Ratiometric pH Measurements Using LysoSensor DND-192

Abstract

A method for the ratiometric pH sensing using LysoSensor DND-192 is presented in this paper. It works in the physiological pH range. It is based on the use of two fluorophores which differ significantly in their lifetimes. As the discrimination of their emissions is performed through two different frequencies, this method can allow significant overlap of the emission spectra. A simple long-pass filter, or a combination of long-and short-pass filters, was used instead of narrow-bandpass devices. Importantly, the measurements were carried out under strong ambient light. The method could be used in a wide variety of applications, such as intracellular measurements, microscopy, bioprocess monitoring, etc.

Fluorescent Indicators for Intracellular pH

“…Many probes are available from Life Technologies for measurement of acidic pH i values. A pyridyl oxazol probe Yellow/Blue DND-160 PDMPO 59 , the anthrathene-based sensor DND-167 60 , DND-189 61 , DND-153 62 , and DND 192 63 , – (Figure ) are dyes that work on the principle of that electronic excited states can be quenched before they fluoresce by electron transfer from amines; this process is known as PeT. , Dyes of this type become more emissive in acidic environments when proximal amines are protonated. Of all the lysosensors shown in Figure , DND-160 59 is unique because it is brightly fluorescent in protonated and deprotonated forms (ϕ ≈ 0.4 for both forms), and its absorbance and fluorescence spectra are significantly blue shifted with isosbestic points at 365 and 470 nm as the pH values are increased.…”

“…Many probes are available from Life Technologies for measurement of acidic pH i values. A pyridyl oxazol probe Yellow/Blue DND-160 PDMPO 59, 119 the anthrathene-based sensor DND-167 60, 120 DND-189 61, DND-153 62, and DND 192 63, [120][121][122] (Figure 7) are dyes that work on the principle of that electronic excited states can be quenched before they fluoresce by electron transfer from amines; this process is known as PeT. 123,124 Dyes of this type become more emissive in acidic environments when proximal amines are protonated.…”

Imaging Lysosomal pH Alteration in Stressed Cells with a Sensitive Ratiometric Fluorescence Sensor

“…The reversed pH responses of rhodamine over coumarin enable imaging of subtle acidification in the range of pH 6.0–4.5 by pronounced changes in ratios of fluorescence intensity (Figure D,E). For instance, I 605 nm / I 485 nm interval between pH 4.5 and pH 5.5 was 70, whereas commercial lysosensors often exhibited moderate ratio changes (around 0.3) per pH unit (Figure D) . Overlay of the optimal sensing range of CM-ROX (pH 6.0–4.5) with lysosome acidity window (pH 6.0–4.5) indicates the applicability of CM-ROX to monitor lysosomal pH changes.…”

“…For instance, I 605 nm / I 485 nm interval between pH 4.5 and pH 5.5 was 70, whereas commercial lysosensors often exhibited moderate ratio changes (around 0.3) per pH unit (Figure 1D). 10 Overlay of the optimal sensing range of CM-ROX (pH 6.0−4.5) with lysosome acidity window (pH 6.0−4.5) indicates the applicability of CM-ROX to monitor lysosomal pH changes.…”

Dynamics of Supercoiled and Linear pBluescript II SK(+) Phagemids Probed with a Long-lifetime Metal-ligand Complex

“…The intensity decays were recovered from the FD data in terms of a multiexponential model using nonlinear least squares analysis Lakowicz et al, 1984;Lakowicz and Gryczinski, 1991;Kang and Lakowicz, 2001;Kang and Kostov, 2002;Kang et al, 2002a,b,c): (2) where the preexponential factor α i is the amplitude of each component, Σα i = 1.0, τ i is the decay time, and n is the number of exponential components. Mean lifetimes were calculated by: (3) where f i is the fractional steady-state contribution of each component to the total emission, and Σf i is normalized to unity.…”