Aleksander S. Golub

Analysis of phosphorescence in heterogeneous systems using distributions of quencher concentration

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.

Po2 measurements in the microcirculation using phosphorescence quenching microscopy at high magnification

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.

Thermostatic animal platform for intravital microscopy of thin tissues.

We describe a novel temperature-controlled, all-on-board design platform for intravital microscopy of thin tissues in small laboratory animals. The apparatus uses transparent heaters and miniature controllers to control independently the temperature of the tissue pedestal and animal heating pad, as well as the animal core temperature. The system ensures a uniform temperature for a thin tissue placed on the surface of a transparent window and maintenance of the animals core temperature without overheating. This platform provides an alternative to warm superfusion solution for in vivo microscopy, under circumstances where a well-controlled temperature is required. All components of the apparatus are commercially available, inexpensive and reliable, thereby simplifying its assembly. The platform is convenient for use with trans- and epi-illumination and does not interfere with movement of the microscope stage.

Phosphorescence quenching microrespirometry of skeletal muscle in situ

We have developed an optical method for the evaluation of the oxygen consumption (Vo(2)) in microscopic volumes of spinotrapezius muscle. Using phosphorescence quenching microscopy (PQM) for the measurement of interstitial Po(2), together with rapid pneumatic compression of the organ, we recorded the oxygen disappearance curve (ODC) in the muscle of the anesthetized rats. A 0.6-mm diameter area in the tissue, preloaded with the phosphorescent oxygen probe, was excited once a second by a 532-nm Q-switched laser with pulse duration of 15 ns. Each of the evoked phosphorescence decays was analyzed to obtain a sequence of Po(2) values that constituted the ODC. Following flow arrest and tissue compression, the interstitial Po(2) decreased rapidly and the initial slope of the ODC was used to calculate the Vo(2). Special analysis of instrumental factors affecting the ODC was performed, and the resulting model was used for evaluation of Vo(2). The calculation was based on the observation of only a small amount of residual blood in the tissue after compression. The contribution of oxygen photoconsumption by PQM and oxygen inflow from external sources was evaluated in specially designed tests. The average oxygen consumption of the rat spinotrapezius muscle was Vo(2) = 123.4 ± 13.4 (SE) nl O(2)/cm(3) · s (N = 38, within 6 muscles) at a baseline interstitial Po(2) of 50.8 ± 2.9 mmHg. This technique has opened the opportunity for monitoring respiration rates in microscopic volumes of functioning skeletal muscle.

Oxygen dependence of respiration in rat spinotrapezius muscle in situ.

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 Po(2) dependence of oxygen consumption, Vo(2), proportional to the rate of Po(2) decrease. Fitting equations obtained from a model of heterogeneous intracellular Po(2) were applied to recover the parameters describing respiration in muscle fibers, with a predicted sigmoidal shape for the dependence of Vo(2) on Po(2). This curve consists of two regions connected by the point for critical Po(2) of the cell (i.e., Po(2) at the sarcolemma when the center of the cell becomes anoxic). The critical Po(2) was below the Po(2) for half-maximal respiratory rate (P(50)) for the cells. In six muscles at rest, the rate of oxygen consumption was 139 ± 6 nl O(2)/cm(3)·s and mitochondrial P(50) was k = 10.5 ± 0.8 mmHg. The range of Po(2) values inside the muscle fibers was found to be 4-5 mmHg at the critical Po(2). The oxygen dependence of respiration can be studied in thin muscles under different experimental conditions. In resting muscle, the critical Po(2) was substantially lower than the interstitial Po(2) of 53 ± 2 mmHg, a finding that indicates that Vo(2) under this circumstance is independent of oxygen supply and is discordant with the conventional hypothesis of metabolic regulation of the oxygen supply to tissue.

Bang-bang model for regulation of local blood flow.

The classical model of metabolic regulation of blood flow in muscle tissue implies the maintenance of basal tone in arterioles of resting muscle and their dilation in response to exercise and/or tissue hypoxia via the evoked production of vasodilator metabolites by myocytes. A century‐long effort to identify specific metabolites responsible for explaining active and reactive hyperemia has not been successful. Furthermore, the metabolic theory is not compatible with new knowledge on the role of physiological radicals (e.g., nitric oxide, NO, and superoxide anion, O2−) in the regulation of microvascular tone. We propose a model of regulation in which muscle contraction and active hyperemia are considered the physiologically normal state. We employ the “bang‐bang” or “on/off” regulatory model which makes use of a threshold and hysteresis; a float valve to control the water level in a tank is a common example of this type of regulation. Active bang‐bang regulation comes into effect when the supply of oxygen and glucose exceeds the demand, leading to activation of membrane NADPH oxidase, release of O2− into the interstitial space and subsequent neutralization of the interstitial NO. Switching arterioles on/off when local blood flow crosses the threshold is realized by a local cell circuit with the properties of a bang‐bang controller, determined by its threshold, hysteresis, and dead‐band. This model provides a clear and unambiguous interpretation of the mechanism to balance tissue demand with a sufficient supply of nutrients and oxygen.

Interstitial PO2Determination by Phosphorescence Quenching Microscopy

Objective: This study introduces the technique of microinjection of phosphor probe into skeletal muscle tissue to determine oxygen tension (Po2) in the interstitium by phosphorescence quenching microscopy.

Interpretation of Phosphorescence Quenching Measurements Made in the Presence of Oxygen Gradients

The quenching of phosphorescence by molecular oxygen has proved to be a useful, noninvasive technique for determining oxygen tension (PO2) or dissolved oxygen concentration ([O2]) in blood vessels and tissues (Vanderkooi et al., 1987; Shonat et al., 1992 & 1995; Torres Filho and Intaglietta, 1993; Torres Filho et al., 1994 & 1996; Kerger et al., 1995; Zheng et al., 1996). The technique can be calibrated under controlled conditions in vitro (Sinaasappel and Ince, 1996), and, for uniformly distributed free oxygen, PO2 is related to phosphorescence lifetime by the Stern-Volmer equation: l/τ=l/τ0 + kqPO2 (1) where τ is the lifetime in the presence of oxygen, τ0 is the lifetime in the absence of oxygen, and kq is the quenching coefficient (which includes the solubility of oxygen in the calibration medium). Under the condition of uniform PO2, the time course of the phosphorescence decay is monoexponential with lifetime τ.

A paradigm shift for local blood flow regulation

a remarkable example of physiological regulation is the coordination between metabolic rate and local blood flow in microscopic volumes of an organ. In skeletal muscle an increase in oxygen consumption (Vo2) over a wide range evokes a proportional (∼5 to 6 × Vo2) increase in blood flow ([29][

The rate of O2 loss from mesenteric arterioles is not unusually high

The O(2) disappearance curve (ODC) recorded in an arteriole after the rapid arrest of blood flow reflects the complex interaction among the dissociation of O(2) from hemoglobin, O(2) diffusivity, and rate of respiration in the vascular wall and surrounding tissue. In this study, the analysis of experimental ODCs allowed the estimation of parameters of O(2) transport and O(2) consumption in the microcirculation of the mesentery. We collected ODCs from rapidly arrested blood inside rat mesenteric arterioles using scanning phosphorescence quenching microscopy (PQM). The technique was used to prevent the artifact of accumulated O(2) photoconsumption in stationary media. The observed ODC signatures were close to linear, in contrast to the reported exponential decline of intra-arteriolar Po(2). The rate of Po(2) decrease was 0.43 mmHg/s in 20-μm-diameter arterioles. The duration of the ODC was 290 s, much longer than the 12.8 s reported by other investigators. The arterioles associated with lymphatic microvessels had a higher O(2) disappearance rate of 0.73 mmHg/s. The O(2) flux from arterioles, calculated from the average O(2) disappearance rate, was 0.21 nl O(2)·cm(-2)·s(-1), two orders of magnitude lower than reported in the literature. The physical upper limit of the O(2) consumption rate by the arteriolar wall, calculated from the condition that all O(2) is consumed by the wall, was 452 nl O(2)·cm(-3)·s(-1). From consideration of the microvascular tissue volume fraction in the rat mesentery of 6%, the estimated respiration rate of the vessel wall was ∼30 nl O(2)·cm(-3)·s(-1). This result was three orders of magnitude lower than the respiration rate in rat mesenteric arterioles reported by other investigators. Our results demonstrate that O(2) loss from mesenteric arterioles is small and that the O(2) consumption by the arteriolar wall is not unusually large.