), while also monitoring the fluorescence emission at dual wavelengths (594 nm and 654 nm). At low pH values, RatioWorks™ pH 165, AM is weakly fluorescent in the far red spectrum and highly fluorescent in the red spectrum. As pH increases, it demonstrates higher fluorescence in the far red spectrum and lower fluorescence in the red spectrum. Attributing to its cell permeability (pKa ~ 7.1), RatioWorks™ pH 165, AM is particularly well suited to quantifying cytosolic pH between 4-9 and is compatible with instrumentation platforms involving any visible-light fixed-wavelength excitation source, such as confocal laser scanning microscopes, flow cytometers, and fluorescence microplate readers. The dual excitation/emission property along with its ratiometric analysis tool makes RatioWorks™ pH 165, AM useful not only for studies requiring accurate pH determinations in cells, but also those employing multiplex immunoassay technologies where background fluorescence can be introduced at shorter wavelengths.

The present study aims to explore the development of RatioWorks™ pH 165, AM, a proprietary fluorescent pH sensor from AAT Bioquest for near-neutral pH conditions in intracellular organelles. The role of RatioWorks™ pH 165, AM as an effective pH indicator is evaluated by analyzing spectral characteristics in HeLa cell lines through pHi measurements with a fluorescence microplate reader. Based on the fluorescence response obtained with Cy3/TRITC and Cy5 filters at different pH regions, it is evident that RatioWorks™ pH 165, AM is an ideal dual excitation/emission fluorescence pH sensor for quantitative pH measurements in intracellular processes. The probe proves to be a competitive pH indicator among other fluorescent-based technologies used in bioassays and applications of these assays for food safety, quality, and efficacy.


Materials and Methods


For the preparation of dye solutions the following buffers were used: Component A (pH 4.5), Component B (pH 5.0), Component C (pH 5.5), Component D (pH 6.0), Component E (pH 6.5), Component F (pH 7.0), Component G (pH 7.5), Component H (pH 8.0), Component I (Nigericin free acid), Component J (DMSO). All the chemicals were of spectroscopic grade quality and were used as received without further purification.


UV-VIS absorption spectra were recorded on a Shimadzu UV-1800 spectrometer. Fluorescence emission spectra were performed on a Varian Cary-Eclipse spectrofluorometer. The fluorescence images of cells were obtained using a Keyence BZ series fluorescence microscope with a Cy3/TRITC-Cy5 filter sets and cells plated in black wall/clear bottom plate. The pH values were measured with a Denver Instruments UB-10 pH meter using a combined glass-calomel electrode. Key parameters for the fluorescence microplate reader (FlexStation 3) include bottom read mode with black wall/clear bottom plate. Signals were examined in the excitation and emission wavelength of 497-578 nm and 594-654 nm, respectively, with a cutoff wavelength between 570-630 nm.

Sample Preparation

Stock solutions (1-5 mM) of RatioWorks™ pH 165, AM were prepared with DMSO. Each test solution (20-50 µM) for fluorescence measurement was prepared from the corresponding stock solution by diluting in 20 mM Hanks and Hepes buffer (HHBS), so that the absorbance of the final solutions at Ex was lower than 0.1. For fluorescence experiments, six dye solutions were prepared in their respective buffers with the following pH values: 4.5, 5, 6, 7, 8, and 9.1.

Cell Culture

HeLa cells were cultured in a DMEM growth medium supplemented with 10% Bovine serum, penicillin (100 µg mL-1), streptomycin (100 µg mL-1) and L-glutamine (2.5 x 10-4 M) at 37°C in a 5:95 CO2-air incubator. The fluorescence images of cells were obtained with a fluorescence microscope. The green emission filter (Pseudo Color), Cy3/TRITC, was used with a range of 570-640 nm, while the red emission filter, Cy5, was in the range of 662-737 nm. The HeLa cells were incubated with RatioWorks™ pH 165, AM dye solutions (10 µL, 120 min). Upon removing the cell culture medium, the cells were washed with phosphate-buffered saline (PBS) and transferred to their respective pH solutions (4.1 to 9.0) with Nigericin (2 µg mL-1). For the purpose of cell imaging and ratiometric analysis, the signal intensity was measured at Ex1/Em1 = 497/594 nm with a cutoff at 570 nm and Ex2/Em2 = 578/654 nm with a corresponding cutoff wavelength of 630 nm. The ratio was calculated by dividing Ex1 over Em1 and Ex2 over Em2.

Cytotoxicity Assay

Cell toxicity was determined using CCK-8 assay. HeLa cell lines were seeded in 96-well microplates in 100 µL of Bovine medium for a stationary culture. After 24 hours of cell attachment, the plates were washed with 100 µL per well PBS. In a fresh medium, different concentrations of RatioWorks™ pH 165 dye solutions (10 µM, 20 µM, 50 µM, and 100 µM) were mixed, added to the cells, and incubated for 6 hours. Cells in a culture medium without fluorescent dyes were used as the control. Subsequently, WST-8™ Solution (10 µL) was added to each well. After 120 min of incubation in a 5% CO2 humidified incubator at 37°C and protected from light, the working solution was pipetted out of the cells, which were then washed and replaced with an HHBS buffer. The fluorescence signal in the cells was observed using a fluorescence microscope with a Cy3/TRITC filter for the acidic pH readings and Cy5 filter for the basic pH readings. The absorbance was determined with a microplate reader at 460 nm. Cell viability was evaluated by monitoring signal intensity at different dye concentrations. The absorbance intensity is directly proportional to the amount of formazan dye produced, which is indicative of the number of living cells in the medium, allowing quantification of cell viability.


Results and Discussion

The pH titration experiments of RatioWorks™ pH 165, AM at 15 µM were performed in various buffer solutions containing 5% DMSO. With a decrease in pH from 10.1 to 4.05, the maximum absorbance at 578 nm was blue shifted to obtain an absorption peak at 478 nm (Figure 1a). A well-defined isosbestic point at around 497 nm was observed in the absorption spectra. The fluorescence emissions of the dye for pH 10.1 to 4.05 obtained by excitation at this point were recorded. At pH 10.1 solution, the probe exhibits only one emission peak at about 654 nm. With an increase in proton concentration, the fluorescence intensity of the probe at 654 nm was reduced and a new emission band at 594 nm concomitantly emerged (Figure 1b).

Optical responses of RatioWorks™ pH 165 (15 µM) at various pH values with buffers in DMSO. (Left) Absorbance Spectra (right) Emission Spectra.

Fluorescence images of live HeLa cells with RatioWorks™ pH 165 at various pH values (4 to 9) with buffers in DMSO. (a) Green channels (Pseudo Color) with Cy3/TRITC filter were collected in 570-640 nm (b) Red channels with Cy5 filter were collected in 662-737 nm.
The above results provide evidence for how ratiometric fluorescent measurements, which are based on the ratio of two fluorescent bands instead of the absolute intensity of one band, make quantitative analyses more accurate and sensitive by minimizing background signals. The fluorophores display dual emission with a change in pH from basic to acidic conditions. This is caused by a combination of excited state intramolecular proton transfer (ESIPT) and an intramolecular charge transfer (ICT), which allows these fluorophores to demonstrate unique sensitivity to subtle variations in their immediate environment that are independent of the probe concentration and other variables. A pKa value of 6.80 was obtained by analyzing the fluorescence ratios at 594 nm and 654 nm. Further observations include color changes (violet to yellow) under visible light, indicating stable equilibrium states. Lastly, the RatioWorks™ pH 165, AM fluorescent indicator shows large Stokes shifts. This improves detection sensitivity by reducing self-quenching that can cause measurement errors.

Cell imaging was initially performed on HeLa cell lines containing a dye concentration of 5-20 µM incubated at 37°C for 30 min and observed at different pH values (4 to 9) using a fluorescence microscope with Cy3/TRITC and Cy5 filter. As pH in the cells increases, the red fluorescence (Cy5 channel) of the dye goes up, while the green fluorescence (Cy3/TRITC channel) goes down. Although the dye works as intended, high concentration of the dye along with a longer incubation time was recommended for consideration in future experiments.

Consequently, in a follow up study, a higher dye concentration of 35 µM incubated at 37°C for 30 min was analyzed keeping every other parameter constant. The results demonstrate a significant improvement in the fluorescence signal intensity in both channels (Figure 2). To quantitatively determine pH values, ratiometric analysis at the dual excitation and emission wavelengths (Ex/Em = 497/594 nm and 578/654 nm, respectively) was carried out (Table 1). The calculated data was in trend with the results obtained from cell image analysis i.e. the ratio of the intensities tend to diminish with an increase in intracellular pH.

Table 1. Ratiometric Analysis of RatioWorks™ pH 165, AM at pH 4.5, 5, 6, 7, 8, and 9

630.0 7.93.8
721.7 6.13.5

Various incubation time periods (30, 60, and 90 min) were explored to optimize optical properties of RatioWorks™ pH 165, AM (Figure 3a, b). Keeping the dye concentration constant (30-50 µM), the same procedure was applied at pH values 4.5 and 9.1. The results demonstrate an increase in signal intensity in both Cy3/TRITC and Cy5 channels, indicating that longer incubation time improves image quality.

Effect of incubation time (30, 60, and 90 min) on fluorescence images of live HeLa cells with RatioWorks™ pH 165, AM at an exposure time of 1 sec and pH of 4.5 and 9.1, respectively. (a) Green channels (Pseudo Color) with Cy3/TRITC filter were collected in 570-640 nm (b) Red channels with Cy5 filter were collected in 662-737 nm.

Cell cytotoxicity measurement at various concentrations of RatioWorks™ pH 165, AM (10, 20, 50, and 100 µM) using CCK-8 assay at a wavelength of 460 nm and an incubation period of 120 min post WST-8 addition.
Cytotoxicity of RatioWorks™ pH 165, AM dye in HeLa cells was studied using CCK-8 assay. Figure 4 shows the toxicity measurements after two hours of cellular internalization of the probe at concentrations of 10 µM, 20 µM, 50 µM, and 100 µM against a control (DMSO) at its respective concentrations. The results indicate low toxicity of the dye up to a concentration of 50 µM where the cell viability remained more than 90% with respect to DMSO. While at a dye concentration of 100 µM, the cell viability was reduced significantly as cells tend to exhibit adverse effects. Based on the findings, it can be concluded that a tolerable dye concentration will go up to 50 µM, based on the cell type and application. The low cytotoxic nature of RatioWorks™ pH 165, AM indicator makes it an ideal tool for handling pH fluctuations in live cell fluorescence imaging.


A novel dual excitation/emission fluorescent pH indicator has been developed by AAT Bioquest for sensitive and selective determination of intracellular pH in cellular processes. RatioWorks™ pH 165, AM displays a distinguished ratiometric fluorescent response with well separated emission bands and a large stokes shift that favors resolution fluorescence imaging. With its ratiometric fluorescence imaging abilities, the probe is able to eliminate the influences of the microenvironment, localized probe distribution, and instrumental parameters. This explains the enhanced accuracy of RatioWorks™ pH 165, AM in monitoring molecular events by responding to near-neutral pH environments in live cells. In contrast to other fluorescein derivatives, RatioWorks™ pH 165, AM dye exhibits responses in the red and far red spectrum, making it ideal for multiplex immunoassays with GFP, where the dye prevents spectrum interference and saturation. This feature is a clear distinction of RatioWorks™ pH 165, AM from other fluorescent based assay technologies used in similar applications.



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