Preparation method of probe for measuring oxygen content based on fluorescence ratio technology

Abstract

The invention discloses a preparation method of a probe for measuring oxygen content based on a fluorescence ratio technology, and belongs to biomedical polymer materials. According to the probe for determining the oxygen content based on the fluorescence ratio technology, the ruthenium metal complex with oxygen responsiveness and the reference probe dye with an oxygen-free sensing function are modified on the surface of biomedical polymer or bovine serum albumin, and the fluorescence intensity change of the selective wavelength of a double-probe system on the surface can be used as a quantitative basis; through ratio calibration, interference of single fluorescence signal change caused by probe concentration change, excitation intensity change, instrument efficiency, photobleaching and non-uniform distribution of the probe at the action part in the testing process can be eliminated, and a more accurate monitoring result can be obtained. The polymer and the protein can solve the problems of system water solubility and fluorophore dispersibility, and enhance the in-vivo circulation half-life period of the probe. The probe disclosed by the invention has a wide prospect in the application of oxygen distribution biological imaging in organisms and oxygen concentration quantitative characterization.

Description

Technical Field

[0001] This invention pertains to biomedical polymer materials, specifically relating to an oxygen content measurement probe and its preparation method, and more specifically to a probe for measuring oxygen content based on fluorescence ratio technology and its preparation method. Background Technology

[0002] Oxygen concentration sensitive probes are based on the oxygen quenching principle of oxygen quenching, which dissipates the energy of excited-state fluorescent molecules by oxygen molecules to suppress photoluminescence. This optical sensing technology, which uses changes in fluorescence signals as the detection basis, overcomes the shortcomings of traditional oxygen monitoring methods and exhibits advantages such as high sensitivity, reversibility, high selectivity, and high spatiotemporal resolution. In the biomedical field, the oxygen concentration in blood and tissues in the human body can serve as an important parameter for clinical diagnosis. Many pathological tissues, such as solid tumors, cardiovascular diseases, inflammation, and stroke, have obvious hypoxic (hypoxic) environments. Therefore, in recent years, the detection of hypoxia or the oxygen status in vivo has received increasing attention. (Hsieh M, Tsai C, Liao M, et al. Quantitative susceptibility mapping-based microscopy of magnetic resonancevenography (QSM-mMRV) for in vivo morphologically and functionally assessing cerebromicrovasculature in rat stroke model[J]. PLoS One, 2016, 11(3):e0149602.). Oxygen concentration-sensitive probes and optical microscopy techniques enable visualization of oxygen distribution in two-dimensional or three-dimensional environments within living organisms, as well as measurement of oxygen partial pressure in blood vessels of various tissue types, including tumors, the brain, retina, and the heart. Among the widely used hypoxia fluorescent probes, several transition metal complexes, such as ruthenium, iridium, and europium complexes, show great potential in biological hypoxia imaging due to their high sensitivity, stability, and reversibility. In particular, ruthenium complexes exhibit unique advantages in oxygen sensing research due to their large Stokes shift, strong photobleaching resistance, and high oxygen sensitivity. (Guan S, Yang D, Weng Y, et al. Excitation-dependent theranostic nanosheet for cancer treatment[J]. Advanced Healthcare Materials, 2018, 7(10): 1701-123.) In fluorescence sensing strategies, ratiometric fluorescence sensing has higher accuracy because it has at least two emission signals, and the output feedback of the ratio of the two fluorescence intensities is used as the input signal. Because it has been shown to be insensitive to ambient light, scattered light and instrument fluctuations, ratiometric sensing can respond more specifically to the analyte.(Wang C, Chen Y, Yang Q, et al. Silica-supported dual-dye nanoprobes for ratiometric hypoxiasensing[J]. Materials Chemistry Frontiers, 2021, 5(1): 458-464.) In summary, by combining the oxygen responsiveness of ruthenium complexes, a probe for measuring oxygen content based on fluorescence ratiometry is designed, which is of great significance for studying diagnostic methods for diseases in vivo. Summary of the Invention

[0003] The purpose of this invention is to provide a probe for measuring oxygen content based on fluorescence ratio technology and its preparation method. The probe for measuring oxygen content based on fluorescence ratio technology can achieve bioimaging of oxygen distribution in arteries, veins, and lesion tissues in vivo, and quantitatively characterize the oxygen concentration in vivo by calculating the light intensity ratio.

[0004] The probe for measuring oxygen content based on fluorescence ratio technology provided by this invention consists of an oxygen-responsive coordination compound molecule, a reference probe dye without oxygen sensing function, and a polymer or protein.

[0005] In the above-mentioned oxygen content determination probe, the oxygen-responsive coordination compound molecule is a ruthenium metal complex with ruthenium as the central atom. It is chemically bonded to a reference probe dye without oxygen sensing function in a certain proportion and then grafted onto the surface of the polymer or protein to form a complex.

[0006] The reference probe dye without oxygen sensing function has an absorption spectrum that overlaps to some extent with the emission spectrum of the ruthenium metal complex, which meets the requirements of the fluorescence resonance energy transfer process. The ratio calibration of the two serves as the basis for quantifying oxygen content.

[0007] The polymer or protein is used to address the system's water solubility and fluorophore dispersibility, and to enhance the probe's circulating half-life in vivo.

[0008] The ruthenium metal complex has ligands that are bidentate or tridentate ligands of bipyridine and / or phenanthroline types with carboxyl and / or amino groups.

[0009] The bidentate ruthenium metal complex has the structure defined in claim 4 of Chinese Patent (CN201710383101.5) entitled "An oxygen concentration-responsive polymer photoluminescent coating and its preparation and application".

[0010] The tridentate ruthenium metal complex with ligands has the following structural formula:

[0011]

[0012] In each of these R groups, R is independently at least one of H, CH3, COOH, and NH2, and at least one or two of them are amino or carboxyl functional groups.

[0013] The ruthenium metal complex can specifically be [Ru(dcbpy)3] 2+ Cl2、[Ru(bpy)3(NH2)2 2+ Cl2、[Ru(dcbpy)2(bpy) 2+ Cl2、[Ru(tpy)2 2+ Cl2.

[0014] The excitation wavelength range of the reference probe dye with the oxygen-free sensing function is 550nm-650nm.

[0015] The reference probe dye contains at least one of amino, carboxyl, and hydroxyl functional groups.

[0016] The reference probe dye may specifically be a fluoroboron dipyrrole dye or a sulfonated and non-sulfonated anthocyanin dye (such as Cy5.5).

[0017] The molar ratio of the ruthenium metal complex to the reference probe dye during bonding is 10:1–1:10, specifically 5:1–1:5.

[0018] The dual-probe system, synthesized by bonding the ruthenium metal complex with a reference probe dye, is grafted onto a biomedical polymer or bovine serum albumin, wherein the biomedical polymer is polyethylene glycol and its derivatives, F127 and its derivatives, or dextran and its derivatives.

[0019] The average molecular weight of the polyethylene glycol is 600-35000;

[0020] The average molecular weight of F127 is 1000-20000;

[0021] The average molecular weight of the dextran is 800-600,000;

[0022] The aforementioned biomedical polymer also contains modifying groups, specifically at least one of amino, carboxylic acid, NHS, methyl, sulfonate, and thiol groups.

[0023] The probe for determining oxygen content based on fluorescence ratio technology was prepared by a method including the following steps:

[0024] By linking a ruthenium metal complex with a reference probe dye, a dual probe system synthesized by linking the ruthenium metal complex with the reference probe dye is obtained. The dual probe system is then grafted onto the surface of a polymer or protein to obtain a probe for measuring oxygen content based on fluorescence ratio technology.

[0025] In the above method, the bonding is achieved through amidation or esterification reactions, and the reaction temperature can be 0-37℃ and the time can be 2-48h.

[0026] The bonding is performed in deionized water or PBS solution;

[0027] The molar ratio of the ruthenium metal complex to the reference probe dye is 10:1–1:10, specifically 5:1–1:5;

[0028] The grafting is achieved through amidation, esterification, and activation esterification. The reaction temperature can be 0-37℃, specifically 0-4℃, 10℃, 20℃, or 25-37℃, and the reaction time can be 2-48h.

[0029] The molar ratio of the dual probe system to the polymer or protein is 1:1 to 1:50.

[0030] Furthermore, the catalyst used in the amidation reaction can be EDC / NHS, and the catalyst used in the esterification reaction can be DMAP / DCC.

[0031] The application of the probes for measuring oxygen content based on fluorescence ratio technology in bioimaging of oxygen distribution and quantitative characterization of oxygen concentration in organisms is also within the scope of protection of this invention.

[0032] The application of the probes used to measure oxygen content based on fluorescence ratio technology in the study of hypoxia in blood vessels and tissues at lesion sites in vivo also falls within the scope of protection of this invention.

[0033] Compared to existing oxygen concentration-sensitive probes, the oxygen content measurement probe of this invention, based on fluorescence ratio technology, modifies the surface of a biomedical polymer or bovine serum albumin with an oxygen-responsive ruthenium metal complex and a non-oxygen-sensing reference probe dye. The selective wavelength fluorescence intensity changes of the dual-probe system can serve as a quantitative basis. Through ratio calibration, interference from variations in probe concentration, excitation intensity, instrument efficiency, photobleaching, and uneven probe distribution at the site of action can be eliminated, resulting in more accurate monitoring results. The use of polymers and proteins can address the system's water solubility and fluorophore dispersion, and enhance the probe's circulating half-life in vivo. These beneficial effects make this invention promising for applications in bioimaging of oxygen distribution and quantitative characterization of oxygen concentration in vivo. Attached Figure Description

[0034] Figure 1 The fluorescence spectra of the oxygen content determination ratio imaging probe aqueous solution prepared in Example 1 of the present invention at different oxygen concentrations are shown.

[0035] Figure 2The curve showing the relationship between the fluorescence intensity ratio of the two probes and the oxygen concentration in the oxygen content determination ratio imaging probe prepared in Example 2 of this invention.

[0036] Figure 3 The fluorescence spectra of the oxygen content measurement ratio imaging probe prepared in Example 3 of the present invention at different oxygen concentrations in blood.

[0037] Figure 4 The circular dichroism spectrum of BSA alone and the oxygen content determination ratio imaging probe prepared in Example 4 of this invention.

[0038] Figure 5 The oxygen content measurement ratio imaging probe prepared in Example 5 of this invention is used for ratio imaging of the distribution of oxygen concentration in the arteries and veins of the cerebral cortex and brain tissue of MCAO model mice under in vivo confocal microscopy. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] The aminofluoroboron dipyrrole structure in the following examples is shown below:

[0042]

[0043] Product name: Aminofluoroboron dipyrrole, purchased from Shaanxi Xinyan Bomei Biotechnology Co., Ltd.

[0044] Example 1

[0045] First, dichlorobis(4-methylisopropylphenyl)ruthenium(II) and 2,2'-bipyridine-5,5'-dicarboxylic acid were dispersed in anhydrous methanol at a molar ratio of 1:6 and refluxed at 80-100℃. After stirring under a nitrogen atmosphere for 40 hours, the reaction was stopped, filtered, and the filtrate was collected. The solvent was removed by rotary evaporation, and the product was recrystallized from acetone to obtain the ruthenium metal complex [Ru(dcbpy)3]. 2+ Cl2.

[0046] Will [Ru(dcbpy)3] 2+Cl2 (0.035 mmol), EDC (0.04 mmol), and NHS (0.08 mmol) were dissolved in 10 mL of deionized water. After stirring in an ice-water bath at 4 °C for 1 h, aminofluoroboron dipyrrole (BDP-NH2, 0.04 mmol) was added. The reaction mixture was then transferred to room temperature and stirred in the dark for 24 h. The product BDP-Ru(dcbpy)3 was purified by thin-layer chromatography. BDP-Ru(dcbpy)3 (0.064 mmol), mPEG3500 (2 mmol), DCC (0.016 mmol), and DMAP (0.006 mmol) were dissolved in 15 mL of DCM. The mixture was stirred in the dark at 37 °C for 48 h. The filtrate was filtered, rotary evaporated, dispersed in water, dialyzed, and lyophilized to obtain the final product BDP-Ru(dcbpy)3-mPEG.

[0047] Take 2 mL of a 0.25 mg / mL, uniformly dispersed BDP-Ru(dcbpy)3-mPEG aqueous solution and add it to a four-way quartz cell. Purge with gases of varying oxygen content for approximately 15 minutes. After purging, use a fluorescence spectrophotometer to measure the fluorescence spectrum under excitation light at a wavelength of 470 ± 10 nm. Ensure the atmosphere reaches equilibrium before proceeding to the next step of the experiment.

[0048] Figure 1 The fluorescence spectra of BDP-Ru(dcbpy)3-mPEG aqueous solution under different oxygen concentrations are shown.

[0049] Figure 1 This reflects that the fluorescence intensity of the ratio fluorescent oxygen concentration probe varies under different oxygen partial pressure environments, enabling the function of judging changes in oxygen partial pressure based on changes in fluorescence.

[0050] Example 2

[0051] Ruthenium trichloride trihydrate and 2,2'-bipyridine were added to DMF solvent at a molar ratio of 1:3. The reaction temperature was 80-120℃, and the reaction was stopped after stirring for 24 hours under a nitrogen atmosphere. The filtrate was filtered, the solvent was evaporated, and the solution was recrystallized from isopropanol to obtain the ruthenium dichloride (2,2'-bipyridine) complex Ru(bpy)2Cl2.

[0052] Ru(bpy)₂Cl₂ was reacted with 2,2'-bipyridine-4,4'-diamine at a molar ratio of 1:1 using DMF as the solvent. The reaction was carried out at 90-120℃ under a nitrogen atmosphere and stirred for 24 hours. The insoluble matter was then filtered off, the solvent was removed, and the mixture was dried to obtain [Ru(bpy)₃(NH₂)₂]. 2+ Cl2.

[0053] [Ru(bpy)3(NH2)2] 2+Cl2 (0.02 mmol) and Cy5.5-NHS (0.02 mmol) were dissolved in 20 mL of deionized water and stirred at room temperature for 12 h. The mixture was then transferred to an ice-water bath and EDC (60.035 mmol) and NHS (0.07 mmol) were added. The mixture was stirred for 1 h. Then, carboxyglucan (Dextran-COOH, 0.4 mmol) with an average molecular weight of 6000 was added to the above system. The mixture was reacted at 30 °C in the dark for 48 h. After dialyzing, the mixture was lyophilized to obtain Cy5.5-Ru(bpy)3(NH2)2-Dextran.

[0054] Take 2 mL of a 0.25 mg / mL, uniformly dispersed Cy5.5-Ru(bpy)3(NH2)2-Dextran aqueous solution into a four-way quartz cell, introduce gases with different oxygen contents, and use a fluorescence spectrophotometer to test the relationship between the ratio of the fluorescence of the two probes and the oxygen concentration using excitation light at a wavelength of 488±10 nm.

[0055] Figure 2 The curve showing the relationship between the fluorescence intensity ratio of the two probes corresponding to Cy5.5-Ru(bpy)3(NH2)2-Dextran and oxygen concentration indicates the change in fluorescence intensity I corresponding to the emission peaks of the ruthenium complex and the reference probe during changes in oxygen concentration. Ru / I Cy The decay follows a perfectly linear decreasing relationship.

[0056] Example 3

[0057] [Ru(dcbpy)2(bpy)] 2+ Cl2 (0.019 mmol), EDC (0.03 mmol), and NHS (0.06 mmol) were dissolved in 15 mL of deionized water and stirred in an ice-water bath at 4 °C for 0.5 h. Cy5-NH2 (0.038 mmol) was added, and the reaction was transferred to room temperature and stirred in the dark for 24 h. EDC and NHS were dispersed into the above system and stirred for 1 h. After activating the carboxyl group, mPEG2000-NH2 (0.8 mmol) was added, and the reaction was carried out in the dark for 24 h. The mixture was dialyzed and freeze-dried to obtain Cy5-Ru(dcbpy)2(bpy)-mPEG.

[0058] According to Chinese Patent (CN201710383101.5), an oxygen concentration-responsive polymer photoluminescent coating and its preparation and application are synthesized [Ru(dcbpy)2(bpy)]. 2+ Cl2.

[0059] Blood was collected from the medial canthal venous plexus of adult male SD rats. Cy5-Ru(dcbpy)2(bpy)-mPEG was dissolved in the rat blood using 10% sodium citrate as a solvent, and the oxygen responsiveness of the probe in the blood was tested. 2 mL of a 0.25 mg / mL sample was placed in a four-way quartz cell, and gases with different oxygen concentrations were introduced. The fluorescence emission spectrum was measured using a fluorescence spectrophotometer with excitation light at 470 nm.

[0060] Figure 3 The fluorescence spectra of Cy5-Ru(dcbpy)2(bpy)-mPEG in blood at different oxygen concentrations are shown.

[0061] Figure 3 This indicates that the fluorescence intensity of the ratio-fluorescent oxygen concentration probe varies under different oxygen partial pressure environments in blood, and the pattern is consistent with that in aqueous solution. This allows the probe to determine changes in oxygen partial pressure in blood vessels within a living organism based on changes in fluorescence.

[0062] Example 4

[0063] [Ru(bpy)3(NH2)2] 2+ Cl2 (0.04 mmol), EDC (0.04 mmol), NHS (0.02 mmol) were dissolved in 15 mL of PBS (0.0067 M) at pH 7.4. After stirring in an ice-water bath at 4 °C for 1 h, Cy5 (0.02 mmol) was added, and the mixture was stirred in the dark for 28 h. [Ru(bpy)3(NH2)2 2+ After the Cl2 and Cy5 bonds are completed, EDC (0.08 mmol) and NHS (0.16 mmol) are dispersed into the above system and stirred for 1 h. Bovine serum albumin (BSA, 1 mmol) is added and reacted in the dark for 36 h. After dialyzing, the ratiometric protein probe Cy5-Ru(bpy)3(NH2)2-BSA is obtained by lyophilization.

[0064] Take 1 mL of a 0.0125 mg / mL, uniformly dispersed aqueous solution of Cy5-Ru(bpy)3(NH2)2-BSA and test its protein structure using a circular dichroism spectroscopy instrument.

[0065] Figure 4 The circular dichroism spectra of BSA alone and Cy5-Ru(bpy)3(NH2)2-BSA modified with dual probes are shown. The results indicate that the conformation of BSA is not affected by the probe modification.

[0066] Example 5

[0067] The terpyridine ruthenium complex [Ru(tpy)2] 2+Cl2 (0.04 mmol), EDC (0.045 mmol), and NHS (0.09 mmol) were dissolved in 25 mL of deionized water and stirred in an ice-water bath at 4 °C for 1 h. BDP-NH2 (0.04 mmol) was added, and the reaction was transferred to room temperature and stirred in the dark for 24 h. EDC (0.04 mmol) and NHS (0.08 mmol) were then dispersed in the above system and stirred for 1 h. After activating the carboxyl groups, aminochitosan with a molecular weight of 10000 (Dextran-NH2, 1.2 mmol) was added, and the reaction was carried out in the dark for 48 h. The mixture was dialyzed and lyophilized to obtain BDP-Ru(tpy)2-Dextran.

[0068] According to the reference (Toyao T, Saito M, Dohshi S, et al. Development of a Ru complex-incorporated MOF photocatalyst for hydrogen production under visible-light irradiation[J]. Chemical Communications, 2014, 50(51):6779-6781.), [Ru(tpy)2] was synthesized. 2+ Cl2.

[0069] Adult male C57BL / 6J mice weighing 25-30g were selected as experimental subjects. A persistent middle cerebral artery occlusion (MCAO) model was established in mice using the suture occlusion method, with the suture insertion depth being 10±0.5mm. Signs of successful modeling included: flexion or decreased grasping ability of the left forelimb, and spontaneous leftward rotation or pitching. Mice in each group were fixed to a stereotaxic head frame. The skull was routinely prepared and disinfected, and a 1.5mm incision was made in the midline of the skull. After separating the skin, subcutaneous tissue, and periosteum layer by layer, a craniotomy was performed, removing a 2.5mm×2.5mm bone flap from each parietal bone. BDP-Ru(tpy)2-Dextran (100mg / kg) was injected via the tail vein 1-6 hours later. Figure 5 (The results in the middle are measured after 6 hours) Fluorescence imaging in the microcirculation of the bilateral cranial windows was then observed under a two-photon confocal microscope (excitation wavelength 488nm).

[0070] Figure 5 This study presents ratioographic imaging of oxygen concentration distribution in the arteries and veins of the cerebral cortex and brain tissue of MCAO model mice using BDP-Ru(tpy)2-Dextran under in vivo confocal microscopy. Comparison of oxygen concentration changes between the healthy and ischemic sides demonstrates the accuracy and practicality of the probe based on fluorescence ratioography for oxygen content measurement.

[0071] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A probe for determining oxygen content based on fluorescence ratio technology consists of an oxygen-responsive coordination compound molecule, a reference probe dye without oxygen sensing function, and a polymer or protein. In the oxygen content determination probe, the oxygen-responsive coordination compound molecule is a ruthenium metal complex with ruthenium as the central atom. It is chemically bonded to a reference probe dye without oxygen sensing function in a certain proportion and then grafted onto the surface of the polymer or protein to form a complex.

2. The probe according to claim 1, characterized in that, The ruthenium metal complex has ligands that are bipyridine and / or phenanthroline bidentate or tridentate ligands with carboxyl and / or amino groups. The reference probe dye without oxygen sensing function has an absorption spectrum that overlaps to some extent with the emission spectrum of the ruthenium metal complex, which meets the requirements of the fluorescence resonance energy transfer process. The ratio of the two is used as the basis for quantifying oxygen content. The polymer or protein is used to address the system's water solubility and fluorophore dispersibility, and to enhance the probe's circulating half-life in vivo.

3. The probe according to claim 1 or 2, characterized in that, The tridentate ruthenium metal complex with ligands has the following structural formula: In each of these R groups, R is independently at least one of H, CH3, COOH, and NH2, and at least one or two of them are amino or carboxyl functional groups.

4. The probe according to claim 1 or 2, characterized in that, The excitation wavelength range of the reference probe dye with the oxygen-free sensing function is 550nm-650nm. The reference probe dye contains at least one of amino, carboxyl, and hydroxyl functional groups; Specifically, the reference probe dye is a fluoroboron dipyrrole dye or a sulfonated and non-sulfonated anthocyanin dye.

5. The probe according to claim 1 or 2, characterized in that, The molar ratio of the ruthenium metal complex to the reference probe dye during bonding is 10:1–1:

10.

6. The probe according to claim 1 or 2, characterized in that, The dual-probe system synthesized by bonding the ruthenium metal complex with the reference probe dye is grafted onto a biomedical polymer or bovine serum albumin, wherein the biomedical polymer is polyethylene glycol and its derivatives, F127 and its derivatives, and dextran and its derivatives. The biomedical polymer also contains a modifying group, which may be at least one of amino, carboxylic acid, NHS, methyl, sulfonate, and thiol groups.

7. A method for preparing the probe according to any one of claims 1-6, comprising the following steps: bonding a ruthenium metal complex to a reference probe dye to obtain a dual probe system synthesized by bonding the ruthenium metal complex to the reference probe dye; grafting the dual probe system onto the surface of a polymer or protein to obtain a probe for determining oxygen content based on fluorescence ratio technology.

8. The method according to claim 7, characterized in that, The bonding is achieved through amidation or esterification reactions at a temperature of 0-37°C for a time of 2-48 hours. The bonding is performed in deionized water or PBS solution; The molar ratio of the ruthenium metal complex to the reference probe dye is 10:1–1:10; The grafting is achieved through amidation, esterification, and activated esterification reactions, with the reaction temperature ranging from 0 to 37°C and the reaction time from 2 to 48 hours. The molar ratio of the dual probe system to the polymer or protein is 1:1 to 1:

50.

9. The application of the probe according to any one of claims 1-6 in bioimaging of oxygen distribution and quantitative characterization of oxygen concentration in living organisms.

10. The application of the probe according to any one of claims 1-6 in the study of hypoxia in blood vessels and tissues at lesions in vivo.

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