Novel fluorescent oxygen indicator for intracellular oxygen measurements (original) (raw)
Singlet Oxygen Sensor Green®: Photochemical Behavior in Solution and in a Mammalian Cell
Photochemistry and Photobiology, 2011
The development of efficient and selective luminescent probes for reactive oxygen species, particularly for singlet molecular oxygen, is currently of great importance. In this study, the photochemical behavior of Singlet Oxygen Sensor Green Ò (SOSG), a commercially available fluorescent probe for singlet oxygen, was examined. Despite published claims to the contrary, the data presented herein indicate that SOSG can, in fact, be incorporated into a living mammalian cell. However, for a number of reasons, caution must be exercised when using SOSG. First, it is shown that the immediate product of the reaction between SOSG and singlet oxygen is, itself, an efficient singlet oxygen photosensitizer. Second, SOSG appears to efficiently bind to proteins which, in turn, can influence uptake by a cell as well as behavior in the cell. As such, incorrect use of SOSG can yield misleading data on yields of photosensitized singlet oxygen production, and can also lead to photooxygenation-dependent adverse effects in the system being investigated.
A Highly Efficient Chemiluminescence Probe for the Detection of Singlet Oxygen in Living Cells
Angewandte Chemie, 2017
Singlet oxygen is among the reactive oxygen species (ROS) with the shortest lifetimes in aqueous media because of its extremely high reactivity.T herefore,d esigning sensors for detection of 1 O 2 is perhaps one of the most challenging tasks in the field of molecular probes.H erein, we report ah ighly selective and sensitive chemiluminescence probe (SOCL-CPP) for the detection of 1 O 2 in living cells.The probe reacts with 1 O 2 to form ad ioxetane that spontaneously decomposes under physiological conditions through achemiexcitation pathway to emit green light with extraordinary intensity. SOCL-CPP demonstrated promising ability to detect and image intracellular 1 O 2 produced by aphotosensitizer in HeLa cells during photodynamic therapy(PDT) mode of action. Our findings make SOCL-CPP the most effective knownc hemiluminescence probe for the detection of 1 O 2 .W ea nticipate that our chemiluminescence probe for 1 O 2 imaging would be useful in PDT-related applications and for monitoring 1 O 2 endogenously generated by cells in response to different stimuli.
Ratiometric Fluorescent Probes for Detection of Intracellular Singlet Oxygen
Organic Letters, 2013
We have developed a series of molecular probes for the fluorescent detection of singlet dioxygen (1 O 2). The probes, based on asymmetrically substituted 1,3-diarylisobenzofurans, undergo the [2 þ 4] cycloaddition reaction with 1 O 2 , producing ratiometric fluorescent responses. Two-photon fluorescence microscope experiments demonstrated the biological utility of the probes for the visualization of endogenous 1 O 2 in macrophage cells.
Journal of Applied Physiology, 2005
In the present work, a novel method for detecting hypoxia in tumors, phosphorescence quenching, was used to evaluate tissue and tumor oxygenation. This technique is based on the concept that phosphorescence lifetime and intensity are inversely proportional to the oxygen concentration in the tissue sample. We used the phosphor Oxyphor G2 to evaluate the oxygen profiles in three murine tumor models: K1735 malignant melanoma, RENCA renal cell carcinoma, and Lewis lung carcinoma. Oxygen measurements were obtained both as histograms of oxygen distribution within the sample and as an average oxygen pressure within the tissue sampled; the latter allowing real-time oxygen monitoring. Each of the tumor types examined had a characteristic and consistent oxygen profile. K1735 tumors were all well oxygenated, with a peak oxygen pressure of 37.8 ± 5.1 Torr; RENCA tumors had intermediate oxygen pressures, with a peak oxygen pressure of 24.8 ± 17.9 Torr; and LLC tumors were all severely hypoxic, w...
Oxyphor R2 and G2: phosphors for measuring oxygen by oxygen-dependent quenching of phosphorescence
2002
Oxygen-dependent quenching of phosphorescence is a useful and essentially noninvasive optical method for measuring oxygen in vivo and in vitro. Calibration of the phosphors is absolute, and once phosphors have been calibrated in one laboratory the same constants can be used by anyone else as long as the measurement is done under the same conditions. Two new phosphors, one based on Pd-meso-tetra-(4-carboxyphenyl)porphyrin and the other on Pd-meso-tetra-(4-carboxyphenyl)tetrabenzoporphyrin, are very well suited to in vivo oxygen measurements. Both phosphors are Generation 2 polyglutamic Pd-porphyrin-dendrimers, bearing 16 carboxylate groups on the outer layer. These phosphors are designated Oxyphor R2 and Oxyphor G2, respectively. Both are highly soluble in biological fluids such as blood plasma and their ability to penetrate biological membranes is very low. The maxima in the absorption spectra are at 415 and 524 nm for Oxyphor R2 and 440 and 632 nm for Oxyphor G2, while emissions are near 700 and 800 nm, respectively. The calibration constants of the phosphors are essentially independent of pH in the physiological range (6.4 to 7.8). In vivo application is demonstrated by using Oxyphor G2 to noninvasively determine the oxygen distribution in a subcutaneous tumor growing in rats.
Microvascular and mitochondrial PO2 simultaneously measured by oxygen-dependent delayed luminescence
2012
Measurement of tissue oxygenation is a complex task and various techniques have led to a wide range of tissue PO 2 values and contradictory results. Tissue is compartmentalized in microcirculation, interstitium and intracellular space and current techniques are biased towards a certain compartment. Simultaneous oxygen measurements in various compartments might be of great benefit for our understanding of determinants of tissue oxygenation. Here we report simultaneous measurement of microvascular PO 2 (mPO 2 ) and mitochondrial PO 2 (mitoPO 2 ) in rats. The mPO 2 measurements are based on oxygen-dependent quenching of phosphorescence of the near-infrared phosphor Oxyphor G2. The mitoPO 2 measurements are based on oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Favorable spectral properties of these porphyrins allow simultaneous measurement of the delayed luminescence lifetimes. A dedicated fiber-based time-domain setup consisting of a tunable pulsed laser, 2 red-sensitive gated photomultiplier tubes and a simultaneous sampling dataacquisition system is described in detail. The absence of cross talk between the channels is shown and the feasibility of simultaneous mPO 2 and mitoPO 2 measurements is demonstrated in rat liver in vivo. It is anticipated that this novel approach will greatly contribute to our understanding of tissue oxygenation in physiological and pathological circumstances. Schematic representation of the measuring concept.
2010
Extracellular oxygen concentrations influence cell metabolism and tissue function. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods show limited spatial resolution and/or require custom made systems. This study describes a new optimised approach for quantitative extracellular oxygen detection, providing an off-the-shelf system with high spatial resolution and an improved lifetime determination over previous techniques, while avoiding systematic photon pile-up. Fluorescence lifetime detection of an oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]2+, was measured using a Becker&Hickl time-correlated single photon counting (TCSPC) card with excitation provided by a multi-photon laser. This technique was able to identify a subpopulation of isolated chondrocyte cells, seeded in three-dimensional agarose gel, displaying a significant spatial oxygen gradient. Thus this technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.
Highly Efficient Chemiluminescence Probe for the Detection of Singlet Oxygen in Living Cells
Angewandte Chemie (International ed. in English), 2017
Singlet oxygen is among the reactive oxygen species (ROS) with the shortest life-times in aqueous media because of its extremely high reactivity. Therefore, designing sensors for detection of (1) O2 is perhaps one of the most challenging tasks in the field of molecular probes. Herein, we report a highly selective and sensitive chemiluminescence probe (SOCL-CPP) for the detection of (1) O2 in living cells. The probe reacts with (1) O2 to form a dioxetane that spontaneously decomposes under physiological conditions through a chemiexcitation pathway to emit green light with extraordinary intensity. SOCL-CPP demonstrated promising ability to detect and image intracellular (1) O2 produced by a photosensitizer in HeLa cells during photodynamic therapy (PDT) mode of action. Our findings make SOCL-CPP the most effective known chemiluminescence probe for the detection of (1) O2 . We anticipate that our chemiluminescence probe for (1) O2 imaging would be useful in PDT-related applications and...
Can redox-sensitive fluorescent probes measure intracellular redox potential?
The generation of ratiometric, glutathione specific, redox-sensitive variants of fluorescent probes enable real-time monitoring of intracellular redox changes by making it possible to cancel out most or all of the possible variability caused by instrument efficiency and effective dye content. The probes permit an indirect measure of the glutathione intracellular redox potential EG, achieved by using the Nernst equation after the measurement of intracellular glutathione redox state. However, EG is strongly dependent on pH and on the total concentration of glutathione, and since their intracellular spatial distribution is not isotropic, the measurement of EG may be affected by systematic errors. Here, we show that these errors are not negligible and we suggest possible methods to cancel them out when measuring the redox state of the glutathione redox couple. PACS 87.16.Xa -Signal transduction and intracellular signaling. PACS 87.64.Kv -Fluorescence. PACS 87.85.Pq -Biomedical imaging.
An Effective, Versatile, and Inexpensive Device for Oxygen Uptake Measurement
Journal of clinical medicine, 2017
In the last ten years, the use of fluorescent probes developed to measure oxygen has resulted in several marketed devices, some unreasonably expensive and with little flexibility. We have explored the use of the effective, versatile, and inexpensive Redflash technology to determine oxygen uptake by a number of different biological samples using various layouts. This technology relies on the use of an optic fiber equipped at its tip with a membrane coated with a fluorescent dye (www.pyro-science.com). This oxygen-sensitive dye uses red light excitation and lifetime detection in the near infrared. So far, the use of this technology has mostly been used to determine oxygen concentration in open spaces for environmental studies, especially in aquatic media. The oxygen uptake determined by the device can be easily assessed in small volumes of respiration medium and combined with the measurement of additional parameters, such as lactate excretion by intact cells or the membrane potential ...