A continuous distribution approach, instead of the traditional mono- and multiexponential analysis, for determining quencher concentration in a heterogeneous system has been developed. A mathematical model of phosphorescence decay inside a volume with homogeneous concentration of phosphor and heterogeneous concentration of quencher was formulated to obtain pulse-response fitting functions for four different distributions of quencher concentration: rectangular, normal (Gaussian), gamma, and multimodal. The analysis was applied to parameter estimates of a heterogeneous distribution of oxygen tension (PO2) within a volume. Simulated phosphorescence decay data were randomly generated for different distributions and heterogeneity of PO2 inside the excitation/emission volume, consisting of 200 domains, and then fit with equations developed for the four models. Analysis using a monoexponential fit yielded a systematic error (underestimate) in mean PO2 that increased with the degree of heterogeneity. The fitting procedures based on the continuous distribution approach returned more accurate values for parameters of the generated PO2 distribution than did the monoexponential fit. The parameters of the fit (M = mean; sigma = standard deviation) were investigated as a function of signal-to-noise ratio (SNR = maximum signal amplitude/peak-to-peak noise). The best-fit parameter values were stable when SNR > or = 20. All four fitting models returned accurate values of M and sigma for different PO2 distributions. The ability of our procedures to resolve two different heterogeneous compartments was also demonstrated using a bimodal fitting model. An approximate scheme was formulated to allow calculation of the first moments of a spatial distribution of quencher without specifying the distribution. In addition, a procedure for the recovery of a histogram, representing the quencher concentration distribution, was developed and successfully tested.

A scanning phosphorescence quenching microscopy technique, designed to prevent accumulated O(2) consumption by the method, was applied to Po(2) measurements in mesenteric tissue. In an attempt to further increase the accuracy of the measurements, albumin-bound probe was topically applied to the tissue and an objective-mounted pressurized bag was used to reduce the oxygen transport bypass through the thin layer of fluid over the mesentery. Po(2) was measured at multiple sites perpendicular to the blood/wall interface in the vicinity of 84 mesenteric arterioles (7-39 microm in diameter) at distances of 5, 15, 30, 60, 120, and 180 microm in seven anesthetized Sprague-Dawley rats, thereby creating Po(2) profiles. Interstitial Po(2) above and immediately beside arterioles was found to agree with known intravascular values. No significant difference in Po(2) profiles was found between small and large arterioles, indicating a small longitudinal Po(2) gradient in the precapillary mesenteric microvasculature. In addition, the Po(2) profiles were used to calculate oxygen consumption in the mesenteric tissue (56-65 nl O(2) x cm(-3) x s(-1)). Correction of these values for contamination with ambient oxygen yielded an oxygen consumption rate of 60-68 nl O(2) x cm(-3) x s(-1), the maximal limit for consumption in the mesentery. The results were compared with measurements made by other workers in regard to the employed techniques.

We have applied the phosphorescence lifetime technique (Vanderkooi, J. M., G. Maniara, T. J. Green, and D. F. Wilson. J. Biol. Chem. 262: 5476-5482, 1987) to determine oxygen tension in single capillaries of the hamster retractor muscle. Palladium meso-tetra(4-carboxyphenyl)porphine (10 mg/ml, pH 7.40, bound to bovine serum albumin) was used as the phosphorescent oxygen sensor. Our measurement system consisted of a microscope configured for epi-illumination, a strobe flash lamp, a 430-nm bandpass excitation filter, and a 630-nm cut-on emission filter. A rectangular diaphragm was used to limit the illumination field to 10 microns x 10 microns, and an end-window photomultiplier tube was used to detect the phosphorescence signal, which was then input to an analog-to-digital board in a personal computer. In vitro calibrations were carried out at 37 degrees C on samples flowing through a glass capillary tube (diameter, 300 microns) at four different O2 concentrations (0, 2.5, 5, and 7.5%). In vivo tests were carried out on arterioles, capillaries, and venules of the retractor muscle of anesthetized hamsters. The phosphorescent compound was administered by injection into a jugular vein (20 mg/kg). Phosphorescence decay curves were analyzed by a new model of heterogeneous oxygen distribution in the excitation/emission volume. Mean Po2 values and the local Po2 gradients within the excitation/ emission volume were calculated from phosphorescence life-times obtained from individual decay curves. The time course of Po2 obtained during 0.5-s measurement periods (5 decay curves at 0.1-s intervals) at a given site along a capillary indicated the presence of a gradient in Po2 within the plasma space between and near red blood cells. Similar Po2 gradients were also detected in arterioles and venules. Mean Po2 values for arterioles, capillaries, and venules over the 0.5-s observation period were 27 +/- 5, 14 +/- 2, and 11 +/- 3 (SD) mmHg, respectively. The magnitude of the Po2 gradient in the arterioles, capillaries, and venules was 6 +/- 1, 4 +/- 1, and 2 +/- 1 mmHg/micron, respectively.

Golub AS, Pittman RN. Oxygen dependence of respiration in rat spinotrapezius muscle in situ. Am J Physiol Heart Circ Physiol 303: H47-H56, 2012. First published April 20, 2012 doi:10.1152/ajpheart.00131.2012.-The oxygen dependence of respiration in striated muscle in situ was studied by measuring the rate of decrease of interstitial PO 2 [oxygen disappearance curve (ODC)] following rapid arrest of blood flow by pneumatic tissue compression, which ejected red blood cells from the muscle vessels and made the ODC independent from oxygen bound to hemoglobin. After the contribution of photo-consumption of oxygen by the method was evaluated and accounted for, the corrected ODCs were converted into the PO2 dependence of oxygen consumption, V O2, proportional to the rate of PO2 decrease. Fitting equations obtained from a model of heterogeneous intracellular PO2 were applied to recover the parameters describing respiration in muscle fibers, with a predicted sigmoidal shape for the dependence of V O2 on PO2. This curve consists of two regions connected by the point for critical PO2 of the cell (i.e., PO2 at the sarcolemma when the center of the cell becomes anoxic). The critical PO 2 was below the PO2 for half-maximal respiratory rate (P50) for the cells. In six muscles at rest, the rate of oxygen consumption was 139 Ϯ 6 nl O2/cm 3 ·s and mitochondrial P50 was k ϭ 10.5 Ϯ 0.8 mmHg. The range of PO2 values inside the muscle fibers was found to be 4 -5 mmHg at the critical PO2. The oxygen dependence of respiration can be studied in thin muscles under different experimental conditions. In resting muscle, the critical PO2 was substantially lower than the interstitial PO2 of 53 Ϯ 2 mmHg, a finding that indicates that V O2 under this circumstance is independent of oxygen supply and is discordant with the conventional hypothesis of metabolic regulation of the oxygen supply to tissue. skeletal muscle; respiratory rate; interstitial PO2; oxygen disappearance curve; phosphorescence quenching method; cell PO2 gradient THE COORDINATION OF OXYGEN demand and supply in skeletal muscle and in the heart is carried out by a mechanism not yet completely understood. Current cardiovascular texts propose the century-old hypothesis of metabolic control of capillary blood flow as the accepted theory of local autoregulation (review Ref. 38). According to this hypothesis, a decline of oxygen delivery leads to a decrease of intracellular PO 2 , an evoked release of a metabolic vasodilator into the extracellular space, the dilation of arterioles, and, eventually, an increase in the flow velocity of blood and number of perfused capillaries. A key aspect of this model is the oxygen dependence of cellular metabolism, making the mitochondria or entire cell sensitive to an inadequate oxygen supply (11).The oxygen dependence of respiration for isolated mitochondria and cells is represented by a hyperbolic curve empirically described by Hill's equation (Eq. 10) (29,39,46,59,60) with the parameters V m (maximal respiratory rate), Hill coefficient ϭ 1 to 1.4...

In phosphorescence quenching microscopy (PQM), the multiple excitation of a reference volume produces the integration of oxygen consumption artifacts caused by individual flashes. We analyzed the performance of two types of PQM instruments to explain reported data on Po2 in the microcirculation. The combination of a large excitation area (LEA) and high flash rate produces a large oxygen photoconsumption artifact manifested differently in stationary and flowing fluids. A LEA instrument strongly depresses Po2 in a motionless tissue, but less in flowing blood, creating an apparent transmural Po2 drop in arterioles. The proposed model explains the mechanisms responsible for producing apparent transmural and longitudinal Po2 gradients in arterioles, a Po2 rise in venules, a hypothetical high respiration rate in the arteriolar wall and mesenteric tissue, a low Po2 in lymphatic microvessels, and both low and uniform tissue Po2. This alternative explanation for reported paradoxical results of Po2 distribution in the microcirculation obviates the need to revise the dominant role of capillaries in oxygen transport to tissue. Finding a way to eliminate the photoconsumption artifact is crucial for accurate microscopic oxygen measurements in microvascular networks and tissue. The PQM technique that employs a small excitation area (SEA) together with a low flash rate was specially designed to avoid accumulated oxygen photoconsumption in flowing blood and lymph. The related scanning SEA instrument provides artifact-free Po2 measurements in stationary tissue and motionless fluids. Thus the SEA technique significantly improves the accuracy of microscopic Po2 measurements in the microcirculation using the PQM.