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 &tau;0. In the presence of oxygen, the phosphorescence lifetime (&tau;) 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 (&tau;) of the phosphorescence to oxygen concentration [O2] (or oxygen partial pressure pO2):
<img src="/files/ftp_upload/1731/1731equation.jpg" 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 &tau;. 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 &tau; 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 &pi;-extended porphyrins. &pi;-Extended porphyrins are porphyrins in which pyrrole rings are fused with external aromatic fragments. &pi;-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&deg;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 &tau;0 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 &mu;M 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&deg;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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+method+for+measurement+of+dioxygen+concentration+based+on+quenching+of+phosphorescence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+phosphorescence:+A+novel+method+for+measuring+the+distribution+of+oxygen+in+perfused+tissue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+phosphorescent+probes+for+oxygen+Imaging+in+biological+systems.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+versatile+synthesis+of+substituted+tetrabenzoporphyrins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rothemund+and+Adler-Longo+Reactions+revisited:+Synthesis+of+tetraphenylporphyrins+under+equilibrium+conditions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+metalloporphyrin-based+dendritic+nanoprobes+for+two-photon+microscopy+of+oxygen.">Design of metalloporphyrin-based dendritic nanoprobes for two-photon microscopy of oxygen.</a> J. Porphyrins and Phthalocyanines. 12, 1261-1269 (2008).</li></ol>

<table border="1" cellspacing="0" cellpadding="0">
 <tr><td>Abbreviation</td>
 <td>Full name</td>
 </tr>
 <tr><td>NMP</td>
 <td>N-methylpyrrolidinone</td>
 </tr>
 <tr><td>TFA</td>
 <td>trifluoroacetic acid</td>
 </tr>
 <tr><td>DIPEA</td>
 <td>diisopropylethylamine</td>
 </tr>
 <tr><td>HBTU</td>
 <td>2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate</td>
 </tr>
 <tr><td>DMSO</td>
 <td>dimethylsulfoxide</td>
 </tr>
 <tr><td>CDMT</td>
 <td>CDMT=1-chloro-3,5-dimethoxytriazine</td>
 </tr>
 <tr><td>NMM</td>
 <td>NMM=N-methylmorfoline</td>
 </tr>
</table>

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.

Watch this Scientific Journal Video about Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems at JoVE.com

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

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 ...

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13.8K Görüntüleme.  University of Pennsylvania. Biz fosforesans giderici ve biyolojik sistemlerde oksijen görüntüleme için porfirin-tabanlı dendritik nanosensors gözden tasarım ilkeleri oksijen ölçümleri sunuyoruz.

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

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

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We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

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

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

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, ...

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.