Oxyphor R2 and G2: Phosphors for Measuring Oxygen by Oxygen-dependent Quenching of Phosphorescence

Analytical Biochemistry. Nov, 2002  |  Pubmed ID: 12423638

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.

Influence of Nonplanarity and Extended Conjugation on Porphyrin Basicity

Inorganic Chemistry. Dec, 2002  |  Pubmed ID: 12495329

Thermodynamic basicities of several new nonplanar water soluble tetraaryltetracyclohexano- (Ar(4)TCHP) and tetraaryltetrabenzoporphyrins (Ar(4)TBP) have been measured and correlated with their structural parameters. While the degrees of nonplanarity in these porphyrins are similar, Ar(4)TCHPs exhibit significantly higher basicities than Ar(4)TBPs and than planar tetraarylporphyrins. Low basicities of distorted Ar(4)TBPs are believed to be due to extended pi-conjugation, which causes delocalization of the core electron density in these porphyrins and reduces negative charges at the protonation site.

Oxygen Distributions in Tissue Measured by Phosphorescence Quenching

Advances in Experimental Medicine and Biology. 2003  |  Pubmed ID: 12580425

Luminescent Zn and Pd Tetranaphthaloporphyrins

Inorganic Chemistry. Jul, 2003  |  Pubmed ID: 12844293

Zn and Pd complexes of meso-tetraphenyltetranaphthaloporphyrins (Ph(4)TNP) exhibit strong infrared absorption bands and luminesce in solutions at room temperature. S1 --> S0 fluorescence (lambda(max) = 732 nm, phi = 5.3%) is the predominant emission in the case of ZnPh(4)TNP (1). This emission is in part due to the delayed fluorescence (phi = 1.1%). Phosphorescence (T1 --> S0) of 1 (lambda(max) = 973 nm) is very weak (phi = 0.04%) and occurs with lifetime of about 440 micros in deoxygenated DMF. In the case of PdPh(4)TNP (2), almost no S1 --> S0 fluorescence could be observed, while the main emission detected was T1 --> S0 phosphorescence (lambda(max) = 938 nm). The phosphorescence of 2 occurs with lifetime of about 65 micros and (phi=6.5%) in deoxygenated DMF solution. Metalloporphyrins 1 and 2 are promising near infrared dyes biomedical applications.

Novel Route to Functionalized Tetraaryltetra[2,3]naphthaloporphyrins Via Oxidative Aromatization

The Journal of Organic Chemistry. Sep, 2003  |  Pubmed ID: 12968910

A novel general route to substituted meso-tetraaryltetra[2,3]naphthaloporphyrins (Ar(4)TNP) and meso-tetraaryloctamethoxytetra[2,3]naphthaloporphyrins (Ar(4)(MeO)(8)TNP) via oxidative aromatization of nonaromatically fused porphyrin precursors is described. Ar(4)(MeO)(8)TNPs exhibit more red-shifted absorption bands than Ar(4)TNPs and differ dramatically in solubility. The first X-ray crystallographic structure of tetranaphthaloporphyrin, i.e., PdAr(4)TNP (Ar = 4-MeO(2)CC(6)H(4)), revealed that the degree of nonplanar distortion of this macrocycle is only slightly higher than that of the homologous tetrabenzoporphyrins (Ar(4)TBP).

Monitoring the Dynamics of Tissue Oxygenation in Vivo by Phosphorescence Quenching

Advances in Experimental Medicine and Biology. 2003  |  Pubmed ID: 15174594

Self-assembly of Amphiphilic Dendritic Dipeptides into Helical Pores

Nature. Aug, 2004  |  Pubmed ID: 15306805

Natural pore-forming proteins act as viral helical coats and transmembrane channels, exhibit antibacterial activity and are used in synthetic systems, such as for reversible encapsulation or stochastic sensing. These diverse functions are intimately linked to protein structure. The close link between protein structure and protein function makes the design of synthetic mimics a formidable challenge, given that structure formation needs to be carefully controlled on all hierarchy levels, in solution and in the bulk. In fact, with few exceptions, synthetic pore structures capable of assembling into periodically ordered assemblies that are stable in solution and in the solid state have not yet been realized. In the case of dendrimers, covalent and non-covalent coating and assembly of a range of different structures has only yielded closed columns. Here we describe a library of amphiphilic dendritic dipeptides that self-assemble in solution and in bulk through a complex recognition process into helical pores. We find that the molecular recognition and self-assembly process is sufficiently robust to tolerate a range of modifications to the amphiphile structure, while preliminary proton transport measurements establish that the pores are functional. We expect that this class of self-assembling dendrimers will allow the design of a variety of biologically inspired systems with functional properties arising from their porous structure.

Flexibility in Proteins: Tuning the Sensitivity to O2 Diffusion by Varying the Lifetime of a Phosphorescent Sensor in Horseradish Peroxidase

Photochemistry and Photobiology. Jul-Aug, 2004  |  Pubmed ID: 15339214

The heme in horseradish peroxidase (HRP) was replaced by phosphorescent Pt-mesoporphyrin IX (PtMP), which acted as a phosphorescent marker of oxygen quenching and allowed comparison with another probe, Pd-mesoporphyrin IX (Khajehpour et al. (2003) Proteins 53, 656-666). Benzohydroxamic acid (BHA), a competitive inhibitor of the enzyme, was also used to monitor its effects on phosphorescence quenching. With the addition of BHA, in the presence of oxygen, the phosphorescence intensity of the protein increased. In contrast, the addition of BHA, in the absence of oxygen, reduced the phosphorescence intensity of the protein. K(d) = 18 microM when BHA binds to PtMP-HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O(2) to the protein interior and (2) BHA itself quenches the phosphorescence. Consistent with this, the oxygen quenching of the phosphorescence of PtMP-HRP gave a quenching constant of k(q) = 234 mm Hg(-1) s(-1) in the absence of BHA and k(q) = 28.7 mm Hg(-1) s(-1) in the presence of BHA. The quenching rate of BHA is 4000 s(-1). The relative quantum yield of the phosphorescence of the Pt derivative is about six times that of the Pd derivative, whereas the phosphorescence lifetime is approximately eight times shorter. The high quantum yield and suitable lifetime make Pt-porphyrins appropriate as sensors of O(2) diffusion and flexibility in heme proteins.

Novel Versatile Synthesis of Substituted Tetrabenzoporphyrins

The Journal of Organic Chemistry. Jan, 2004  |  Pubmed ID: 14725469

A novel general synthetic route to tetraaryltetrabenzoporphyrins (Ar(4)TBP) with various peripheral functional groups is developed. The procedure includes (i) Barton-Zard condensation of 1-nitro- or 1-phenylsulfonylcyclohexenes with isocyanoacetic acid esters, (ii) condensation of the resulting 4,5,6,7-tetrahydroisoindoles with aromatic aldehydes to give fused tetraaryltetracylohexenoporphyrins (Ar(4)TCHP), and (iii) aromatization of the metal complexes of Ar(4)TCHP's into the corresponding Ar(4)TBP's. Cu and Zn complexes of Ar(4)TBP's are further demetalated to give the corresponding Ar(4)TBP free bases. The overall yields for the sequence range from 15% to 40%, making the method suitable for the preparation of gram quantities of Ar(4)TBP's in a single run. The scope of the method, the selection of the peripheral substituents, the choice of the metal ions, and their influence on the yields of aromatization are discussed. The basic spectroscopic properties of newly synthesized Ar(4)TBP's and Ar(4)TCHP's are reported together with the first X-ray crystallographic structure of the NiAr(4)TBP complex.

Phosphorescence Lifetime Imaging in Turbid Media: the Inverse Problem and Experimental Image Reconstruction

Applied Optics. Jan, 2004  |  Pubmed ID: 14765914

Three-dimensional phosphorescence lifetime imaging is a novel method for the mapping of oxygen concentration in biological tissues. We present reconstruction techniques for recovering phosphorescent objects in highly scattering media based on the telegraph equation and two regularization methods, i.e., the Tikhonov-Phillips regularization and the maximum entropy method. Theoretical results are experimentally validated, and the reconstructed images of phosphorescent objects rendering oxygen maps in a layer are presented.

Oxygen Distribution in Murine Tumors: Characterization Using Oxygen-dependent Quenching of Phosphorescence

Journal of Applied Physiology (Bethesda, Md. : 1985). Apr, 2005  |  Pubmed ID: 15579567

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.

Arylamide Dendrimers with Flexible Linkers Via Haloacyl Halide Method

Organic Letters. Apr, 2005  |  Pubmed ID: 15844900

[reaction: see text] Soluble arylamide dendrons with flexible linkers, peripheral ester or carboxyl groups (R), and focal amino or halogen functionalities (F) were synthesized from aryl glycineamide (AG) building blocks. The AG blocks were prepared in high yields from trivial starting materials by Fischer's haloacyl halide method, which also could be extended to the dendrimer synthesis itself. The G2 AG dendrons were coupled to a Pd porphyrin core, demonstrating outstanding encapsulation efficiency in aqueous solutions.

Synthesis of Symmetrical Tetraaryltetranaphtho[2,3]porphyrins

The Journal of Organic Chemistry. Jun, 2005  |  Pubmed ID: 15932297

A new method of synthesis of meso-tetraaryltetranaphtho[2,3]porphyrins (Ar4TNP) has been developed. Ar4TNPs with peripheral functional groups are obtained by oxidative aromatization of meso-tetraarylporphyrins in which pyrrole units are fused with either octahydro- or dihydronaphthalene moieties. These precursor porphyrins are synthesized in four to five steps from readily available starting materials, such as naphthalene or 1,4-benzoquinone. The pathway originating in dihydronaphthalene, i.e., the "dialine" route, was found to be superior to the alternative "octaline"route in that it (1) enables the shortening of the overall reaction sequence, (2) has a broader scope in terms of the peripheral substitution in Ar4TNPs, and (3) affords higher yields of the target porphyrins. Pd complexes of the synthesized Ar4TNPs exhibit remarkably strong absorption bands at 710-720 nm (epsilon approximately 200,000 M(-1) cm(-1)) and phosphoresce at room temperature with moderate quantum yields (phi = 2-3%, lambda(max) = 900-1000 nm). The absorption maxima of naphthoporphyrins substituted with eight methoxy groups (Ar4TNP(OMe)8) were found to be about 15-20 nm red shifted compared to the corresponding maxima of unsubstituted Ar4TNPs. The X-ray crystallographic data suggest that these spectral shifts are caused not by the differences in nonplanar distortions of the macrocycles but by the purely electronic effects of the substituents.

Phosphorescent Oxygen Sensor with Dendritic Protection and Two-photon Absorbing Antenna

Journal of the American Chemical Society. Aug, 2005  |  Pubmed ID: 16104764

Imaging oxygen in 3D with submicron spatial resolution can be made possible by combining phosphorescence quenching technique with multiphoton laser scanning microscopy. Because Pt and Pd porphyrin-based phosphorescent dyes, traditionally used as phosphors in biological oxygen measurements, exhibit extremely low two-photon absorption (2PA) cross-sections, we designed a nanosensor for oxygen, in which a 2P absorbing antenna is coupled to a metalloporphyrin core via intramolecular energy transfer (ET) with the purpose of amplifying the 2PA induced phosphorescence of the metalloporphyrin. The central component of the device is a polyfunctionalized Pt porphyrin, whose triplet state emission at ambient temperatures is strong, occurs in the near infrared and is sensitive to O2. The 2PA chromophores are chosen in such a way that their absorption is maximal in the near infrared (NIR) window of tissue (e.g., 700-900 nm), while their fluorescence is overlapped with the absorption band(s) of the core metalloporphyrin, ensuring an efficient antenna-core resonance ET. The metalloporphyrin-antenna construct is embedded inside the protecting dendritic jacket, which isolates the core from interactions with biological macromolecules, controls diffusion of oxygen and makes the entire sensor water-soluble. Several Pt porphyrin-coumarin based sensors were synthesized and their photophyics studied to evaluate the proposed design.

Oxygen Distribution and Vascular Injury in the Mouse Eye Measured by Phosphorescence-lifetime Imaging

Applied Optics. Sep, 2005  |  Pubmed ID: 16149347

Maps of the oxygen distribution in the retina of the mouse eye were obtained by phosphorescence-lifetime imaging. Phosphor dissolved in the blood was excited by modulated light and phosphorescence imaged through microscope optics with an intensified-CCD camera. Phosphorescence lifetimes and oxygen pressures were calculated for each pixel of the images. The resolution was sufficient to permit the detection of anomalies that result in reduced oxygen pressures in individual retinal capillaries. High-resolution maps of oxygen distribution in the retina can provide greater understanding of the role of oxygen and vascular function in diseases of the eye.

Synthesis and Luminescence of Soluble Meso-unsubstituted Tetrabenzo- and Tetranaphtho[2,3]porphyrins

The Journal of Organic Chemistry. Nov, 2005  |  Pubmed ID: 16268634

[Structure: see text]. Syntheses of soluble tetrabenzoporphyrins (TBP) and tetranaphtho[2,3]porphyrins (TNP), with multiple substituents in the conjugated aromatic rings but bearing no substituents in the meso-positions, is reported. Both types of porphyrins were obtained by direct aromatization of precursor porphyrins, annealed with either cyclohexene or dihydronaphthalene fragments. TBPs and TNPs possess powerful absorption bands in the near-infrared (lambda = 610-710 nm, epsilon = 100,000-300,000 M(-1) cm(-1)) and exhibit strong luminescence. Free bases and Zn complexes fluoresce with quantum yields of up to 50%, whereas Pd and Pt complexes phosphoresce in solutions at ambient temperatures. Remarkably, the phosphorescence quantum yields of Pd and Pt TBPs reach as high as 20-50%, which places them among the brightest near-infrared phosphors known to date.

Imaging Oxygen Pressure in the Retina of the Mouse Eye

Advances in Experimental Medicine and Biology. 2005  |  Pubmed ID: 16594148

The phosphorescence lifetime imaging system previously used to image oxygen in the retina of the cat eye was modified to allow imaging of phosphorescence lifetimes in the much smaller mouse eye. Following the lead of Shonat and coworkers, a frequency domain approach was used in which the excitation light source was modulated in a 50% on: 50% off square wave while the gate of the intensified CCD camera was similarly modulated but delayed with respect to the excitation. These were analyzed by fitting the intensity at each pixel to a sinusoid. The phase of the phosphorescence relative to the excitation was determined and from the phase shift and frequency, the phosphorescence lifetime was calculated. The Stern-Volmer relationship was then used to calculate the oxygen pressure at each pixel of the image array. High resolution maps of phosphorescence lifetime and oxygen pressure in the retina of the mouse eye have been attained. The retinal veins draining into the optic head appear as large, highly phosphorescent vessels against a lower phosphorescence background with a network of smaller vessels. The oxygen pressure in the retinal veins is typically from 20 to 30 mm Hg while the background has somewhat higher oxygen pressures. Experiments are underway to resolve the oxygen in the choroid from that in the retina. The arteries on the retinal surface can be observed, but their small diameter, relatively high oxygen pressures (> 90 mm Hg), and surrounding tissue with much lower oxygen pressures, makes accurate determination of the oxygen pressure a challenge.

Feasibility of Diffuse Optical Imaging with Long-lived Luminescent Probes

Optics Letters. Apr, 2006  |  Pubmed ID: 16625910

Long-lived near-infrared phosphors with high quantum yields have recently become available, making it possible to image oxygen distributions in tissue in three dimensions. By numerical simulations we demonstrate that, by using phosphorescent probes with appropriate oxygen quenching constants, one can image hypoxic phantoms in scattering media with adequate spatial resolution, employing simple time-gated measurements. The approach developed will guide experimental imaging of phosphorescence lifetime and oxygen pressure in living tissue.

Oxygen Pressures in the Interstitial Space and Their Relationship to Those in the Blood Plasma in Resting Skeletal Muscle

Journal of Applied Physiology (Bethesda, Md. : 1985). Dec, 2006  |  Pubmed ID: 16888050

This study compared oxygen pressures (Po(2)), measured by oxygen-dependent quenching of phosphorescence, in the intravascular (blood plasma) space in the muscle with those in the interstitial (pericellular) space. Our hypothesis was that the capillary wall would not significantly impede oxygen diffusion from the blood plasma to the pericellular space. A new near-infrared oxygen sensitive probe, Oxyphor G3, was used to obtain oxygen distributions in the interstitial space. Oxyphor G3 is a Pd-tetrabenzoporphyrin encapsulated inside generation 2 poly-arylglycine (AG) dendrimer. The periphery of the dendrimer is modified with oligoethylene glycol residues (average molecular weight 350) to make the probe water soluble and biologically inert. Oxyphor G3 was injected into thigh muscle using a 30-gauge needle. Histograms of the Po(2) in the interstitial space were measured in awake and anesthetized animals and compared with those for Oxyphor G2 in the intravascular (blood plasma) space. For awake mice, the lowest 10% of Po(2) values for the interstitial and intravascular spaces (believed to represent capillary bed) were not significantly different [23.8 (SD 4.5) and 25 Torr (SD 4.3), respectively], whereas, in isoflurane-anesthetized mice, there was a small but significant (P = 0.01) difference [20.4 (SD 6.3) and 27.9 Torr (SD 3.5), respectively]. The peak values for the histograms for the interstitial space in awake and isoflurane-anesthetized mice were 40.8 (SD 7.5) and 36.9 Torr (SD 8.3), respectively, whereas those for the intravascular space were 52.2 (SD 4.9) and 55.9 Torr (SD 8.4), respectively, showing no significant difference due to isoflurane anesthesia. The histograms for the intravascular space were significantly wider, with more contribution at higher Po(2) values. A different anesthetic, ketamine plus xylazine injected intraperitoneally, caused a marked decrease in the tissue Po(2) values in both spaces, with the time course and extent of the decrease dependent on the time after injection and variable among mice. It was, therefore, not further used.

Imaging Oxygen Pressure in the Rodent Retina by Phosphorescence Lifetime

Advances in Experimental Medicine and Biology. 2006  |  Pubmed ID: 16927680

Simultaneous Fluorometry and Phosphorometry of Langendorff Perfused Rat Heart: Ex Vivo Animal Studies

Optics Letters. Oct, 2006  |  Pubmed ID: 17001378

Fluorescence imaging of intrinsic fluorophores of tissue is a powerful method to assess metabolic changes at the cellular and intracellular levels. At the same time, exogenous phosphorescent probes can be used to accurately measure intravascular tissue oxygenation. Heart failure is the leading cause of death in America. A rat heart can potentially model the human heart to study failures or other abnormalities optically. We report simultaneous fluorescence and phosphorescence measurements performed on a rat heart. We have used two different optical systems to acquire fluorescence signals of flavoprotein and nicotinamide adenine dinucleotide--the two intrinsic fluorophores of mitochondria--and the phosphorescence signal of an intravascular oxygen probe to extract intracellular and intravascular metabolism loads, respectively.

Tomographic Imaging of Oxygen by Phosphorescence Lifetime

Applied Optics. Nov, 2006  |  Pubmed ID: 17086268

Imaging of oxygen in tissue in three dimensions can be accomplished by using the phosphorescence quenching method in combination with diffuse optical tomography. We experimentally demonstrate the feasibility of tomographic imaging of oxygen by phosphorescence lifetime. Hypoxic phantoms were immersed in a cylinder with scattering solution equilibrated with air. The phantoms and the medium inside the cylinder contained near-infrared phosphorescent probe(s). Phosphorescence at multiple boundary sites was registered in the time domain at different delays (t(d)) following the excitation pulse. The duration of the excitation pulse (t(p)) was regulated to optimize the contrast in the images. The reconstructed integral intensity images, corresponding to delays t(d), were fitted exponentially to give the phosphorescence lifetime image, which was converted into the three-dimensional image of oxygen concentrations in the volume. The time-independent diffusion equation and the finite element method were used to model the light transport in the medium. The inverse problem was solved by the recursive maximum entropy method. We provide what we believe to be the first example of oxygen imaging in three dimensions using long-lived phosphorescent probes and establish the potential of these probes for diffuse optical tomography.

Energy and Electron Transfer in Enhanced Two-photon-absorbing Systems with Triplet Cores

The Journal of Physical Chemistry. A. Aug, 2007  |  Pubmed ID: 17608457

Enhanced two-photon-absorbing (2PA) systems with triplet cores are currently under scrutiny for several biomedical applications, including photodynamic therapy (PDT) and two-photon microscopy of oxygen. The performance of so far developed molecules, however, is substantially below expected. In this study we take a detailed look at the processes occurring in these systems and propose ways to improve their performance. We focus on the interchromophore distance tuning as a means for optimization of two-photon sensors for oxygen. In these constructs, energy transfer from several 2PA chromophores is used to enhance the effective 2PA cross section of phosphorescent metalloporphyrins. Previous studies have indicated that intramolecular electron transfer (ET) can act as an effective quencher of phosphorescence, decreasing the overall sensor efficiency. We studied the interplay between 2PA, energy transfer, electron transfer, and phosphorescence emission using Rhodamine B-Pt tetrabenzoporphyrin (RhB-PtTBP) adducts as model compounds. 2PA cross sections (sigma2) of tetrabenzoporphyrins (TBPs) are in the range of several tens of GM units (near 800 nm), making TBPs superior 2PA chromophores compared to regular porphyrins (sigma2 values typically 1-2 GM). Relatively large 2PA cross sections of rhodamines (about 200 GM in 800-850 nm range) and their high photostabilities make them good candidates as 2PA antennae. Fluorescence of Rhodamine B (lambda(fl) = 590 nm, phi(fl) = 0.5 in EtOH) overlaps with the Q-band of phosphorescent PtTBP (lambda(abs) = 615 nm, epsilon = 98 000 M(-1) cm(-1), phi(p) approximately 0.1), suggesting that a significant amplification of the 2PA-induced phosphorescence via fluorescence resonance energy transfer (FRET) might occur. However, most of the excitation energy in RhB-PtTBP assemblies is consumed in several intramolecular ET processes. By installing rigid nonconducting decaproline spacers (Pro10) between RhB and PtTBP, the intramolecular ETs were suppressed, while the chromophores were kept within the Förster r0 distance in order to maintain high FRET efficiency. The resulting assemblies exhibit linear amplification of their 2PA-induced phosphorescence upon increase in the number of 2PA antenna chromophores and show high oxygen sensitivity. We also have found that PtTBPs possess unexpectedly strong forbidden S0 --> T1 bands (lambda(max) = 762 nm, epsilon = 120 M-1 cm-1). The latter may overlap with the laser spectrum and lead to unwanted linear excitation.

Selective Transport of Water Mediated by Porous Dendritic Dipeptides

Journal of the American Chemical Society. Sep, 2007  |  Pubmed ID: 17784763

Oxygen Pressures in the Interstitial Space of Skeletal Muscle and Tumors in Vivo

Advances in Experimental Medicine and Biology. 2008  |  Pubmed ID: 18290314

A new Oxyphor (Oxyphor G3) has been used to selectively determine the oxygen pressure in interstitial (pericellular) spaces. Oxyphor G3 is a Pd-tetrabenzoporphyrin, encapsulated inside generation 2 poly-arylglycine (AG) dendrimer, and therefore is a true near infrared oxygen sensor, having a strong absorption band at 636nm and emission near 800nm. The periphery of the dendrimer is modified with oligoethylene glycol residues (Av. MW 350) to make the probe water soluble and biologically inert. Oxyphor G3 was injected along "tracks" in the tissue using a small needle (30gage or less) and remained in the pericellular space, allowing oxygen measurements for several hours with a single injection. The oxygen pressure distributions (histograms) were compared with those for Oxyphor G2 in the intravascular (blood plasma) space. In normal muscle, in the lower oxygen pressure region of the histograms (capillary bed) the oxygen pressure difference was small. At higher oxygen pressures in the histograms there were differences consistent with the presence of high flow vessels with oxygen pressures substantially above those of the surrounding interstitial space. In tumors, the oxygen pressures in the two spaces were similar but with large differences among tumors. In mice, anesthesia with ketamine plus xylazine markedly decreased oxygen pressures in the interstitial and intravascular spaces compared to awake or isoflurane anesthetized mice.

Influence of Optical Heterogeneities on Reconstruction of Spatial Phosphorescence Lifetime Distributions

Optics Letters. Apr, 2008  |  Pubmed ID: 18414531

We have previously demonstrated that phosphorescence lifetime imaging (PLI) allows for unbiased determination of absolute oxygen levels in homogeneously absorbing/scattering media. We computationally show that tomographic PLI can perform equally well in heterogeneous environments relying only on surface measurements of phosphorescence.

Synthesis of 5,15-diaryltetrabenzoporphyrins

The Journal of Organic Chemistry. Jun, 2008  |  Pubmed ID: 18452337

A general method of synthesis of 5,15-diaryltetrabenzoporphyrins (Ar 2TBPs) has been developed, based on 2 + 2 condensation of dipyrromethanes followed by oxidative aromatization. Two pathways to Ar 2TBPs were investigated: the tetrahydroisoindole pathway and the dihydroisoindole pathway. In the tetrahydroisoindole pathway, precursor 5,15-diaryltetracyclohexenoporphyrins (5,15-Ar 2TCHPs) were assembled from cyclohexeno-fused meso-unsubstituted dipyrromethanes and aromatic aldehydes or, alternatively, by way of the classical MacDonald synthesis. In the first case, scrambling was observed. Aromatization by tetracyclone was more effective than aromatization by DDQ but failed in the cases of porphyrins with electron-withdrawing substituents in the meso-aryl rings. The dihydroisoindole pathway was found to be much superior to the tetrahydroisoindole pathway, and it was developed into a general preparative method, consisting of (1) the synthesis of 4,7-dihydroisoindole and its transformation into meso-unsubstituted dipyrromethanes, (2) the synthesis of 5,15-diaryloctahydrotetrabenzoporphyrins (5,15-Ar 2OHTBPs), and (3) their subsequent aromatization by DDQ. Ar 2TBP free bases exhibit optical absorption spectra similar to those of meso-unsubstituted tetrabenzoporphyrins and fluoresce with high quantum yields. Pd complex of Ph 2TBP was found to be highly phosphorescent at room temperature.

Oxygen Microscopy by Two-photon-excited Phosphorescence

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Aug, 2008  |  Pubmed ID: 18663708

High-resolution images of oxygen distributions in microheterogeneous samples are obtained by two-photon laser scanning microscopy (2P LSM), using a newly developed dendritic nanoprobe with internally enhanced two-photon absorption (2PA) cross-section. In this probe, energy is harvested by a 2PA antenna, which passes excitation onto a phosphorescent metalloporphyrin via intramolecular energy transfer. The 2P LSM allows sectioning of oxygen gradients with near diffraction-limited resolution, and lifetime-based acquisition eliminates dependence on the local probe concentration. The technique is validated on objects with a priori known oxygen distributions and applied to imaging of pO(2) in cells.

Effects of Structural Deformations on Optical Properties of Tetrabenzoporphyrins: Free-bases and Pd Complexes

The Journal of Physical Chemistry. A. Aug, 2008  |  Pubmed ID: 18665576

A recently developed method of synthesis of pi-extended porphyrins made it possible to prepare a series of tetrabenzoporphyrins (TBP) with different numbers of meso-aryl substituents. The photophysical parameters of free-bases and Pd complexes of meso-unsubstituted TBP's, 5,15-diaryl-TBP's (Ar2TBP's) and 5,10,15,20-tetraaryl-TBP's (Ar4TBP's) were measured. For comparison, similarly meso-arylsubstituted porphyrins fused with nonaromatic cyclohexeno-rings, i.e. Ar(n)-tetracyclohexenoporphyrins (Ar(n)TCHP's, n = 0, 2, 4), were also synthesized and studied. Structural information was obtained by ab initio (DFT) calculations and X-ray crystallography. It was found that: 1) Free-base Ar4TBP's are strongly distorted out-of-plane (saddled), possess broadened, red-shifted spectra, short excited-state lifetimes and low fluorescence quantum yields (tau(fl) = 2-3 ns, phi(fl) = 0.02-0.03). These features are characteristic of other nonplanar free-base porphyrins, including Ar4TCHP's. 2) Ar2TBP free-bases possess completely planar geometries, although with significant in-plane deformations. These deformations have practically no effect on the singlet excited-state properties of Ar2TBP's as compared to planar meso-unsubstituted TBP's. Both types of porphyrins retain strong fluorescence (tau(fl) = 10-12 ns, phi(fl) = 0.3-0.4), and their radiative rate constants (k(r)) are 3-4 times higher than those of planar H2TCHP's. 3) Nonplanar deformations dramatically enhance nonradiative decay of triplet states of regular Pd porphyrins. For example, planar PdTCHP phosphoresces with high quantum yield (phi(phos) = 0.45, tau(phos) = 1118 micros), while saddled PdPh4TCHP is practically nonemissive. In contrast, both ruffled and saddled PdAr(n)TBP's retain strong phosphorescence at ambient temperatures (PdPh2TBP: tau(phos) = 496 micros, phi(phos) = 0.15; PdPh4TBP: tau(phos) = 258 micros, phi(phos) = 0.08). It appears that pi-extension is capable of counterbalancing deleterious effects of nonplanar deformations on triplet emissivity of Pd porphyrins.

Dynamic Quenching of Porphyrin Triplet States by Two-Photon Absorbing Dyes: Towards Two-Photon-Enhanced Oxygen Nanosensors

Journal of Photochemistry and Photobiology. A, Chemistry. 2008  |  Pubmed ID: 19030124

Two-photon-enhanced dendritic nanoprobes are being developed for two-photon (2P) laser scanning microscopy of oxygen [1]. In these molecular constructs, phosphorescence of metalloporphyrins is coupled to two-photon absorption (2PA) of electronically separate antenna dyes via intramolecular Förster-type resonance energy transfer (FRET). In the originally developed probes, competing electron transfer (ET) between the antennae and the long-lived triplet states of metalloporphyrins partially quenched the phosphorescence, reducing the probe's sensitivity and dynamic range. The rate of such ET can be reduced by tuning the redox potentials of the chromophores. In order to identify the optimal metalloporphyrin-2P antenna pairs, we performed screening of several phosphorescent Pt porphyrins (FRET acceptors) and 2P dyes (FRET donors) using dynamic quenching of phosphorescence. Phosphorescence lifetimes of Pt porphyrins were measured as a function of the dye concentration in organic solutions. The obtained Stern-Volmer quenching constants were correlated with the corresponding ET driving forces (DeltaG(ET)), calculated using the Rehm-Weller equation. FRET-pairs with minimal quenching rates were identified. The developed approach allows convenient screening of candidate-compounds for covalent assembly of 2P-enhanced triplet nanodevices. Systematic electrochemical measurements in a series of Pt porphyrins with varying peripheral substitution and conjugation pathways are presented.

Design of Metalloporphyrin-Based Dendritic Nanoprobes for Two-Photon Microscopy of Oxygen

Journal of Porphyrins and Phthalocyanines. Dec, 2008  |  Pubmed ID: 19763243

Metalloporphyrin-based phosphorescent nanoprobes are being developed for two-photon microscopy of oxygen. In these molecular constructs generation of porphyrin triplet states upon two-photon excitation is induced upon the intramolecular Förster-type resonance energy transfer from a covalently attached 2P antenna. In the earlier developed prototypes, electron transfer between the antenna and the metalloporphyrin strongly interfered with the phosphorescence, reducing the sensitivity and the dynamic range of the sensors. By tuning the distances between the antenna and the core and adjusting their redox potentials the unwanted electron transfer could be prevented. An array of phosphorescent Pt porphyrins (energy transfer acceptors) and 2P dyes (energy transfer donors) was screened using dynamic quenching of phosphorescence, and the FRET-pair with the minimal ET rate was identified. This pair, consisting of Coumarin-343 and Pt meso-tetra-(4-alkoxyphenyl)porphyrin, was used to construct a probe in which the antenna fragments were linked to the termini of G3 poly(arylglycine) (AG) dendrimer with PtP core. The folded dendrimer formed an insulating layer between the porphyrin and the antenna, simultaneously controlling the rate of oxygen quenching (Stern-Volmer oxygen quenching constant). Modification of the dendrimer periphery with oligoethyleneglycol residues made the probe's signal insensitive to the presence of proteins and other macromolecular solutes.

Optical Monitoring of Oxygen Tension in Cortical Microvessels with Confocal Microscopy

Optics Express. Dec, 2009  |  Pubmed ID: 20052157

Evaluating cerebral oxygenation is of critical importance for the understanding of brain function and several neuropathologies. Although several techniques exist for measuring cerebral oxygenation in vivo, the most widely accepted techniques offer limited spatial resolution. We have developed a confocal imaging system for minimally invasive measurement of oxygen tension (pO(2)) in cerebral microvessels with high spatial and temporal resolution. The system relies on the phosphorescence quenching method using exogenous porphyrin-based dendritic oxygen probes. Here we present high-resolution phosphorescence images of cortical microvasculature and temporal pO(2) profiles from multiple locations in response to varied fraction of inspired oxygen and functional activation.

Dendritic Phosphorescent Probes for Oxygen Imaging in Biological Systems

ACS Applied Materials & Interfaces. Jun, 2009  |  Pubmed ID: 20072726

Oxygen levels in biological systems can be measured by the phosphorescence quenching method using probes with controllable quenching parameters and defined biodistributions. We describe a general approach to the construction of phosphorescent nanosensors with tunable spectral characteristics, variable degrees of quenching, and a high selectivity for oxygen. The probes are based on bright phosphorescent Pt and Pd complexes of porphyrins and symmetrically pi-extended porphyrins (tetrabenzoporphyrins and tetranaphthoporphyrins). pi-Extension of the core macrocycle allows tuning of the spectral parameters of the probes in order to meet the requirements of a particular imaging application (e.g., oxygen tomography versus planar microscopic imaging). Metalloporphyrins are encapsulated into poly(arylglycine) dendrimers, which fold in aqueous environments and create diffusion barriers for oxygen, making it possible to regulate the sensitivity and the dynamic range of the method. The periphery of the dendrimers is modified with poly(ethylene glycol) residues, which enhance the probe's solubility, diminish toxicity, and help prevent interactions of the probes with the biological environment. The probe's parameters were measured under physiological conditions and shown to be unaffected by the presence of biomacromolecules. The performance of the probes was demonstrated in applications, including in vivo microscopy of vascular pO(2) in the rat brain.

Precise Detection of PH Inside Large Unilamellar Vesicles Using Membrane-impermeable Dendritic Porphyrin-based Nanoprobes

Analytical Biochemistry. May, 2009  |  Pubmed ID: 19248752

Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu(3), which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe's pK is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe's pK was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu(3) was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu(3) was found to be superior to the commercially available pH indicators.

Simultaneous Imaging of Cerebral Partial Pressure of Oxygen and Blood Flow During Functional Activation and Cortical Spreading Depression

Applied Optics. Apr, 2009  |  Pubmed ID: 19340106

We developed a novel imaging technique that provides real-time two-dimensional maps of the absolute partial pressure of oxygen and relative cerebral blood flow in rats by combining phosphorescence lifetime imaging with laser speckle contrast imaging. Direct measurement of blood oxygenation based on phosphorescence lifetime is not significantly affected by changes in the optical parameters of the tissue during the experiment. The potential of the system as a novel tool for quantitative analysis of the dynamic delivery of oxygen to support brain metabolism was demonstrated in rats by imaging cortical responses to forepaw stimulation and the propagation of cortical spreading depression waves. This new instrument will enable further study of neurovascular coupling in normal and diseased brain.

Neutrophil Alpha-defensins Cause Lung Injury by Disrupting the Capillary-epithelial Barrier

American Journal of Respiratory and Critical Care Medicine. May, 2010  |  Pubmed ID: 20093642

The involvement of neutrophil activation in the sentinel, potentially reversible, events in the pathogenesis of acute lung injury (ALI) is only partially understood. alpha-Defensins are the most abundant proteins secreted by activated human neutrophils, but their contribution to ALI in mouse models is hindered by their absence from murine neutrophils and the inability to study their effects in isolation in other species.

Two-photon Microscopy of Oxygen: Polymersomes As Probe Carrier Vehicles

The Journal of Physical Chemistry. B. Nov, 2010  |  Pubmed ID: 20462225

Oxygen concentration distributions in biological systems can be imaged by the phosphorescence quenching method in combination with two-photon laser scanning microscopy. In this paper, we identified the excitation regime in which the signal of a two-photon-enhanced phosphorescent probe (Finikova, O. S.; Lebedev, A. Y.; Aprelev, A.; Troxler, T.; Gao, F.; Garnacho, C.; Muro, S.; Hochstrasser, R. M.; Vinogradov, S. A. ChemPhysChem 2008, 9, 1673-1679) is dependent quadratically on the excitation power (quadratic regime), and performed simulations that relate the photophysical properties of the probe to the imaging resolution. Further, we characterized polymersomes as a method of probe encapsulation and delivery. Photophysical and oxygen sensing properties of the probe were found unchanged when the probe is encapsulated in polymersomes. Polymersomes were found capable of sustaining high probe concentrations, thereby serving to improve the signal-to-noise ratios for oxygen detection compared to the previously employed probe delivery methods. Imaging of polymersomes loaded with the probe was used as a test-bed for a new method.

Pi-extended Dipyrrins Capable of Highly Fluorogenic Complexation with Metal Ions

Journal of the American Chemical Society. Jul, 2010  |  Pubmed ID: 20583759

The synthesis and properties of a new family of pi-extended dipyrrins capable of forming brightly fluorescent complexes with metal ions are reported. The metal complexes possess tunable spectral bands and exhibit different emission properties depending on the mode of metal coordination.

Palmitic Acid Acutely Inhibits Acetylcholine- but Not GLP-1-stimulated Insulin Secretion in Mouse Pancreatic Islets

American Journal of Physiology. Endocrinology and Metabolism. Sep, 2010  |  Pubmed ID: 20606076

Fatty acids, acetylcholine, and GLP-1 enhance insulin secretion in a glucose-dependent manner. However, the interplay between glucose, fatty acids, and the neuroendocrine regulators of insulin secretion is not well understood. Therefore, we studied the acute effects of PA (alone or in combination with glucose, acetylcholine, or GLP-1) on isolated cultured mouse islets. Two different sets of experiments were designed. In one, a fixed concentration of 0.5 mM of PA bound to 0.15 mM BSA was used; in the other, a PA ramp from 0 to 0.5 mM was applied at a fixed albumin concentration of 0.15 mM so that the molar PA/BSA ratio changed within the physiological range. At a fixed concentration of 0.5 mM, PA markedly inhibited acetylcholine-stimulated insulin release, the rise of intracellular Ca(2+), and enhancement of cAMP production but did not influence the effects of GLP-1 on these parameters of islet cell function. 2-ADB, an IP(3) receptor inhibitor, reduced the effect of acetylcholine on insulin secretion and reversed the effect of PA on acetylcholine-stimulated insulin release. Islet perfusion for 35-40 min with 0.5 mM PA significantly reduced the calcium storage capacity of ER measured by the thapsigargin-induced Ca(2+) release. Oxygen consumption due to low but not high glucose was reduced by PA. When a PA ramp from 0 to 0.5 mM was applied in the presence of 8 mM glucose, PA at concentrations as low as 50 microM significantly augmented glucose-stimulated insulin release and markedly reduced acetylcholine's effects on hormone secretion. We thus demonstrate that PA acutely reduces the total oxygen consumption response to glucose, glucose-dependent acetylcholine stimulation of insulin release, Ca(2+), and cAMP metabolism, whereas GLP-1's actions on these parameters remain unaffected or potentiated. We speculate that acute emptying of the ER calcium by PA results in decreased glucose stimulation of respiration and acetylcholine potentiation of insulin secretion.

Two-photon High-resolution Measurement of Partial Pressure of Oxygen in Cerebral Vasculature and Tissue

Nature Methods. Sep, 2010  |  Pubmed ID: 20693997

Measurements of oxygen partial pressure (pO(2)) with high temporal and spatial resolution in three dimensions is crucial for understanding oxygen delivery and consumption in normal and diseased brain. Among existing pO(2) measurement methods, phosphorescence quenching is optimally suited for the task. However, previous attempts to couple phosphorescence with two-photon laser scanning microscopy have faced substantial difficulties because of extremely low two-photon absorption cross-sections of conventional phosphorescent probes. Here we report to our knowledge the first practical in vivo two-photon high-resolution pO(2) measurements in small rodents' cortical microvasculature and tissue, made possible by combining an optimized imaging system with a two-photon-enhanced phosphorescent nanoprobe. The method features a measurement depth of up to 250 microm, sub-second temporal resolution and requires low probe concentration. The properties of the probe allowed for direct high-resolution measurement of cortical extravascular (tissue) pO(2), opening many possibilities for functional metabolic brain studies.

Highly Non-planar Dendritic Porphyrin for PH Sensing: Observation of Porphyrin Monocation

Inorganic Chemistry. Nov, 2010  |  Pubmed ID: 20882973

Metal-free porphyrin-dendrimers provide a convenient platform for the construction of membrane-impermeable ratiometric probes for pH measurements in compartmentalized biological systems. In all previously reported molecules, electrostatic stabilization (shielding) of the core porphyrin by peripheral negative charges (carboxylates) was required to shift the intrinsically low porphyrin protonation pK(a)'s into the physiological pH range (pH 6-8). However, binding of metal cations (e.g., K(+), Na(+), Ca(2+), Mg(2+)) by the carboxylate groups on the dendrimer could affect the protonation behavior of such probes in biological environments. Here we present a dendritic pH nanoprobe based on a highly non-planar tetraaryltetracyclohexenoporphyrin (Ar(4)TCHP), whose intrinsic protonation pK(a)'s are significantly higher than those of regular tetraarylporphyrins, thereby eliminating the need for electrostatic core shielding. The porphyrin was modified with eight Newkome-type dendrons and PEGylated at the periphery, rendering a neutral water-soluble probe (TCHpH), suitable for measurements in the physiological pH range. The protonation of TCHpH could be followed by absorption (e.g., ε(Soret)(dication)∼270,000 M(-1) cm(-1)) or by fluorescence. Unlike most tetraarylporphyrins, TCHpH is protonated in two distinct steps (pK(a)'s 7.8 and 6.0). In the region between the pK(a)'s, an intermediate species with a well-defined spectroscopic signature, presumably a TCHpH monocation, could be observed in the mixture. The performance of TCHpH was evaluated by pH gradient measurements in large unilamellar vesicles. The probe was retained inside the vesicles and did not pass through and/or interact with vesicle membranes, proving useful for quantification of proton transport across phospholipid bilayers. To interpret the protonation behavior of TCHpH we developed a model relating structural changes on the porphyrin macrocycle upon protonation to its basicity. The model was validated by density functional theory (DFT) calculations performed on a planar and non-planar porphyrin, making it possible to rationalize higher protonation pK(a)'s of non-planar porphyrins as well as the easier observation of their monocations.

Oxygen-dependent Quenching of Phosphorescence Used to Characterize Improved Myocardial Oxygenation Resulting from Vasculogenic Cytokine Therapy

Journal of Applied Physiology (Bethesda, Md. : 1985). May, 2011  |  Pubmed ID: 21292844

This study evaluates a therapy for infarct modulation and acute myocardial rescue and utilizes a novel technique to measure local myocardial oxygenation in vivo. Bone marrow-derived endothelial progenitor cells (EPCs) were targeted to the heart with peri-infarct intramyocardial injection of the potent EPC chemokine stromal cell-derived factor 1α (SDF). Myocardial oxygen pressure was assessed using a noninvasive, real-time optical technique for measuring oxygen pressures within microvasculature based on the oxygen-dependent quenching of the phosphorescence of Oxyphor G3. Myocardial infarction was induced in male Wistar rats (n = 15) through left anterior descending coronary artery ligation. At the time of infarction, animals were randomized into two groups: saline control (n = 8) and treatment with SDF (n = 7). After 48 h, the animals underwent repeat thoracotomy and 20 μl of the phosphor Oxyphor G3 was injected into three areas (peri-infarct myocardium, myocardial scar, and remote left hindlimb muscle). Measurements of the oxygen distribution within the tissue were then made in vivo by applying the end of a light guide to the beating heart. Compared with controls, animals in the SDF group exhibited a significantly decreased percentage of hypoxic (defined as oxygen pressure ≤ 15.0 Torr) peri-infarct myocardium (9.7 ± 6.7% vs. 21.8 ± 11.9%, P = 0.017). The peak oxygen pressures in the peri-infarct region of the animals in the SDF group were significantly higher than the saline controls (39.5 ± 36.7 vs. 9.2 ± 8.6 Torr, P = 0.02). This strategy for targeting EPCs to vulnerable peri-infarct myocardium via the potent chemokine SDF-1α significantly decreased the degree of hypoxia in peri-infarct myocardium as measured in vivo by phosphorescence quenching. This effect could potentially mitigate the vicious cycle of myocyte death, myocardial fibrosis, progressive ventricular dilatation, and eventual heart failure seen after acute myocardial infarction.

Evaluation of Phototoxicity of Dendritic Porphyrin-based Phosphorescent Oxygen Probes: an in Vitro Study

Photochemical & Photobiological Sciences : Official Journal of the European Photochemistry Association and the European Society for Photobiology. Jun, 2011  |  Pubmed ID: 21409208

Biological oxygen measurements by phosphorescence quenching make use of exogenous phosphorescent probes, which are introduced directly into the medium of interest (e.g. blood or interstitial fluid) where they serve as molecular sensors for oxygen. The byproduct of the quenching reaction is singlet oxygen, a highly reactive species capable of damaging biological tissue. Consequently, potential probe phototoxicity is a concern for biological applications. Herein, we compared the ability of polyethyleneglycol (PEG)-coated Pd tetrabenzoporphyrin (PdTBP)-based dendritic nanoprobes of three successive generations to sensitize singlet oxygen. It was found that the size of the dendrimer has practically no effect on the singlet oxygen sensitization efficiency in spite of the strong attenuation of the triplet quenching rate with an increase in the dendrimer generation. This unexpected result is due to the fact that the lifetime of the PdTBP triplet state in the absence of oxygen increases with dendritic generation, thus compensating for the concomitant decrease in the rate of quenching. Nevertheless, in spite of their ability to sensitize singlet oxygen, the phosphorescent probes were found to be non-phototoxic when compared with the commonly used photodynamic drug Photofrin in a standard cell-survival assay. The lack of phototoxicity is presumably due to the inability of PEGylated probes to associate with cell surfaces and/or penetrate cellular membranes. In contrast, conventional photosensitizers bind to cell components and act by generating singlet oxygen inside or in the immediate vicinity of cellular organelles. Therefore, PEGylated dendritic probes are safe to use for tissue oxygen measurements as long as the light doses are less than or equal to those commonly employed in photodynamic therapy.

Measuring Oxygen in Living Tissue: Intravascular, Interstitial, and "tissue" Oxygen Measurements

Advances in Experimental Medicine and Biology. 2011  |  Pubmed ID: 21445769

Oxygen dependent quenching of phosphorescence has been used to measure the oxygen pressure in both the vasculature of the microcirculation and the interstitial spaces of resting muscle tissue. Oxygen sensitive molecules were either dissolved in the blood (intravascular space) or micro-injected into the interstitial space and the distributions, histograms, of the oxygen pressure were measured. The mean oxygen pressures are higher in the blood than in the interstitial space but the oxygen pressures in the lowest 10% of the two spaces were not significantly different, indicating there is minimal (< 1 mm Hg) oxygen gradient between the two spaces in the capillary bed.

Simultaneous Two-photon Imaging of Oxygen and Blood Flow in Deep Cerebral Vessels

Nature Medicine. Jul, 2011  |  Pubmed ID: 21642977

Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO(2)) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO(2) can be accurately measured in the brain at depths up to 300 μm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO(2) in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO(2) in capillaries and by a biphasic PO(2) response in the neuropil, consisting of an 'initial dip' and a rebound. 2PLM of PO(2) opens new avenues for studies of brain metabolism and blood flow regulation.

Single Cell Responses to Spatially Controlled Photosensitized Production of Extracellular Singlet Oxygen

Photochemistry and Photobiology. Sep-Oct, 2011  |  Pubmed ID: 21668871

The response of individual HeLa cells to extracellularly produced singlet oxygen was examined. The spatial domain of singlet oxygen production was controlled using the combination of a membrane-impermeable Pd porphyrin-dendrimer, which served as a photosensitizer, and a focused laser, which served to localize the sensitized production of singlet oxygen. Cells in close proximity to the domain of singlet oxygen production showed morphological changes commonly associated with necrotic cell death. The elapsed postirradiation "waiting period" before necrosis became apparent depended on: (1) the distance between the cell membrane and the domain irradiated, (2) the incident laser fluence and, as such, the initial concentration of singlet oxygen produced and (3) the lifetime of singlet oxygen. The data imply that singlet oxygen plays a key role in this process of light-induced cell death. The approach of using extracellularly generated singlet oxygen to induce cell death can provide a solution to a problem that often limits mechanistic studies of intracellularly photosensitized cell death: it can be difficult to quantify the effective light dose, and hence singlet oxygen concentration, when using an intracellular photosensitizer.

"Overshoot" of O₂ is Required to Maintain Baseline Tissue Oxygenation at Locations Distal to Blood Vessels

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2011  |  Pubmed ID: 21940458

In vivo imaging of cerebral tissue oxygenation is important in defining healthy physiology and pathological departures associated with cerebral disease. We used a recently developed two-photon microscopy method, based on a novel phosphorescent nanoprobe, to image tissue oxygenation in the rat primary sensory cortex in response to sensory stimulation. Our measurements showed that a stimulus-evoked increase in tissue pO₂ depended on the baseline pO₂ level. In particular, during sustained stimulation, the steady-state pO₂ at low-baseline locations remained at the baseline, despite large pO₂ increases elsewhere. In contrast to the steady state, where pO₂ never decreased below the baseline, transient decreases occurred during the "initial dip" and "poststimulus undershoot." These results suggest that the increase in blood oxygenation during the hemodynamic response, which has been perceived as a paradox, may serve to prevent a sustained oxygenation drop at tissue locations that are remote from the vascular feeding sources.

Two New "protected" Oxyphors for Biological Oximetry: Properties and Application in Tumor Imaging

Analytical Chemistry. Nov, 2011  |  Pubmed ID: 21961699

We report the synthesis, calibration, and examples of application of two new phosphorescent probes, Oxyphor R4 and Oxyphor G4, optimized specifically for in vivo oxygen imaging by phosphorescence quenching. These "protected" dendritic probes can operate in either albumin-rich (blood plasma) or albumin-free (interstitial space) environments at all physiological oxygen concentrations, from normoxic to deep hypoxic conditions. Oxyphors R4 and G4 are derived from phosphorescent Pd-meso-tetra-(3,5-dicarboxyphenyl)-porphyrin (PdP) or Pd-meso-tetra-(3,5-dicarboxyphenyl)-tetrabenzoporphyrin (PdTBP), respectively, and possess features common for protected dendritic probes, i.e., hydrophobic dendritic encapsulation of phosphorescent metalloporphyrins and hydrophilic PEGylated periphery. The new Oxyphors are highly soluble in aqueous environments and do not permeate biological membranes. The probes were calibrated under physiological conditions (pH 6.4-7.8) and temperatures (22-38 °C), showing high stability, reproducibility of signals, and lack of interactions with biological solutes. Oxyphor G4 was used to dynamically image intravascular and interstitial oxygenation in murine tumors in vivo. The physiological relevance of the measurements was demonstrated by dynamically recording changes in tissue oxygenation during application of anesthesia (isofluorane). These experiments revealed that changes in isofluorane concentration significantly affect tissue oxygenation.

Frontiers in Optical Imaging of Cerebral Blood Flow and Metabolism

Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism. Jan, 2012  |  Pubmed ID: 22252238

In vivo optical imaging of cerebral blood flow (CBF) and metabolism did not exist 50 years ago. While point optical fluorescence and absorption measurements of cellular metabolism and hemoglobin concentrations had already been introduced by then, point blood flow measurements appeared only 40 years ago. The advent of digital cameras has significantly advanced two-dimensional optical imaging of neuronal, metabolic, vascular, and hemodynamic signals. More recently, advanced laser sources have enabled a variety of novel three-dimensional high-spatial-resolution imaging approaches. Combined, as we discuss here, these methods are permitting a multifaceted investigation of the local regulation of CBF and metabolism with unprecedented spatial and temporal resolution. Through multimodal combination of these optical techniques with genetic methods of encoding optical reporter and actuator proteins, the future is bright for solving the mysteries of neurometabolic and neurovascular coupling and translating them to clinical utility.Journal of Cerebral Blood Flow & Metabolism advance online publication, 18 January 2012; doi:10.1038/jcbfm.2011.195.

Monitoring Cardiopulmonary Function and Progression Toward Shock: Oxygen Micro-sensor for Peripheral Tissue

Advances in Experimental Medicine and Biology. 2012  |  Pubmed ID: 22259105