Intracellular measurement of oxygen by quenching of fluorescence of pyrenebutyric acid☆ (original) (raw)
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Novel fluorescent oxygen indicator for intracellular oxygen measurements
Journal of Biomedical Optics, 2002
Photo-Optical Instrumentation Engineers (SPIE). One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this publication for a fee or for commercial purposes, and modification of the contents of the publication are prohibited. Access to this work was provided by the University of Maryland, Baltimore County (UMBC) ScholarWorks@UMBC digital repository on the Maryland Shared Open Access (MD-SOAR) platform.
Measurements of pertinent concentrations of oxygenin vivo
Magnetic Resonance in Medicine, 1991
A new method able to measure the concentration of oxygen in complex biological systems, including in vivo, has been developed using low-frequency EPR and newly characterized free radicals that are very sensitive to the concentration of oxygen. The free radicals (fusinite and lithium phthalocyanine) are very stable in tissues (for at least 150 days), apparently nontoxic, and can reflect oxygen concentrations that are less than the K,,, of cytochrome oxidase (0. I p M or lower). Their biological stability is indicated by the fact that repeated measurements with fusinite of the concentration of oxygen in skeletal muscle have been made in the same animal for more than 150 days without any change in sensitivity or signs of toxicity. o
Measurement of the intracellular concentration of oxygen in a cell perfusion system
Magnetic Resonance in Medicine, 1994
OJ was measured in the embedding material (alginate) in a typical apparatus for conducting studies of viable cells with NMR, using low frequency EPR. In suspension cultures respiration was independent of [O,] in the perfusing media down to about 1 pM while in alginate beads, the comparable value was 70 pM, indicating that the alginate was a very substantial barrier to the free diffusion of oxygen. With knowledge of [O,] in the various compartments, [OJ in the perfusing medium can be increased and the full power of NMR can be used to provide information on metabolism under various conditions. These results also provide evidence supporting the feasibility and usefulness of EPR techniques using nitroxides to measure [O,] in macroscopic samples such as NMR perfusion tubes. This technique is rapid, apparently nonperturbing, and enables one to differentiate between the concentrations of oxygen in different compartments.
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
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.
Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene
The Journal of General Physiology, 1975
Quenching of pyrene fluorescence by oxygen was used to determine oxygen diffusion coeffÉcients in phospholipid dispersions and erythrocyte plasma membranes. The fluorescence intensity and lifetime of pyrene in both artificial and natural membranes decreases about 80 % in the presence of 1 atm O,, while the fluorescence excitation and emission spectra and the absorption spectrum are unaltered. Assuming the oxygen partition coefficient between membrane and aqueous phase to be 4.4, the diffusion coefficients for oxygen at 37°C are 1.51 X 10 -5 cm2/s in dimyristoyl lecithin vesicles, 9.32 X 10 -6 cm~/s in dipalmitoyl lecithin vesicles, and 7.27 X I0 -s em2/s in erythroeyte plasma membranes. The heats of activation for oxygen diffusion are low (< 3 kcal/degree-mol). A dramatic increase in the diffusion constant occurs at the phase transition of dimyristoyl and dipalmitoyl lecithin, which may result from an increase in either the oxygen diffusion coefficient, partition coefficient, or both. The significance of the change in oxygen diffusion below and above the phase transition for biological membranes is discussed.
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...