Name: Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping (Video) | JoVE Journal
Uploaded: 2010-08-26T17:00:00.000+00:00
Duration: 9 min 40 s
Description: Learn ESR microscopy for three-dimensional oxygen mapping in live cyanobacteria cells. This method enables micron-scale imaging of oxygen gradients and metabolic activity under varying light conditions.

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This work was partially supported by grant no. 213/09 from the Israeli Science Foundation, grant no. 2005258 from the BSF foundation, grant no. 201665 from the European Research Council (ERC), and by the Russell Berrie Nanotechnology Institute at the Technion. We acknowledge the help of Prof. Noam Adir and Faris Salame from the Schulich Faculty of Chemistry at the Technion regarding the supply and handling of the cyanobacteria. The help and the support of Svetlana Yoffis from the Technion Micro-Nano Fabrication Unit is greatly appreciated.

1.	Overview of ESR Micro-imaging
First, we provide a brief explanation of ESR, ESR microscopy, and the various components of our system, and then we will describe the actual imaging experiments.
Electron spin resonance is a spectroscopic technique in which electromagnetic radiation at a specific frequency is absorbed by molecules with unpaired electron spins, placed under an external static magnetic field (Figure 1). ESR is employed in broad areas of science, such as chemistry, biology, physics, and materials science, for the detection and identification of free radicals and paramagnetic centers. It is a powerful method for studying the environment of paramagnetic molecules in live species and provides information about acidity (pH), viscosity, oxygen, and reactive oxygen species concentrations3.
For heterogeneous samples, ESR spectral information can be obtained in a spatially resolved manner (i.e, by obtaining an image), through the use of magnetic field gradients4. This is very similar to the more common method of magnetic resonance imaging (MRI) that mainly observes proton spins. Up until now, such ESR imaging techniques were applied for live specimens with relatively large size of a few centimeters and in mm-scale resolution. (For example see Figure 2, taken from reference 5.) A relatively recent development in ESR imaging is the extension of its capabilities from looking at small animals at millimeter-scale resolution to the measurements of millimeter- and sub-millimeter-size samples with micron-scale resolution. This field is known as ESR microscopy, which today can provide 3D ESR images with a resolution approaching 1 micron 2 (see representative examples in Figure 3).
An ESR microscope is essentially similar to a conventional ESR spectrometer. It has a magnet for generating the static field, a microwave system for spin excitation and signal detection, a probe for holding the sample, and a computerized console to control the acquisition process and data handling. Other components unique the ESR imaging in general and existing also in ESR microcopy are magnetic field gradient sources, which are part of the electronic system, and gradient coils that are located in the imaging probe. More details about our specific system are shown in the protocol movie and described in reference 2.
2.	ESR Micro-imaging Sample Preparation
This stage describes the method for sample preparation for the ESR micro-imaging experiment. At the end of this stage cells are placed on the bottom of a specially-prepared glass ESR microscopy sample container together with a trityl radical6 buffer solution. This protocol describes the measurement of cyanobacteria cells and therefore, for other types of cells, proper adjustments may be needed at the sample preparation stage.
<ol>
<li>	First, a few squares of absorbent paper with a size of ~400 400 μm are taken and inserted into an Eppendorf tube which is subsequently filled with 1.2 mL of the cyanobacteria suspension (at a concentration of 40 mg/mL).</li>
<li>The suspension is centrifuged for 2 minutes at 6000 RPM in a microcentrifuge.</li>
<li>Following this, the supernatant buffer is completely removed except for ~50 μL which are left to avoid cyanobacteria dehydration. As a result of this process, the absorbent paper is now saturated by the cyanobacteria cells.</li>
<li>Using fine tweezers, a few fibers are extracted from the paper and placed on the bottom of a cup-like specially prepared glass sample holder7. Following that a 3 mM of trityl in BG-11 solution8, 9 (see Scheme 1) is added to the sample holder by the aid of a fine syringe. The holder is then sealed using UV curable glue, leaving a small air outlet open. 
<table border="1">
<tbody>
 <tr>
 <td> Stock 4</td>
 <td>Stock 3</td>
 <td>Stock 2</td>
 <td>Stock 1</td>
 </tr>
 <tr>
 <td> H3BO3 2.86g/liter</td>
 <td>K2HPO4 :3H2O 4.0g/liter</td>
 <td>MgSO4 : 7H2O 7.5g/liter</td>
 <td>Na2Mg EDTA 0.1g/liter</td>
 </tr>
 <tr>
 <td>MnCl2 :4H2O 1.81g/liter</td>
 <td> </td> 
 <td> </td> 
 <td>Ferric ammonium citrate 0.6g/liter</td>
 </tr>
 <tr>
 <td>ZnSO4 : 7H2O 0.222g/liter</td>
 <td> </td> 
 <td> </td>
 <td>Citric acid :1H2O 0.6g/liter</td>
 </tr>
 <tr>
 <td>CuSO4 :5H2O 0.079g/liter</td>
 <td> </td>
 <td> </td>
 <td>CaCl2 : 2H2O 3.6g/liter</td>
 </tr>
 <tr>
 <td>COCl2 :6H2O 0.050g/liter</td>
 <td> </td>
 <td> </td>
 <td> </td>
 </tr>
 <tr>
 <td>NaMoO4: 2H2O 0.391g/liter</td>
 <td> </td>
 <td> </td>
 <td> </td>
 </tr>
 </tbody>
</table>
 Scheme 1. Preparation of BG-11 medium.
</li> 
</ol>
3. 	ESR Micro-imaging Experiments
<ol>
<li>	To begin the imaging experiment, turn on the ESR micro-imaging system and insert the sample into the resonator that goes inside the imaging probe. </li>
<li>Now, using the computer control software, set the system on "Tune" mode and find the resonance microwave frequency of the probe, which will be used for the ESR measurements. </li>
<li>Following that, set the static magnetic field on the value that matches the applied microwave frequency, set the timing parameters for the pulse sequence and observe the ESR signal to make sure that the system functions well and the sample is well-prepared.</li>
<li>Then, set the imaging parameters, such as the number of pixels, the strength of the gradients, and the length of the gradient pulses to their required values. </li>
<li>Following setup, collect three 3D ESR images by a Hahn echo imaging pulse sequence (Figure 4) with interpulse separation, values of 500, 600, and 700 ns.</li>
<li>Light projected at the sample is turned on or off depending on the required experimental conditions.</li>
<li> 	During acquisition, data is automatically saved. Theses raw data files are then processed via Matlab software script to provide images of the trityl radical concentration and the relaxation time T2 map, which is translated into an oxygen concentration image via pre-existing calibration.</li>
</ol>
4. Representative Results 
The outcomes of the experiment are several three-dimensional ESR micro-images recorded at different τ values. Typical raw data images are provided in Figure 5. The top three images, measured under dark conditions, are very similar except for reduction in signal intensity. On the other hand, the image pattern changes under light irradiation due to the different relaxation times in different parts of the sample. This data can be processed1 to obtain an amplitude image, as shown in Figure 6 and also images of the relaxation time, T2 (Figure 7). The T2 images are translated into oxygen concentration values via a pre-existing calibration curve that links the oxygen concentration to the relaxation time via the equation: <img src="/files/ftp_upload/2122/2122eq.jpg" alt="Chemical kinetics equation, diagram of enzymatic reaction rate, T2(C,D)-dependent kinetics analysis" data-altupdatedat="1788235205000" data-alt="Equation 1">
Here, T20 is the spin-spin relaxation time of the probe under anoxic conditions (depending on the probe's concentration, C, and its diffusion coefficient, D), and k is a proportionality constant. In most cases, the diffusion coefficient does not vary much for live samples (although, if needed, it can in principle be directly evaluated also by ESR 6, 10), and the spin concentration is obtained during the imaging process. Therefore, this relation can be used to directly measure oxygen concentration.
Going back to Figure 6, it is evident from the amplitude image that the cyanobacteria cells were located mainly on the right side of the sample holder. Furthermore, based on Figure 7, it is clear that light initiates the production of O2 and causes a significant increase in the solution's O2 concentration, mainly in the voxels near the cyanobacteria.
<img src="/files/ftp_upload/2122/2122fig1.jpg" alt="Electron spin resonance diagram, energy vs magnetic field, showing ΔE=hv with microwave frequency." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1: Energy levels in electron spin resonance.
 
<img src="/files/ftp_upload/2122/2122fig2.jpg" alt="Static MRI showing anatomy, tumor highlighted; pO2 map diagram indicating oxygen levels (mmHg)." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2: Typical oxygen concentration image of a Tumor bearing mouse. The image on the left shows the anatomical information, based on an MRI image. A stable free organic radical was injected to the mouse and its ESR characteristics provide the oxygen concentration at its environment (right). ESR-based results are superimposed on MRI anatomical image. Field of view is 32 mm.
<img src="/files/ftp_upload/2122/2122fig3.jpg" alt="Optical and ESR images showing STU letters; electron spin resonance imaging and analysis." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3: Two examples of high resolution micro-scale ESR images of photolithographically-generated sample with N@C60 powder (left) and LiPc paramagnetic crystals (right)
<img src="/files/ftp_upload/2122/2122fig4.jpg" alt="Pulse sequence diagram, spin echo process, MW pulses, Gx, Gy, Gz gradients, NMR spectroscopy." data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4: Typical Hahn imaging pulse sequence showing the microwave (MW) and gradient, Gx, Gy and Gz pulses.
<img src="/files/ftp_upload/2122/2122fig5.jpg" alt="Transient absorption spectroscopy; 6 data sets showing τ=500-700ns, under dark/light conditions; diagram." data-altupdatedat="1788235205000" data-alt="Figure 5"> 
Figure 5: Typical raw-data ESR micro-images: a,b, and c are raw data of the Cyanobacterium sample with no light illumination measured for τ = 500,600,700 ns, respectively. Items d,e, and are the same as a, b, and c but with light illumination. Intensity is plotted in arbitrary scale (but is consistent within each set of three dark or light raw data images)
<img src="/files/ftp_upload/2122/2122fig6.jpg" alt="Spectroscopy heatmap; concentration gradient from 0 to 25 µM; chemical distribution analysis." data-altupdatedat="1788235205000" data-alt="Figure 6"> 
Figure 6: Amplitude image corresponding to the radical concentration in the solution (arbitrary scale).
<img src="/files/ftp_upload/2122/2122fig7.jpg" alt="Dark-light comparison in fluorescence lifetime imaging microscopy (FLIM) results; color-coded maps." data-altupdatedat="1788235205000" data-alt="Figure 7"> 
Figure 7: T2 images and the corresponding [O2] values under dark (left) and light (right) conditions.

<ol><li>Halevy, R., Tormyshev, V., Blank, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-imaging+of+Oxygen+Concentration+near+Live+Photosynthetic+Cells+by+Electron+Spin+Resonance%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Micro-imaging of Oxygen Concentration near Live Photosynthetic Cells by Electron Spin Resonance.</a> Biophysical Journal. , Forthcoming (2010).</li>
<li>Blank, A., Suhovoy, E., Halevy, R., Shtirberg, L., Harneit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ESR+imaging+in+solid+phase+down+to+sub-micron+resolution%3A+methodology+and+applications%5BTitle%5D)+AND+%22J+Phys+Chem%22%5BJournal%5D)">ESR imaging in solid phase down to sub-micron resolution: methodology and applications.</a> J Phys Chem. 11, 6689-6699 (2009).</li>
<li>Gallez, B., Swartz, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+EPR%3A+when%2C+how+and+why%3F%5BTitle%5D)+AND+%22NMR+Biomed%22%5BJournal%5D)">In vivo EPR: when, how and why?</a> NMR Biomed. 17, 223-225 (2004).</li>
<li>Eaton, G. R., Eaton, S. S., Ohno, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aEaton%2C+GR+%22EPR+imaging+and+in+vivo+EPR%22">EPR imaging and in vivo EPR</a>. , CRC Press. Boca Raton. (1991).</li>
<li>Matsumoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+imaging+of+tumor+oxygenation+and+microvascular+permeability+using+Overhauser+enhanced+MRI%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Simultaneous imaging of tumor oxygenation and microvascular permeability using Overhauser enhanced MRI.</a> Proc Natl Acad Sci USA. 106, 17898-17903 (2009).</li>
<li>Talmon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Diffusion+in+Porous+Media+by+PGSE%5BTitle%5D)+AND+%22ESR.+J+Phys+Chem%22%5BJournal%5D)">Molecular Diffusion in Porous Media by PGSE.</a> ESR. J Phys Chem. , Forthcoming (2010).</li>
<li>Halevy, R., Talmon, Y., Blank, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photolithographic+production+of+glass+sample+holders+for+improved+sensitivity+and+resolution+in+ESR+microscopy%5BTitle%5D)+AND+%22Applied+Magnetic+Resonance%22%5BJournal%5D)">Photolithographic production of glass sample holders for improved sensitivity and resolution in ESR microscopy.</a> Applied Magnetic Resonance. 31, 591-598 (2007).</li>
<li>Allen, M. M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+and+Division+of+Some+Unicellular+Blue-Green+Algae%5BTitle%5D)+AND+%22J+Gen+Microbiol%22%5BJournal%5D)">Growth and Division of Some Unicellular Blue-Green Algae.</a> J Gen Microbiol. 51, 199-199 (1968).</li>
<li>Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generic+Assignments%2C+Strain+Histories+and+Properties+of+Pure+Cultures+of+Cyanobacteria%5BTitle%5D)+AND+%22J+Gen+Microbiol%22%5BJournal%5D)">Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria.</a> J Gen Microbiol. 111, 1-61 (1979).</li>
<li>Blank, A., Talmon, Y., Shklyar, M., Shtirberg, L., Harneit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+measurement+of+diffusion+in+liquid+phase+by+electron+spin+resonance%5BTitle%5D)+AND+%22Chem+Phys+Lett%22%5BJournal%5D)">Direct measurement of diffusion in liquid phase by electron spin resonance.</a> Chem Phys Lett. 465, 147-152 (2008).</li>
</ol>

No conflicts of interest declared.

This protocol shows how ESR micro-imaging can be applied to map oxygen concentration near live small 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 be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve. In the near future we plan on further improving this methodology to provide live sample images with a resolution of a few microns, providing contrast parameters such as super oxide concentration, acidity (pH), probe diffusion coefficient and, of course, oxygen concentration. These capabilities are complementary to the current optical-based methodologies both in terms of contrast type and also of samples characteristics (e.g non-transparent thick samples and, in some cases, intracellular vs. extracellular measurements).

<table><tbody><tr><td>Centrifuge </td><td>Kendro</td><td>Heraus, 75003235</td><td></td></tr><tr><td>Perdeuterated triarylmethyl (trityl) radical</td><td>Novosibirsk</td><td></td><td>Synthesized at Novosibirsk using the method described in reference &lt;sup&gt;6&lt;/sup&gt;.</td></tr><tr><td>BG-11 buffer</td><td></td><td></td><td>For instruction preparation, see Scheme 1 and references 8, 9.</td></tr><tr><td>Syringe</td><td>Hamilton Co</td><td>Microliter 7000.5</td><td></td></tr><tr><td>Ultraviolet Curing</td><td>Norland Products, Inc.</td><td>NOA63, or NOA61.</td><td></td></tr></tbody></table>

electron spin resonance micro-imaging of live species for oxygen mapping

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 ...

The overall goal of the following experiment is to acquire a micron scale, three dimensional images of the oxygen concentration in the immediate environment of live cells by electron spin resonance or ESR microscopy. The protocol will be demonstrated on cyanobacteria, which are common photosynthetic cells. This is achieved by first preparing a sample of cyanobacteria cells inside the ESR microscopy sample holder.Embedded in stable organic radical solution. The sample holder is placed inside the imaging probe, which goes into the static electromagnet of the ESR microscope system. Now, several three dimensional ESR micro images of the organic radical solution are acquired by the ESR microscope.This is carried out for several variants of pulse sequences and often under various external conditions such as under light and dark conditions. Next, the raw data images are processed by the computer in order to extract the three-dimensional spatially resolved relaxation times of the radical. These relaxation times provide a direct measure to the oxygen concentration through pre-existing calibration leading to full three-dimensional oxygen maps of the measured samples under various external conditions.Results are obtained that clearly show the lack of oxygen production by the cyanobacteria when no light is applied. Furthermore, it is shown that under light irradiation, significant oxygen gradient and oxygen heterogeneity are created near the cyanobacteria cells. Hi, I am al from the laboratory of BLA in the S Faculty of Chemistry at the Technion, the Israel Institute of Technology in Haifa.Today we will show you a procedure for mapping oxygen concentrations near life cells in three dimensional manner at high special resolution by the means of fierce arm microscopy. We use this procedure in our laboratory to study a oxygen consumption rate and metabolic activity of cells. So let's get started.Electron spin resonance or ESR is a spectroscopic technique in which electromagnetic radiation at a specific frequency is absorbed by paramagnetic species having unpaired electron spin placed under an external static magnetic field. It is a powerful method for studying the environment of paramagnetic molecules in live species, including the concentrations of oxygen and reactive oxygen species. Here a typical oxygen concentration image of a tumor bearing mouse is shown for heterogeneous samples.The ESR spectral information can be obtained in a spatially resolved manner through the use of magnetic field gradients. A stable, free, organic radical was injected into the mouse and its ESR characteristics provide the oxygen concentration at its environment. The ESR based results are superimposed on an MRI anatomical image.Up until now. Such ESR imaging techniques of live specimens were only carried out with relatively large samples at millimeter scale. Resolution ESR microscopy involves the measurements of millimeter and sub millimeter size samples, providing three dimensional ESR images with a resolution approaching one micron.Here are two examples of high resolution micron scale ESR, images of generated sample with NNC 60 powder and lithium fellow cyanide paramagnetic crystals. An ESR microscope is similar to a conventional ESR spectrometer, having a magnet for generating the static field, a microwave system for spin excitation and signal detection, a probe for holding the sample and a computerized console to control the acquisition process and data handling. Other components of the system include magnetic field gradient drivers, which are part of the electronic system and gradient coils that are located in the imaging probe.The gradient coils also have an aperture to allow for light irradiation of the sample. A controlled temperature moist air is applied to the sample to ensure minimal dehydration during the experiment. The main unique features of ESR microscopy are the small size of the probe and the gradient coils, the higher spin sensitivity of the system and the high strength of the magnetic field gradient drivers Prior to the start of this procedure, prepare a cyanobacteria suspension at a concentration of 40 milligrams per milliliter.To begin ESR micro imaging sample preparation, take a few squares of absorbent paper at a size of approximately 400 by 400 microns using fine tweezers. Then insert them into an einor tube. Subsequently filled the tube with 1.2 milliliters of the cyanobacteria suspension.Then centrifuge the suspension for two minutes at 6, 000 RPM in a micro centrifuge following centrifugation, completely remove the snat except for approximately 50 microliters, which is left to avoid cyanobacteria dehydration. This process results in the saturation of the absorbent paper with the cyanobacteria cells. Next, prepare a specially prepared cup like glass sample holder, extract a few fibers from the paper using fine tweezers, and place them on the bottom of the sample holder.Following that, add three millimolar of tridel and BG 11 solution, which is prepared as described in the written protocol by the eight of a syringe. Then seal the sample holder with UV curable glue by applying the glue between the edges of the glass sample holder and its matching cover glass piece, leaving a small air outlet open. Finally, cure the glue with UV light for two seconds.To begin the imaging experiment, turn on the ESR micro imaging system and insert the sample into the resonator that goes inside the imaging probe. Now using the computer control software, set the system on tune mode and find the resonance microwave frequency of the probe, which will be used for the ESR measurements. Following that, set the static magnetic field on the value that matches the applied microwave frequency.Set the timing parameters for the pulse sequence and observe the ESR signal to make sure that the system functions well and the sample is well prepared. Then set the imaging parameters such as the number of pixels, the strength of the gradients, and the length of the gradient pulses to their required values. Following setup, collect three 3D ESR images by a Han Echo Imaging pulse sequence with interpulse separation tau values of 500 600 and 700 nanoseconds.At this point, turn the light projection at the sample on if required for the experimental conditions throughout the acquisition, the data is automatically saved. Following data acquisition process, the raw data files via MATLAB software script. Using the software generate images of the trill radical concentration and the relaxation time.T two map then translate these images to an oxygen concentration image via a preexisting calibration curve that links the oxygen concentration to the relaxation time. Here T two zero is the spin, spin, relaxation time of the probe under anoxic conditions, depending on the probes concentration C and its diffusion coefficient D.Typical raw data images from several three dimensional ESR imaging experiments performed on cyanobacteria are shown. These images were acquired with different tau values under either dark or light conditions.Under dark conditions, the results are very similar except for decreased intensity for the larger T values due to spin, spin relaxation time T two. Conversely, under light irradiation, the image pattern changes due to different relaxation times throughout the sample. This data is then processed to obtain an amplitude image from this image.It is evident that the cyanobacteria is located mainly on the right side of the sample holder. T two images are translated to oxygen concentration values via a pre-existing calibration curve that links the oxygen concentration to the relaxation time. The results are shown for cyanobacteria in the presence and absence of light.It is clear that oxygen production is initiated with light and causes significant increase in the solution oxygen concentration. This leads to a decrease in the radical T two, mainly in the voxels near the absorbent paper fibers with the cyanobacteria, We have just shown you how to prepare sample containing life cells with stable free radical for mapping oxygen concentration by E ES R microscopy. When doing this procedure, it is important to remember to keep it as short as possible to assure S variability and to avoid sample de duration.So that's it. Thank you for watching us, and good luck with your experiment.

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