Name: Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems (Video) | JoVE Journal
Uploaded: 2010-03-03T17:30:00.000+00:00
Duration: 10 min 38 s
Description: Learn to synthesize and calibrate phosphorescent nanoprobes for oxygen imaging using platinum and palladium porphyrin dendritic sensors. This method enables accurate biological oxygen measurement.

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Support of the grants EB007279 and HL081273 from the NIH USA is gratefully acknowledged.

1. General description of oxygen measurement protocol
(This section does not have any action, but is crucial for understanding the rest of the paper. It can be filmed, for example, as a sequence of a few Power Point slides, accompanied by the voice.)
1.1) The probe is delivered into the medium of interest, for example, injected in the blood or interstitial fluid of an animal. 
1.2) The object (surface of the tissue) is illuminated with the light of appropriate wavelength in order to promote the probe into its excited triplet state. Typically excitation occurs via the one-photon mechanism. However, in the case of special two-photon-enhanced probes, excitation can be done via the two-photon mechanism, which permits using the probes in high-resolution microscopy applications. One-photon excitation generally provides less spatial resolution, but requires simpler instrumentation and can be used in single-point measurements with LED-based fiber-optic phosphorometers. 
1.3) Phosphorescence emitted by the probe originates from the long-lived triplet state. The probe molecule must be specially designed to give high quantum yield of the triplet state and emit phosphorescence instead of fluorescence. While in the triplet state, the probe can experience collisional encounters with molecules of oxygen, which can de-activate the triplet state - a process called "quenching." In the absence of oxygen, the lifetime of the triplet state, and hence of the phosphorescence decay, is τ0. In the presence of oxygen, the phosphorescence lifetime (τ) is shortened as a result of the quenching. There is a direct relationship between the lifetime of the triplet state and the amount of oxygen in the environment. This is the well-known Stern-Volmer relationship, which connects the lifetime (τ) of the phosphorescence to oxygen concentration [O2] (or oxygen partial pressure pO2):
<img src="/files/ftp_upload/1731/1731equation.jpg" alt="Stern-Volmer equation for fluorescence quenching analysis with oxygen concentration." data-altupdatedat="1788235205000" data-alt="equation">
In biological systems, oxygen is by far the most effective quencher of phosphorescence, which makes the phosphorescence quenching method very selective to oxygen. 
1.4) In order to measure the lifetime t, the emitted phosphorescent photons are binned in time. For example, after the excitation pulse, 200 photons might be collected in the first 15 microseconds, 150 photons collected in the next 15 microseconds, and so on, until no photons are collected. The numbers in the bins plotted against time give the phosphorescence decay, which is analyzed to yield the lifetime τ. In imaging, this procedure is applied in every pixel of the image, resulting in phosphorescence lifetime maps. Measurements of lifetimes are insensitive to the heterogeneities of probe distribution throughout the object, which is common for biological samples. Thus, lifetimes report only on oxygen. Lifetimes τ are converted into oxygen concentrations using the Stern-Volmer relationship.
2. Construction of phosphorescent probes
Phosphorescent probes for oxygen measurements in biological systems consist of phosphorescent chromophores (cores), encapsulated into cages constructed of dendrimers - highly regular hyperbranched polymers [3]. Dendrimers protect the cores from interactions with the environment and control the rate of oxygen diffusion to the cores, making it possible to optimize the sensitivity and dynamic range of the probes (constant kq in the Stern-Volmer relationship). The termini of the dendrimers are modified with chemically inert hydrophilic polyethylene glycol (PEG) groups, making the probes highly water-soluble and preventing their interactions with biological macromolecules.
Phosphorescent cores of the probes are Pt or Pd complexes of porphyrins and π-extended porphyrins. π-Extended porphyrins are porphyrins in which pyrrole rings are fused with external aromatic fragments. π-Extension allows tuning of the probes spectroscopic properties to satisfy the requirements of a particular imaging application (e.g. microscopy vs. tomography). As an example, we describe synthesis of Pd tetaaryltetrabenzoporphyrins (PdAr4TBP) [4]. PdAr4TBP's have powerful absorption bands in the far-red part of the spectrum and strong phosphorescence, well suited for biological oxygen measurements.
The synthesis of PdAr4TBP's consists of the following steps:
2.1) Preparation of tetraaryltetracyclohexenoporphyrins (Ar4TCHP) by Lindsey condensation [5] of tetrahydroisoindole with substituted benzaldehydes. To a 0.01 M solution of tetrahydroisoindole (1 eq) in CH2CL2 the aromatic aldehyde (1 eq) was added under argon. The reaction mixture was stirred for 10 min in the dark at R.T. BF3xEt2O (0.2 eq) was added, and the reaction mixture was stirred at rt for an additional 2 h. DDQ (1 eq) was added, and the mixture was left overnight under continuous stirring. The solution was washed with 10% aq Na2SO3, 10% aq Na2CO3, 5% aq HCl and finally with brine. The organic phase was dried over Na2SO4, then concentrated in vacuum. The residue was recrystallized from CH2Cl2 - tert-butyl ether mixture to give Ar4TCHP as green powder. Yields are about 50%. 
2.2) Insertion of Pd to obtain PdAr4TCHPs. Free-base porphyrins (1 eq) were treated with PdCl2 or Pd(OAc)2 (1.2 eq) in refluxing CH3CN in the presence of excess of Et3N (20 eq). The conversion was monitored by UV-vis spectroscopy (solvent CH2Cl2-AcOH) and considered complete after the Soret band of dictation at 468-472 nm disappeared. The mixture was allowed to cool down, diluted with CH2Cl2 and filtered through a thin layer of Celite to remove Pd(0). The solvent was evaporated, and the residue was purified by chromatography on silica gel column using CH2Cl2 as a solvent. Dark red fraction was collected, the solvent was evaporated to give target PdAr4TCHPs in almost quantitative yield.
2.3) PdAr4TBP's were prepared by oxidation of PdAr4TCHPs with the 2-fold excess of DDQ (16 eq) in refluxed THF. During refluxing, the color changed from dark-red to deep-green. The solvent was evaporated, the residue was diluted with CH2Cl2, washed with 10% aq Na2SO3, water and brine. The organic phase was dried over Na2SO4 and concentrated in vacuum. The residue was purified by chromatography on silica gel (eluent CH2Cl2). The first dark-green fraction was collected, concentrated in vacuum to give 85-90% of PdAr4TBP's as blue-green powder.
2.4) Peripheral ester groups of Pd tetrakis-(3,5-dibutoxycarbonylphenyl)-tetrabenzoporphyrin (PdAr4TBP) were hydrolyzed using 10-fold excess of KOH in 1% (v/v) water/THF at rt. The solvent was decanted from the precipitate of the formed potassium salts of carboxylic acid derivatives of PdAr4TBP's. The solids were dissolved in water, stirred for additional 2 h, acidified with conc. HCl to pH~4-5. The obtained precipitate was isolated by centrifugation, washed with water and dried in vacuum. 
Protecting dendrons. Each individual branch of the dendrimer is called dendron. Protecting dendrons are constructed from aryl-glycine (AG) building blocks. AG dendrons can be easily synthesized from inexpensive starting materials and isolated in high purity and yield by chromatography-free methods. Synthesis of generation 2 (G2) AG-dendron consists of the following steps: 
2.4) Synthesis of the building blocks, Boc-protected 3,5-dicarboxyphenyl glycineamide and 3,5-dibutoxycarbonylphenyl glycineamide, using of the Fischer haloacyl halide method.
2.5) Coupling of the building blocks using CDMT/NMM peptide coupling chemistry. 
2.6) Deprotecting the focal amino group by stirring the solution of the G2 dendron in TFA for 2 h at R.T. 
2.7) For assembly of the higher generation dendrons, coupling/deprotection steps are repeated.
Dendrimer assembly is achieved by coupling of the deprotected dendrons to PdAr4TBP (or other porphyrin) using the HBTU/DIPEA coupling chemistry. 
2.8) First, the Boc-protective groups are removed from the dendron. For example, Boc-protected G3 dendron (0.58 g) was dissolved in TFA (15 ml), and the mixture was left to react at R.T. for 2h. The acid was removed by rotary evaporation, yielding the dendron as the TFA-salt. The residue was dissolved in dry NMP (10 ml) and a few drops of DIPEA were added, to quench the traces of TFA. 
2.9) Before starting the coupling reaction, it is critical to completely dissolve the porphyrin. For example, Pt tetracarboxyphenylporphyrin (0.061 g) was dissolved in dry NMP (65 ml) under heating at 140°C for 10 min, under nitrogen flow. 
2.10) The solution was cooled down to room temperature, HBTU (0.211 g) was added, and the mixture was stirred for 5 min. 
2.11) DIPEA (0.35 ml) was added to the mixture in one portion, immediately followed by addition of the solution of TFA-salt of the dendron in NMP (see 2.8), and the mixture was left overnight under stirring.
2.12) The mixture was poured into 3% aq. NaCl (350 ml), and the resulting precipitate was collected by centrifugation and washed with water, MeOH, and Et2O by repetitive suspension/centrifugation to yield the target porphyrin-dendrimer. 
2.13) To hydrolyze the peripheral ester groups, the dendrimer was first treated with NMe4OH (~ 5 mM) in DMSO/MeOH over 20-60 min period, followed by the complete solvent removal. This procedure allowed us to generate dendrimers with sufficient number of carboxylate groups to give aqueous solubility. To complete the hydrolysis, the dendrimer was treated with 0.1N aqueous NaOH (overnight). As a result, pure porphyrin-dendrimer carboxylic acids could be isolated in 80-95% yields. 
Modification of the dendrimer periphery. At this point, starting from the same porphyrin-dendrimer, either one- or two-photon probes can be synthesized. In the first case, all the peripheral carboxyl groups are modified with PEG units - a methodology known as PEGylation. In the latter case, several carboxyl groups are first coupled with two-photon antenna chromophores (Coumarin-343) modified with ethylenediamine (EDA) linkers, followed by PEGylation of the remaining carboxyls [6]. Dendrimers were PEGylated using monomethoxyoligoethyleneglycolamine (m-PEG-NH2, Av. MW 1000) using HBTU/DIPEA coupling chemistry. 
2.14) To a solution of the dendron in NMP (or DMF) (1-10 mM) 1.25-fold excess of HBTU was added, and the reaction mixture was stirred at R.T. for 10 min. 6.25-Fold excess of DIPEA was added, followed by the immediate addition of 1.25-fold excess of m-PEG-NH2. The reaction mixture was stirred for 2 days at rt. Diethyl ether was added to the reaction mixture, the formed precipitate was separated by centrifugation and re-precipitated from THF by addition of diethyl ether. 
2.15) Final purification was achieved with size-exclusion chromatography by the collecting of the first front phase. PEGylated porphyrin-dendrimer was dissolved in a small amount of THF (~15 ml) and loaded on the top of a chromatographic column filled with polystyrene SEC phase (Biorad, Biobeads S-X1). The column was eluted with pure THF, and the dark colored band moving with the front was collected.
2.16) THF was removed on a rotary evaporator, and the remaining material was dried in vacuum. 
3. Probe characterization
Photophysical characterization includes measurements of the probe's absorption and emission spectra, phosphorescence lifetimes τ0 and Stern-Volmer oxygen quenching constants kq under physiological conditions.
3.1) The absorption and emission spectra are obtained under ambient conditions using standard instrumentation (spectrophotometer and steady fluorometer). ~1 μM solutions of the probe is used.
3.2) Next, the Stern-Volmer calibration plot is obtained. This is the most important measurement, because it allows us to relate the lifetime of the probe to the oxygen concentration.
3.3) A solution of the probe in PBS buffer (pH 7.2) is placed in a special cylindrical cuvette, which is positioned inside the temperature controlled chamber, located inside a light-impermeable cage with ports for excitation and emission optical fibers. The fibers are brought into close contact with the cuvette inside the chamber in the right-angle geometry.
3.4) The cuvette is closed with a stopper, into which a highly sensitive Clark-type oxygen electrode (CK-oxygen electrode) is inserted. The stopper also contains two needle ports for inlet and outlet of argon. The electrode is immersed into the solution, whereas the needles merely provide for the flow of gas flow above the solution surface. At first, argon is not connected to the inlet.
3.5) The temperature is set to the desired value (typically 36-37°C) and solution is left under stirring for equilibration.
3.6) The excitation fiber is connected to the excitation port of a digital phosphorometer, operated by a PC. The light source in the phosphorometer is a high-power LED, whose output is controlled by a 333 kHz D/A board. The emission fiber is connected to another optical port of the phosphorometer, which is coupled to a highly infrared-sensitive APD (avalanche photodiode). The output of the diode is amplified and fed into the A/D channel of the same control board, which allows synchronization between the excitation and emission channels.
3.7) The control software allows generation of excitation pulses of any desired length (e.g. 5 or 10 ms), followed by collection of phosphorescence decay during any arbitrary chosen period (typically 2-3 ms). Time required for a single lifetime measurement is typically 0.5-1 s, however, measurements can be obtained as rapidly as 10-20 per second.
3.8) The output of the oxygen electrode is amplified and directed into another A/D board on the same PC. This is a low frequency board (1 kHz max), which is used to record the electrode current at selected time periods. The program for recording the electrode data runs simultaneously with the phosphorometer software.
3.9) Once the solution temperature is equilibrated, both the phosphorometer and the electrode programs are initialized at the same time to perform measurements every 10 s. Their outputs are logged synchronously into two separate files. After that, argon is connected to the inlet port on the cuvette stopper.
3.10) As argon flows over the surface of the stirred solution, it gradually replaces oxygen. This results in a decrease of the electrode current and an increase in the phosphorescence lifetime, which is measured by the phosphorometer. Usually, oxygen is displaced form the solution entirely after about 2 h, during which time the data are fully automatically logged into files.
3.11) After the end of the titration run, the electrode data and the phosphorescence lifetimes are imported into a standard analysis program (e.g. Miocrocal Origin). The electrode current depends linearly on oxygen concentration, and for the electrode used it is practically zero at zero oxygen. At atmospheric pressure, pO2 is known (about 150 mm Hg). Thus, the electrode data can be directly converted to the oxygen scale, and the plot of inverse phosphorescence lifetime vs. pO2 can be constructed. This plot is fitted with a straight line using the least-squares method to give the oxygen quenching constant kq as its slope. Phosphorescence lifetime t0 is obtained either from the same fit (as the inverse of the intercept) or directly from the measurement at zero-oxygen.
3.12) If required, the titration is repeated using a solution of the probe in the presence of albumin - a protein present in the blood plasma in high concentrations, - in order to emulate the conditions met in the real experimental system (blood of an animal in vivo). The obtain Stern-Volmer plots should be identical if the dendrimer is protecting the probe well and PEG groups isolate the probe from contacts with albumin. Otherwise, the probe and the proteins in the blood will interact, and that will lead to an altered Stern-Volmer plot, causing ambiguity in oxygen measurements.
Thus obtained calibration constants are used in imaging experiments where the oxygen concentrations are a priori unknown.

<ol><li>Vanderkooi, J. M., Maniara, G., Green, T. J., Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+optical+method+for+measurement+of+dioxygen+concentration+based+on+quenching+of+phosphorescence%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">An optical method for measurement of dioxygen concentration based on quenching of phosphorescence.</a> J. Biol. Chem. 262, 5476-5482 (1987).</li>
<li>Rumsey, W. L., Vanderkooi, J. M., Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+of+phosphorescence%3A+A+novel+method+for+measuring+the+distribution+of+oxygen+in+perfused+tissue%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Imaging of phosphorescence: A novel method for measuring the distribution of oxygen in perfused tissue.</a> Science. 241, 1649-1651 (1988).</li>
<li>Lebedev, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+phosphorescent+probes+for+oxygen+Imaging+in+biological+systems%5BTitle%5D)+AND+%22Acs+Applied+Materials+and+Interfaces%22%5BJournal%5D)">Dendritic phosphorescent probes for oxygen Imaging in biological systems.</a> Acs Applied Materials and Interfaces. 1, 1292-1304 (2009).</li>
<li>Finikova, O. S., Cheprakov, A. V., Beletskaya, I. P., Carroll, P. J., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+versatile+synthesis+of+substituted+tetrabenzoporphyrins%5BTitle%5D)+AND+%22Journal+of+Organic+Chemistry%22%5BJournal%5D)">Novel versatile synthesis of substituted tetrabenzoporphyrins.</a> Journal of Organic Chemistry. 69, 522-535 (2004).</li>
<li>Lindsey, J. S., Schreiman, I. C., Hsu, H. C., Kearney, P. C., Marguerettaz, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rothemund+and+Adler-Longo+Reactions+revisited%3A+Synthesis+of+tetraphenylporphyrins+under+equilibrium+conditions%5BTitle%5D)+AND+%22Journal+of+Organic+Chemistry%22%5BJournal%5D)">Rothemund and Adler-Longo Reactions revisited: Synthesis of tetraphenylporphyrins under equilibrium conditions.</a> Journal of Organic Chemistry. 52, 827-836 (1987).</li>
<li>Lebedev, A. Y., Troxler, T., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+of+metalloporphyrin-based+dendritic+nanoprobes+for+two-photon+microscopy+of+oxygen%5BTitle%5D)+AND+%22J.+Porphyrins+and+Phthalocyanines%22%5BJournal%5D)">Design of metalloporphyrin-based dendritic nanoprobes for two-photon microscopy of oxygen.</a> J. Porphyrins and Phthalocyanines. 12, 1261-1269 (2008).</li>
</ol>

<table><tbody><tr><td>N-methylpyrrolidinone</td><td></td><td></td><td>NMP</td></tr><tr><td>trifluoroacetic acid</td><td></td><td></td><td>TFA</td></tr><tr><td>diisopropylethylamine</td><td></td><td></td><td>DIPEA</td></tr><tr><td>2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate</td><td></td><td></td><td>HBTU</td></tr><tr><td>dimethylsulfoxide</td><td></td><td></td><td>DMSO</td></tr><tr><td>CDMT=1-chloro-3,5-dimethoxytriazine</td><td></td><td></td><td>CDMT</td></tr><tr><td>NMM=N-methylmorfoline</td><td></td><td></td><td>NMM</td></tr></tbody></table>

synthesis and calibration of phosphorescent nanoprobes for oxygen imaging in biological systems

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

We present the design of phosphorescent probes for oxygen based on platinum and palladium porphyrin DERs. The probes consist of metal porphyrin cores encapsulating dendron and peripheral hydrophilic polyethylene glycol layer construction of the probes and their calibrations will be illustrated in this paper. Hi, my name is Luis Sinks.I work with Professor Sergei OV here in the Department of Biochemistry and biophysics at the University of Pennsylvania. I am Emmanuel Losis, also from the Vina. I'm fat also from the, And I'm Sergei V.Biological Oxygen measurements by phosphorous and quenching make use of exogenous phosphorous and probes, which are introduced directly into the medium of interest.Blood or interstitial fluid probes are the only invasive component of the measurement scheme requiring special attention to their design. Today we'll show you a procedure for the synthesis and calibration of dendritic phosphorescent nano probes for oxygen measurement and biological systems. We use this procedure to synthesize and characterize probes For semi and two-fold oxygen measurements.So let's get started. Before we begin constructing probes, let's first go over the basic theory behind probe phosphorescence. Phosphorescence originates from the long lived triplet state.The probe molecule must be designed to give high quantum yield of the triplet state and to emit phosphorescence instead of fluorescence. Excitation of the probes occurs by the one photon or in the case of special two photon enhanced probes by the two photon mechanism. One photon excitation generally provides less spatial resolution, but requires simpler instrumentation and can be used in single point measurements with LED based fiber optic phos perimeters.While in the triplet state, the probe can experience collisional encounters with molecules of oxygen, which can deactivate the triplet state a process called quenching. Therefore, in the presence of oxygen, the phosphorescence lifetime is shortened. As a result of the quenching, the dependence of the lifetime of the triplet state on the amount of oxygen in the environment is characterized by the stern volmer equation.In in vivo experiments, the probe is delivered into the blood or interstitial fluid of an animal, and the surface of the tissue is illuminated with light of an appropriate wavelength to bring the probe into Its excited triplet state. In order to measure the phosphorescent lifetime, the emitted phosphorescent photons are bi in time. For example, after the excitation pulse, 3, 609 photons might be collected in the first five microseconds, 1, 421 photons collected in the next five microseconds and so on until no photons are collected.The numbers in the bins plotted against the time give the phosphorescence decay, which is analyzed to yield the phosphorescence lifetime. In imaging, this procedure is applied to every pixel of the image resulting in phosphorescent lifetime maps. Measurements of lifetimes are insensitive to the heterogeneities of probe distribution throughout the object, which is common for biological samples.Now let's see how to construct probes. Begin the synthesis procedure by adding an aromatic aldehyde to a 0.01 molar solution of tetra hydro iso indole. Stir the reaction mixture for 10 minutes in the dark at room temperature.Then add boron, tri fluoride dathyl, ate, and keep stirring for an additional two hours. Next, add dichloride cyan, benzo quinone or DDQ, which results in the color. Change from pale red to deep green and leave the mixture overnight under continuous stirring the next day, wash and dry the solution, and then concentrate it in a vacuum.Reg crystallization of the residue gives the target as a green powder. Yields are typically around 50%Next, treat the freebase porphyrin with palladium acetate. Monitor the conversion by UV vista spectroscopy.The conversion is complete after the soap band of the Dion between 468 and 472 nanometers disappears. The porphyrin is isolated by column chromatography on silica gel. To prepare palladium tetra benzo porphyrin oxidize the palladium tetra cyclo porphyrin.During reflexing, the color changes from dark red to deep green evaporate the solvent, dilute the residue with di chloro methane wash, dry and concentrate the organic phase in a vacuum. After chromatography on silica gel isolate palladium tetra benzo porphyrin as a bluegreen powder. Next hydrolyze the peripheral etro groups of the palladium trab benzo porphyrin.First treat the tetra benzo porphyrin Esther with base in tetra hydro purin. Then continue the hydrolysis and aqueous base, precipitate the porphyrin by addition of hydrochloric acid and dry it in a vacuum. This completes the synthesis of the porphyrin.Now let's see how to synthesize entrons. Before the probes are assembled, the entrons, which are the branches of the Denmark need to be pre synthesized. We use aero gly, same DERs, which can be conveniently prepared from inexpensive studying materials using chromatography free methods.Therons with amino groups at their focal points are then attached to the caril groups on the phy, which we just showed you how to make. Then the ester groups on the periphery of the DME are hydrolyzed similarly to the carboxylic groups on the periphery of the porphyrin. At this point, starting from the Porphyrin DME poly carboxylic acid, either one or two photon probes can be synthesized to synthesize the two photon probes.First divergently attach a few two photon antenna fragments to several carboxyl groups on the demer periphery. Now proceed with modification of the remaining carboxylic acid residues on the demer. Start by adding a 1.25 fold excess of HBTU to a highly concentrated solution of the porphyrin rimer and stir the reaction mixture at room temperature for 10 minutes.Now, add di isopropyl ethylamine and methoxy polyethylene glyco lamine. Stir the reaction mixture for two days at room temperature and then add ethyl ether. Separate the form to precipitate by centrifugation and re precipitate it from tetrahedran a few times by adding dathyl ether.Finally, purify the probe by size exclusion chromatography on polystyrene beads using tetrahedran as a solvent. Now let's look at probe characterization and calibration. The absorption and emission spectra of the probe are obtained using one micromolar probe solutions under ambient conditions with a standard spectrophotometer and steady state fluorimeter.Next to obtain the stern Ulmer calibration plot, which allows us to relate the lifetime of the probe to the oxygen concentration, place a solution of the probe into a special cylindrical vete. The vete is positioned inside a temperature controlled chamber inside a light impermeable cage with ports for excitation and emission optical fibers. Close the vete with a stopper, which has an inserted highly sensitive Clark type oxygen electrode.The stopper also has two needle ports for inlet and outlet of Argonne. Set the temperature to 36 to 37 degrees Celsius and leave the solution stirring until it reaches equilibrium. Connect the excitation fiber to the excitation pour of a digital phospho perimeter controlled by a pc.The light source and the phos perimeter is a high power LED, whose output is controlled by a 333 kilohertz digital to analog board. The emission fiber is connected to another optical port of the phos perimeter, which is coupled to an infrared sensitive avalanche photo diode. The output of the diode is amplified and fed into the ad channel of the same control board, allowing synchronization between the excitation and emission channels.The home written control software generates excitation pulses of any desired length, followed by the collection of phosphorescence decay. The output of the oxygen electrode is amplified and directed into another analog digital board on the same pc. This is a low frequency board, one kilohertz max, which is used to record the electrode current at selected time points, typically 10 times a second.Once the solution temperature is equilibrated, both the phos perimeter and the electrode programs are initialized at the same time. To perform measurements every 10 seconds, their outputs are logged synchronously into two separate files. After that, Argonne is connected to the inlet port on the vet stopper.As Argonne flows over the surface of the stirred solution, it gradually replaces oxygen. This results in a decrease of the electrode current and an increase in the phosphorus lifetime, which is measured by the phos perimeter. Usually oxygen is displaced from the solution entirely.After about two hours after the titration run, the electrode data and the phosphorus lifetimes are imported into a standard analysis program, which creates a plot of the inverse phosphorus lifetime versus oxygen partial pressure. This plot is fitted with a straight line using the least squares method to give the oxygen quenching constant as its slope. Phosphorescent lifetime is obtained either from the same fit or directly from the measurement at zero oxygen.The titration can be repeated using a solution of the probe in the presence of albumin, a protein present in the blood plasma in order to emulate the conditions met in the blood of an animal in vivo. The obtained stern Ulmer plots should be identical if the ER is protecting the probe. Well, and the peg groups isolate the probe from contact with albumin.The suggestion here, selected steps in the synthesis of oxygen, then treated probs and their calibration. When doing the calibration. It's important to make sure that the conditions are as close to that of the biological system of interest.It's important to make sure that the probe molecule is unaffected by biomolecules, such as albumin. So that's it. Thanks for watching and good luck with your experience.

synthesis-calibration-phosphorescent-nanoprobes-for-oxygen-imaging

Research

JoVE Journal

Biology

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

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

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Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

We describe the use of a novel, frequency-domain luminescence lifetime camera for mapping 2D O2 distributions with optical sensor foils. The camera system and image analysis procedures are described along with the preparation, calibration and application of sensor foils for visualizing the O2 microenvironment in the rhizosphere of aquatic plants.

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

This paper describes the use of a new, fast optical imager for the macroscopic photoluminescence lifetime imaging of long decay emitting samples. The integration, image acquisition, and analysis procedures are described, along with the preparation and characterization of the sensor materials for the imaging and the application of the imager in studying biological samples.

Fluorescence Lifetime Macro Imager for Biomedical Applications

We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF).

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

This protocol presents a method for fabricating a double-layer microfluidic chip and analyzing microbial growth under rapid oxygen oscillations. The device's performance was validated through oxygen sensing in fluid channels and microbial cultivation.

Creating Rapid Oxygen Oscillations in Microbial Single-cell Growth Analysis using a Microfluidic Double-layer Device

Learn to synthesize and calibrate phosphorescent nanoprobes for oxygen imaging using platinum and palladium porphyrin dendritic sensors. This method enables accurate biological oxygen measurement.

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

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