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.     

Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements.

This study describes the use of two-photon excitation phosphorescence lifetime measurements for quantitative oxygen determination in vivo. Doubling the excitation wavelength of Pd-porphyrin from visible light to the infrared allows for deeper tissue penetration and a more precise and confined selection of the excitation volume due to the nonlinear two-photon effect. By using a focused laser beam from a 1,064-nm Q-switched laser, providing 10-ns pulses of 10 mJ, albumin-bound Pd-porphyrin was effectively excited and oxygen-dependent decay of phosphorescence was observed. In vitro calibration of phosphorescence lifetime vs. oxygen tension was performed. The obtained calibration constants were kq = 356 Torr(-1) x s(-1) (quenching constant) and tau0 = 550 micros (lifetime at zero-oxygen conditions) at 37 degrees C. The phosphorescence intensity showed a squared dependency to the excitation intensity, typical for two-photon excitation. In vivo demonstration of two-photon excitation phosphorescence lifetime measurements is shown by step-wise PO2 measurements through the cortex of rat kidney. It is concluded that quantitative oxygen measurements can be made, both in vitro and in vivo, using two-photon excitation oxygen-dependent quenching of phosphorescence. The use of two-photon excitation has the potential to lead to new applications of the phosphorescence lifetime technique, e.g., noninvasive oxygen scanning in tissue at high spatial resolution. To our knowledge, this is the first report in which two-photon excitation is used in the setting of oxygen-dependent quenching of phosphorescence lifetime measurements.     

Noninvasive imaging of the distribution in oxygen in tissue in vivo using near-infrared phosphors.

A newly developed water-soluble phosphor suitable for measuring oxygen pressure in the blood (Green 2W) was used for noninvasive, in vivo imaging of oxygen distribution in the vascular systems of mice. Oxygen quenches the phosphorescence of Green 2W, measured in the presence of 2% albumin, according to the Stern-volmer relationship. This oxygen-dependent quenching of phosphorescence has been used to obtain digital maps of the oxygen distribution in the tissue vasculature. EMT-6 mammary carcinoma tumors were grown by injecting 1 x 10(6) cells in 0.1-ml carrier into the subcutaneous space over the muscle on the hindquarter. When the tumors were approximately 8 mm in diameter, 300 micrograms of phosphorescence probe (Green 2W; absorption maximum 636 nm) was injected into the tail vein. The mice were immobilized with intraperotoneal Ketamine (133 mg/kg) and Xylazine (10 mg/kg) and illuminated with flashes (< 4-microseconds t1/2) of light of 630 +/- 12 nm. The emitted phosphorescence (790-nm maximum) was imaged an intensified CCD camera. Images were collected beginning at 30, 50, 80, 120, 180, 240, 420, and 2500 microseconds after the flash and used to calculate digital maps of the phosphorescence lifetimes and oxygen pressure. Both the illumination light and the phosphorescence were in the near-infrared region of the spectrum, where tissue has greatly decreased absorbance. The light therefore readily passed through the skin and centimeter thicknesses of tissue. The oxygen maps could be obtained by illuminating from the side of the mouse opposite the camera (and tumor). The tumors were readily observed as regions with oxygen pressures substantially below those of the surrounding tissue. Thus, phosphorescence measurements can noninvasively detect volumes of tissue with below-normal oxygen pressure in the presence of much larger volumes of tissue with normal oxygen pressures. In addition, tissue oxygen pressures can be monitored in real time, even through centimeter thicknesses of tissue.     

Oxygen distribution in murine tumors: characterization using oxygen-dependent quenching of phosphorescence.

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, with a peak oxygen pressure of 1.8 +/- 1.1 Torr. These results correlated well with measurements of tumor cell oxygenation measured by nitroimidazole (EF5) binding and were consistent with assessments of tumor blood flow by contrast enhanced ultrasound and tumor histology. The results show that phosphorescence quenching is a reliable, reproducible, and noninvasive method capable of providing real-time determination of oxygen concentrations within tumors.     

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution¹. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe''s lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature². The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 μm is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.     

Singlet molecular oxygen in photobiochemical systems: IR phosphorescence studies.

Singlet molecular oxygen (1O2) is one of the most active intermediates involved in photosensitized oxygenation reactions in chemical and biological systems. Deactivation of singlet oxygen is accompanied by infrared phosphorescence (1270 nm) which is widely employed for 1O2 detection and study. This review considers techniques for phosphorescence detection, phosphorescence spectra, quantum yields and kinetics under laser excitation, the radiative and real 1O2 lifetimes in organic solvents and water, 1O2 quenching by biomolecules, and estimation of singlet oxygen lifetimes, diffusion lengths and phosphorescence quantum yields in blood plasma, cell cytoplasm, erythrocyte ghosts, retinal rod outer segments and chloroplast thylakoids. The experiments devoted to 1O2 phosphorescence detection in photosensitizer-containing living cells are discussed in detail. Information reviewed is important for understanding the mechanisms of photodestruction in biological systems and various applied problems of photobiology and photomedicine.     

Simplex optimization of the variables affecting the micelle-stabilized room temperature phosphorescence of 6-methoxy-2-naphthylacetic acid and its kinetic determination in human urine

This article reports the kinetic determination of 6-methoxy-2-naphthylacetic acid (6-MNA), the major metabolite of nabumetone, from micelle-stabilized room temperature phosphorescence (MS-RTP) measurements made by using the stopped-flow mixing technique. This methodology allows one to determine analytes in complex matrices without the need for a tedious separation process. It also shortens analysis times substantially. The proposed method uses simplex methodology to optimize the chemical and instrumental variables affecting the phosphorescence. It was applied to the determination of 6-MNA in human urine. The maximum phosphorescence signal is obtained within only 10 s after the sample is prepared. The maximum slope of the kinetic curve, which corresponds to the maximum rate of the phosphorescence development, is measured at lambda(ex)=273 nm and lambda(em)=516 nm. Least-squares regression was used to fit experimental data, and the detection limit, repeatability, and standard deviation for replicate samples were determined.     

Phosphorescent porphyrin probes in biosensors and sensitive bioassays

Platinum(II) and palladium(II) complexes of porphyrins and related tetrapyrrolic pigments emit strong phosphorescence at room temperatures, which is characterized by long lifetimes falling into the sub-millisecond range and long-wave spectral characteristics. These features make the dyes useful as probes for a number of bioanalytical applications, particularly those employing time-resolved fluorescent detection. They can provide high sensitivity and selectivity, together with rather simple instrumental set-up. A number of analytical systems are now under development that are based on the use of phosphorescent porphyrin probes. Experimental results are presented on the following systems: (i) fibre-optic phosphorescence lifetime-based oxygen sensor on the basis of hydrophobic platinum-porphyrins and development of advanced sensing materials and prototype instrumentation; (ii) practical applications of the optical oxygen sensor, including a sensitive immunosensor that employs glucose oxidase labels, a rapid screening method for cell viability in microtitre-plate format, non-destructive measurement of oxygen in packaged foods and reagentless biosensors for metabolites (glucose, lactate); and (iii) the use of water-soluble platinum- and palladium-porphyrins as labels for ultra-sensitive time-resolved phosphorescence immunoassays.     

Measurement of tumor oxygenation using new frequency domain phosphorometers

Oxygen dependent quenching of phosphorescence allows for non-invasive measurements of oxygen in tissue. We have designed and constructed a novel multi-frequency instrument for measurement of phosphorescence lifetimes and developed algorithms for determining the distribution of oxygen (oxygen histogram) in the microvasculature of tissue with good temporal resolution (Vinogradov et al., 2002, Compar. Biochem. A, these proceedings). This technology, in combination with a new water soluble near infra red phosphor (Oxyphor G2), was used to examine the oxygenation of subcutaneous Q7 tumors grown on the flank of Buffalo rats and their response to giving the rats oxygen or carbogen to breathe. Phosphorescence was measured using excitation at 635 nm and emission at >700 nm (the phosphorescence maximum is near 800 nm). The excitation and collection light guides were placed on the surface of the skin of the anesthetized animals separated by approximately 0.8 cm. A 6 x 6 or 7 x 7 grid (approx. 4 cm x 4 cm) was drawn on the flank and oxygen histograms were measured in each square, providing 'images' of the oxygen distribution in the tissue. This procedure determines the tissue oxygen distribution at each position in the grid. Regions of relative hypoxia (associated with the tumor) can be readily localized and the extent of hypoxia quantitatively evaluated.     

On‐Line Control of Glucose Concentration in High‐Yielding Mammalian Cell Cultures Enabled Through Oxygen Transfer Rate Measurements

Glucose control is vital to ensure consistent growth and protein production in mammalian cell cultures. The typical fed‐batch glucose control strategy involving bolus glucose additions based on infrequent off‐line daily samples results in cells experiencing significant glucose concentration fluctuations that can influence product quality and growth. This study proposes an on‐line method to control and manipulate glucose utilizing readily available process measurements. The method generates a correlation between the cumulative oxygen transfer rate and the cumulative glucose consumed. This correlation generates an on‐line prediction of glucose that has been successfully incorporated into a control algorithm manipulating the glucose feed‐rate. This advanced process control (APC) strategy enables the glucose concentration to be maintained at an adjustable set‐point and has been found to significantly reduce the deviation in glucose concentration in comparison to conventional operation. This method has been validated to produce various therapeutic proteins across cell lines with different glucose consumption demands and is successfully demonstrated on micro (15 mL), laboratory (7 L), and pilot (50 L) scale systems. This novel APC strategy is simple to implement and offers the potential to significantly enhance the glucose control strategy for scales spanning micro‐scale systems through to full scale industrial bioreactors.     

Triplet-state detection of labeled proteins using fluorescence recovery spectroscopy

The experimental procedures for detecting the triplet states of chromophores in solutions (cuvettes) by fluorescence recovery spectroscopy (FRS) are described in detail, together with applications in studies of protein structure and protein-cell interactions in the microsecond to millisecond time domain. The experimental configuration has been characterized by measuring the emission intensities and anisotropies of eosin and erythrosin immobilized in poly(methyl methacrylate). The fluorescence data are compared with those from phosphorescence emission measurements and with theoretical predictions. Triplet-state lifetimes were obtained in 5 mM phosphate buffer, pH 7.0, of concanavalin A labeled with eosin, tetramethylrhodamine, and fluorescein and of alpha 2-macroglobulin labeled with the first two probes. In the case of labeled concanavalin A, iodide quenching measurements gave bimolecular rate constants of approximately 10(9) M-1 s-1. The usefulness of FRS for studying protein-cell interactions is exemplified with eosin-labeled concanavalin A bound to living A-431 human epidermoid carcinoma cells. Finally, the advantages and disadvantages of the technique are compared to those of the alternative phosphorescence emission method.     

Calibration of Pd-porphyrin phosphorescence for oxygen concentration measurements in vivo

Sinaasappel, M., and C. Ince. Calibration ofPd-porphyrin phosphorescence for oxygen concentration measurements in vivo. J. Appl. Physiol. 81(5):2297-2303, 1996.Quantitative measurement of oxygenconcentrations in the microvasculature is of prime importance in issuesrelated to oxygen transport to tissue. The introduction of thequenching of the Pd-porphyrin phosphorescence as oxygen sensor in vivoby Wilson et al. (J. Appl. Physiol.74: 580-589, 1993) has provided in this context a major advance inthis area of research. For in vivo application, the dye is coupled toalbumin to restrict the dye to the circulation and to measure oxygen in the physiological range. In this study a phosphorimeter with a gatedphotomultiplier is presented and validated. Furthermore, anonlinear-fit method using the Marquardt-Levenberg algorithm is used tocalculate the decay time. With this new phosphorimeter, calibrationmeasurements were performed to investigate the effects of pH,temperature, and diffusivity. The results present a preparation methodfor albumin coupling of the dye that eliminates the pH dependency ofthe quenching kinetics. Furthermore, the decreased oxygen diffusivityof serum was compared with that of water, and it was shown thatcalibration constants measured in water can be extrapolated to serum.

     

Cluster optimization algorithm based on CPU and GPU hybrid architecture

With the rapid development of network technology and parallel computing, clusters formed by connecting a large number of PCs with high-speed networks have gradually replaced the status of supercomputers in scientific research and production and high-performance computing with cost-effective advantages. The research purpose of this paper is to integrate the Kriging proxy model method and energy efficiency modeling method into a cluster optimization algorithm of CPU and GPU hybrid architecture. This paper proposes a parallel computing model for large-scale CPU/GPU heterogeneous high-performance computing systems, which can effectively describe the computing capabilities and various communication behaviors of CPU/GPU heterogeneous systems, and finally provide algorithm optimization for CPU/GPU heterogeneous clusters. According to the GPU architecture, an efficient method of constructing a Kriging proxy model and an optimized search algorithm are designed. The experimental results in this paper show that the construction of the Kriging proxy model can obtain a 220 times speedup ratio, and the search algorithm can reach an 8 times speedup ratio. It can be seen that this heterogeneous cluster optimization algorithm has high feasibility.

     

A simple correlation for predicting effective diffusivities in immobilized cell systems

A simple correlation method has been developed to predict effective diffusivities of small molecules in heterogeneous materials such as immobilized cell systems. This correlation uses a single diffusivity measurement at one cell volume fraction to predict diffusivities for any other volume fraction of cell. The method has been applied to 20 sets of published diffusivity measurements in immobilized cell systems and accurately predicts affective diffusivities of molecules for the full range of cell fractions. It may also be used to predict effective diffusivities in heterogeneous materials in which the diffusivity of a molecule in each phase and the volume fraction of each phase are known. (c) 1996 John Wiley & Sons, Inc.     

The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration

Oxygen-dependent quenching of phosphorescence has been used to measure the dependence of mitochondrial oxidative phosphorylation on oxygen concentration in suspensions of isolated rat liver mitochondria. An instrument has been designed which simultaneously monitors the phosphorescence lifetime of a fluorophor and the reduction of cytochrome c by dual wavelength spectrophotometry. The phosphorescence lifetime method gives very rapid (less than 100 ms) measure of the oxygen concentration (Vanderkooi, J. M., Maniara, G., Green, T. J., and Wilson, D. F. (1987) J. Biol. Chem. 262, 5476-5482) from concentrations characteristic of air-saturated media to as low as 2 x 10(-8) M. The results may be summarized as follows. For well coupled rat liver mitochondria at pH 7.0 and in the presence of ATP, as the oxygen concentration was lowered, increased cytochrome c reduction was observed to begin at oxygen concentrations greater than 20 microM. For mitochondria in the presence of uncoupler, cytochrome c reduction began at oxygen concentrations less than 1.0 microM. The oxygen dependence of reduction of cytochrome c in well coupled mitochondria treated with ATP was strongly dependent on the pH of the suspending medium. Reduction of cytochrome c began at higher oxygen concentrations as the pH was made more alkaline. The oxygen concentration for half-maximal respiratory rates was much larger for well coupled mitochondria treated with ATP (approximately 0.7 microM) than for mitochondria treated with uncoupler (less than 0.1 microM). It is concluded that the oxygen dependence of mitochondrial oxidative phosphorylation is such that mitochondria could function in their proposed role of tissue oxygen sensors for regulation of such diverse functions as local blood flow and electrical activity in the carotid body.     

Separation of 5'-ribonucleoside monophosphates by ion-pair reverse-phase high-performance liquid chromatography

We have developed computer programs for characterization of ligand-binding systems in terms of continuous affinity distributions of arbitrary shape based on a numerical finite difference method. This method provides an excellent initial estimate of the affinity distribution, which can be further refined by means of nonlinear least-squares curve fitting. The method has been extensively tested for several cases including receptor heterogeneity, cooperativity, and for several examples of experimental design (e.g., ligand concentrations), and various levels of random and systematic experimental errors. The results provide a guide to experimental design, and indicate limits to the resolution obtained by ligand-binding studies, irrespective of the method of analysis.     

Accurate Construction of Photoactivated Localization Microscopy (PALM) Images for Quantitative Measurements

Localization-based superresolution microscopy techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) have allowed investigations of cellular structures with unprecedented optical resolutions. One major obstacle to interpreting superresolution images, however, is the overcounting of molecule numbers caused by fluorophore photoblinking. Using both experimental and simulated images, we determined the effects of photoblinking on the accurate reconstruction of superresolution images and on quantitative measurements of structural dimension and molecule density made from those images. We found that structural dimension and relative density measurements can be made reliably from images that contain photoblinking-related overcounting, but accurate absolute density measurements, and consequently faithful representations of molecule counts and positions in cellular structures, require the application of a clustering algorithm to group localizations that originate from the same molecule. We analyzed how applying a simple algorithm with different clustering thresholds (t_Thresh and d_Thresh) affects the accuracy of reconstructed images, and developed an easy method to select optimal thresholds. We also identified an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction with the clustering algorithm. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions. The main advantage of our method is that it generates a superresolution image and molecule position list that faithfully represents molecule counts and positions within a cellular structure, rather than only summarizing structural properties into ensemble parameters. This feature makes it particularly useful for cellular structures of heterogeneous densities and irregular geometries, and allows a variety of quantitative measurements tailored to specific needs of different biological systems.     

Identifying Transport Behavior of Single-Molecule Trajectories

Models of biological diffusion-reaction systems require accurate classification of the underlying diffusive dynamics (e.g., Fickian, subdiffusive, or superdiffusive). We use a renormalization group operator to identify the anomalous (non-Fickian) diffusion behavior from a short trajectory of a single molecule. The method provides quantitative information about the underlying stochastic process, including its anomalous scaling exponent. The classification algorithm is first validated on simulated trajectories of known scaling. Then it is applied to experimental trajectories of microspheres diffusing in cytoplasm, revealing heterogeneous diffusive dynamics. The simplicity and robustness of this classification algorithm makes it an effective tool for analysis of rare stochastic events that occur in complex biological systems.     

Recovery of radial PO(2) profiles from phosphorescence quenching measurements in microvessels

Work by previous investigators has indicated that a substantial amount of oxygen diffuses from the precapillary circulation. These losses imply that there should be radial gradients of oxygen tension (PO(2)) in arterioles, leading to a non-uniform distribution of oxygen within these microvessels. We have employed the phosphorescence quenching method to measure oxygen, allowing us to evaluate the heterogeneity of PO(2) inside short segments of microvessels. The phosphorescence decay curve contains information about the distribution of oxygen within the excited volume and the distribution can be represented as a histogram, by decomposing the decay curve into several components with weights proportional to the volume fraction of plasma with different PO(2), under the condition of a high signal-to-noise ratio. Furthermore, the histogram can be converted into a radial profile of PO(2), based on the assumptions of a circular vascular lumen, axisymmetric distribution of oxygen and monotonic PO(2) profile. Albumin-bound Pd-porphyrin phosphor was infused into the circulation of hamsters and excited by flash illumination at 10 Hz, with a square region of excitation light just covering the entire lumen, (i.e. width of region equaled luminal diameter) of microvessels in the hamster mesentery. A set of 50 curves (5 s of data) was averaged to obtain a decay curve with low noise. Curves were analyzed with the above histogram procedure, and this analysis allowed us to distinguish between PO(2) values originating from intra and extravascular subvolumes. The intravascular PO(2) in these microvessels was very heterogeneous, which could be explained by the existence of significant radial PO(2) gradients. The radial PO(2) gradients were estimated to be approximately 1 mmHg/microm.