Novel fluorescent oxygen indicator for intracellular oxygen measurements (original) (raw)
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AJP: Regulatory, Integrative and Comparative Physiology, 2006
Papkovsky DB. Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging. The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptormediated Ca 2ϩ influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid, and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas, such as cardiovascular and neurological research, metabolic diseases, and cancer. metalloporphyrin; mitochondrial function; uncoupling
High resolution imaging of intracellular oxygen concentration by phosphorescence lifetime
Scientific Reports
Optical methods using phosphorescence quenching by oxygen are suitable for sequential monitoring and non-invasive measurements for oxygen concentration (OC) imaging within cells. Phosphorescence intensity measurement is widely used with phosphorescent dyes. These dyes are ubiquitously but heterogeneously distributed inside the whole cell. The distribution of phosphorescent dye is a major disadvantage in phosphorescence intensity measurement. We established OC imaging system for a single cell using phosphorescence lifetime and a laser scanning confocal microscope. This system had improved spatial resolution and reduced the measurement time with the high repetition rate of the laser. By the combination of ubiquitously distributed phosphorescent dye with this lifetime imaging microscope, we can visualize the OC inside the whole cell and spheroid. This system uses reversible phosphorescence quenching by oxygen, so it can measure successive OC changes from normoxia to anoxia. Lower regio...
Intracellular measurement of oxygen by quenching of fluorescence of pyrenebutyric acid☆
Biochimica Et Biophysica Acta - General Subjects, 1972
We have examined pyrenebutyric acid as a possible fluorescent probe to determine intracellular concentrations of oxygen. Isolated rat liver cells rapidly take up pyrenebutyric acid, and the fluorescence of the cells is quenched by oxygen to the same extent as is free pyrenebutyric acid. Therefore, this technique appears to be applicable to the physiological range of oxygen concentrations. The observation of Vaughan and Weber I that oxygen concentrations in the physiological range would quench the fluorescence of pyrenebutyric acid in solution suggested to us that this principle might be used to measure intracellular PO2. Such a technique could eliminate the disadvantages encountered with techniques using electrodes, namely tissue damage and alteration of oxygen tensions by consumption of oxygen, providing that the following criteria could be fulfilled: (a) Penetration of the fluorescent probe into the cell. (b) Absence of metabolic damage as determined by respiration rate. (c) The quenching of the fluorescence of the intracellular probe by oxygen should occur according to a known relationship, preferably Stern-Volmer 2 .
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.