Name: in situ monitoring of diffusion of guest molecules in porous media using electron paramagnetic resonance imaging
Uploaded: 2016-09-02T14:00:00.000+00:00
Duration: 6 min 34 s
Description: Watch this Scientific Journal Video about In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging at JoVE.com

The authors thank Prof. Peter Imming and Diana M&#252;ller for synthesis of the Trityl spin probe and Prof. Sebastian Polarz, Martin Wessig and Andreas Schachtschneider for the synthesis of the porous materials. Financial support by the DFG (DR 743/7-1) and within the SPP 1570 is gratefully acknowledged.

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Ethanol is harmful if swallowed or inhaled and it is flammable.
1. Optimize the Continuous Wave (CW) EPR Parameters
<ol>
	<li>Prepare 40 &#181;l of IPSL in ethanol (p.a.) at a concentration of 1 mM.</li>
	<li>Take a pipette controller and fill a capillary with the IPSL solution to a filling height of 2 cm. Pull the solution 1 cm further into the capillary so that there is an air gap below the solution. Seal the capillary on both ends with capillary tube sealing compound. The air gap prevents diffusion of components of the sealing compound into the sample.</li>
	<li>Wrap two strips of polytetrafluoroethylene (PTFE) tape of about 5 cm length around the capillary at a distance of 1 cm from the upper and lower end of the capillary.</li>
	<li>Put the capillary into an EPR sample tube (4 mm inner diameter). Make sure that the PTFE tape keeps the capillary fixed in the center axis of the sample tube. Push the capillary down to the bottom of the sample tube.</li>
	<li>Put the sample into the resonator and center the spin label solution within the resonator.</li>
	<li>Tune the spectrometer for critical coupling by following the instructions in the spectrometer manual.</li>
	<li>Preliminary Spectrometer Settings
	<ol>
		<li>Use the microwave frequency to set the center field B using the formula 
		<img alt="Equation 1" src="/files/ftp_upload/54335/54335eq1.jpg" /> 
		&#8203;where g&#160;&#8776; 2.003&#160;is a rough estimate for the g factor of the unpaired radical in the nitroxide spin label, h is the Planck constant and &#956;B is the Bohr magneton.</li>
		<li>Set up a new experiment &#34;field_sweep&#34; with the magnetic field as abscissa and the signal intensity as ordinate. Use the following parameters: Centerfield as calculated in the previous step, sweep width: 400 G, modulation amplitude: 0.8 G, modulation frequency: 100 kHz, microwave attenuation: 30 dB, number of points: 2,048, number of scans: 1, scan time: 80 sec, time constant: 50 msec.</li>
		<li>Activate the Setup Scan mode. For the Setup Scan Time Constant, select the lowest value that the spectrometer offers. Adjust the receiver gain to a value where the displayed signal fills 80% of the displayed intensity range, so that even with noise no data point has a higher intensity than 80% of the maximum. Disable Setup Scan afterwards.</li>
		<li>Press the &#34;Run&#34; button.</li>
		<li>Read the field value of the zero crossing of the central peak from the spectrum obtained. Set the center field to that value.</li>
		<li>Take the horizontal line tool and measure the spectrum width from the point where the leftmost peak starts to rise above the baseline level to the point where the rightmost peak returns to the baseline level.</li>
		<li>Set the sweep width to three times the spectrum width.</li>
	</ol>
	</li>
	<li>Recalculate the Spectrometer Parameters
	<ol>
		<li>Calculate the sweep time: sweep width/sweep speed. Use a sweep speed of 5 G/sec.</li>
		<li>Calculate the minimum number of data points: 10 * sweep width/line width.</li>
		<li>Calculate the conversion time: sweep time/number of data points.</li>
		<li>Calculate the time constant: 0.1 * line width * scan time/sweep width.</li>
	</ol>
	</li>
	<li>Measure a Saturation Curve to Determine the Optimum Microwave Power
	<ol>
		<li>Set the microwave attenuation to 10 dB and adjust the receiver gain as described in step 1.7.3.</li>
		<li>Set the microwave attenuation to 50 dB and record a spectrum. If the signal to noise ratio is less than 5:1, increase the number of scans. Repeat this step until the signal to noise ratio is 5:1 or greater.</li>
		<li>Create a new experiment &#34;saturation&#34; using the magnetic field as abscissa 1, the microwave power as abscissa 2 and the signal intensity as ordinate. Copy all settings from the &#34;field_sweep&#34; experiment from step 1.9.2. For abscissa 2, set the start value of the microwave attenuation to 10 dB, the increment value to 1 dB and the number of points to 41 to cover a range from 10 dB to 50 dB. Run the experiment.</li>
		<li>Create a spreadsheet for the saturation curve. Insert the microwave attenuation in dB into the first column.</li>
		<li>Calculate the square root of the microwave power in a.u. in the second column with the formula 
		<img alt="Equation 2" src="/files/ftp_upload/54335/54335eq2.jpg" /> 
		&#8203;where x is the microwave attenuation in dB from the first column.</li>
		<li>Use the spectrometer software to measure the peak to peak intensity of the central spectral line for each microwave attenuation in the experiment. Write that intensity into the third column in the spreadsheet.</li>
		<li>Plot the square root of the microwave power against the peak to peak intensity (column 3 against column 2) to get the saturation curve. Include the origin (0,0) in the plot.</li>
		<li>Identify the linear regime of the saturation curve. The optimal microwave power is the highest microwave power that is still in the linear regime. Use the corresponding attenuation setting for all further experiments.</li>
	</ol>
	</li>
</ol>
2. Determine the Magnetic Field Gradient Strength and the Time Resolution
<ol>
	<li>Create a new experiment in the spectrometer software with the magnetic field as abscissa 1 and the signal intensity as ordinate. Enable the gradient coil controls.</li>
	<li>Copy all spectrometer settings from the previous experiment as determined in 1.8 and 1.9.8.</li>
	<li>Set the magnetic field gradient strength to 170 G/cm in the direction of the sample axis pointing upwards.</li>
	<li>Calculate the sweep width sw = sw0 + FOV&#183;G, where&#160;sw0 is the sweep width determined in 1.8.4 in the absence of a magnetic field gradient, FOV is the field of view (2.5 cm) and G&#160;is the magnetic field gradient strength.</li>
	<li>Calculate the estimated pixel size = line width / G, using the line width of the spectrum recorded in 1.9.3 in the absence of a magnetic field gradient.</li>
	<li>Calculate the sweep time = sw / sweep speed. Use the same sweep speed as in 1.8.1.</li>
	<li>Calculate the minimum number of data points required using the higher value of 
	i. N1= 10 * sweep width / line width 
	ii. N2&#160;= 10 * field of view / (G * pixel size).</li>
	<li>Calculate the conversion time: sweep time/number of data points.</li>
	<li>Calculate the time constant: 0.1 * line width * scan time / sweep width or lower.</li>
	<li>Set the parameters calculated in 2.3 through 2.9 and press the &#34;Run&#34; button.</li>
	<li>Measure the noise level of the baseline as well as the peak to peak intensity of the central line with the vertical line tool. Calculate the signal to noise ratio.</li>
	<li>If the signal to noise ratio is less than 5:1, double the number of scans in the &#34;scan&#34; panel of the spectrometer parameters and repeat steps 2.1.3 through 2.11.</li>
</ol>
3. Prepare the Sample
Caution: Wear safety glasses.
Note: Keep the aerogel completely submerged in solvent at all times. See Figure 2 for a photograph and schematic.
<ol>
	<li>Fill a Petri dish of 10 cm diameter with ethanol (p.a.) up to a height of 5 mm.</li>
	<li>Put the aerogel into the Petri dish and cut off a cylindrical piece of 5 mm to 1 cm in length.</li>
	<li>Prepare a piece of heat shrink tubing that is about 1 cm longer than the aerogel cylinder.</li>
	<li>Use a glass tubing cutter to break a sample tube of 2 mm inner diameter to create two pieces of 4 cm length. Both pieces should have two open ends. 
	Caution: Wear safety glasses and appropriate gloves when operating the heat gun. Do not direct the airflow towards skin or clothing and keep it away from flammable materials.</li>
	<li>Insert one of the sample tube pieces 5 mm deep into one end of the heat shrink tubing. Use a heat gun to carefully heat this end of the heat shrink tubing without shrinking the rest of the tubing. The heat shrink tubing should now be fixed at the glass tube.
	<ol>
		<li>Submerge this combination of glass tube and heat shrink tubing in the Petri dish of the aerogel. Carefully push the piece of aerogel from step 3.2 into the open end of the heat shrink tubing.</li>
	</ol>
	</li>
	<li>Fill a test tube with ethanol (p.a.) up to a height of 7 cm. Transfer the sample from the Petri dish into the test tube. While doing so, make sure that the open end of the heat shrink tubing is oriented to the top. Make sure that the aerogel is completely submerged in ethanol.</li>
	<li>Insert the second 4 cm long piece of sample tube from step 3.4 into the open end of the heat shrink tubing. Do not apply force, gravity should be enough to close the gaps between the aerogel and the sample tube pieces. Put the test tube with the sample into a beaker.</li>
	<li>Fill a pressure cooker with at least 500 ml ethanol and add a stir bar.</li>
	<li>Put the beaker containing the sample on a trivet inside the pressure cooker. 
	Caution: Perform the next step under a fume hood and continue to wear safety glasses.</li>
	<li>Cook and stir the samples at a pressure setting of 1 bar above ambient pressure on the magnetic stirrer. The temperature must reach at least 90 &#176;C. Let it cool down as soon as the pressure is reached and the pressure valve releases ethanol vapor. If the heat shrink tubing did not shrink, repeat this step. 
	Note: Immediately clean the pressure cooker with water to minimize the effect of ethanol on the seal valves. At this point, the prepared sample can be stored in ethanol for several months.</li>
</ol>
4. Prepare the Spectrometer
<ol>
	<li>Create a 2d experiment using the magnetic field as abscissa 1, time as abscissa 2 and signal intensity as ordinate, so that a magnetic field sweep is recorded for each time step. Enable the gradient coil controls.</li>
	<li>Set the time delay between measurements to zero. Set the other parameters as determined in section 2. Set the number of points for the time axis to 20 hr/sweep time. Set the microwave bridge to perform a fine tune after each slice scan.</li>
	<li>Follow the steps in section 1.7 to tune the spectrometer to the empty resonator.</li>
</ol>
5. Prepare the Sample for Measurement
Note: The only time critical steps of this protocol are 5.3 through 6.2, which is from the start of the diffusion process with the addition of the spin label until the time the data acquisition in the spectrometer starts. Perform these steps without introducing any delays.
<ol>
	<li>Put a finger on top of the sample from section 3 to keep the ethanol solution from flowing out on the bottom. Then use a syringe to remove some ethanol from the bottom 5 mm of the lower sample tube and seal that end with tube sealing compound. Make sure that there is an air bubble of 2 mm in height above the sealing compound.</li>
	<li>Remove all ethanol from the sample tube above the aerogel except for 3 mm just above the aerogel using a Pasteur capillary pipette.</li>
	<li>Inject 20 &#181;l of spin label solution in ethanol on top of the aerogel. Make sure to not create an air bubble on top of the aerogel. Mark the current time as start of the diffusion process.</li>
	<li>Place the sample in a sample tube with 4 mm inner diameter. Use PTFE tape to center the sample.</li>
	<li>Use a felt-tip pen to mark the outer sample tube at a position of 68 mm above the upper edge of the aerogel. This helps in correctly centering the sample in the resonator and puts the center of the resonator 1 mm below the upper edge of the aerogel.</li>
</ol>
6. Perform the Diffusion Experiment
<ol>
	<li>Place the sample in the resonator so that the marking from 5.5 aligns with the top of the PTFE holder of the resonator and tune the spectrometer for critical coupling as is described in the operating manual of the spectrometer.</li>
	<li>Use the Setup Scan mode to set the receiver gain as described in 1.7.3 while the gradient coils are still turned off.</li>
	<li>Start the experiment that has been set up in section 4. Write down the current time. Either wait 20 hr for the experiment to finish or stop the experiment when the recorded signal does not change over the course of 4 hr or more. Save the result.</li>
</ol>
7. Perform Additional Experiments Needed for Data Analysis
Note: Conduct the experiments in 7.1 and 7.2 with the same sample directly after the diffusion experiment and without moving the sample.
<ol>
	<li>Record the Point Spread Function for Deconvolution
	<ol>
		<li>Switch to the &#8220;field sweep&#8221; experiment from step 1.7.2. Copy all settings from the experiment in step 6.</li>
		<li>Record a spectrum and measure the signal to noise ratio. If it is less than 20:1, increase the number of scans and repeat this step. Otherwise save the spectrum.</li>
	</ol>
	</li>
	<li>Perform a 2d Imaging Experiment
	<ol>
		<li>Create a new experiment on the spectrometer with the magnetic field as abscissa 1, the angle of the magnetic field gradient as abscissa 2 and the signal intensity as ordinate. Copy the parameters from step 6. Set the imaging plane to the YZ plane, which is the plane including the direction of the static magnetic field B0&#160;and the sample axis.</li>
		<li>Set the number of angles N of the gradient direction to N = FOV/desired pixel size or higher.</li>
		<li>Start the measurement and save the result.</li>
		<li>Repeat the steps in 7.1 and save the result.</li>
	</ol>
	</li>
	<li>Measure the Resonator Sensitivity Profile
	<ol>
		<li>Prepare another sample of spin probe in solution by repeating steps 1.1 through 1.5, but this time add 4 cm of the solution into the capillary instead of 2 cm.</li>
		<li>Follow the steps in section 2 to record a spectrum of the sample with a magnetic field gradient in the direction of the sample axis. For step 2.3, use a field of view of 3 cm. Save the result.</li>
		<li>Repeat the measurement in the absence of the magnetic field gradient and save the result.</li>
	</ol>
	</li>
</ol>
8. Data Analysis
<ol>
	<li>Reconstruct the 2d Imaging Experiment
	<ol>
		<li>Load the 2d imaging experiment from 7.2.3 into the primary viewport of the spectrometer software.</li>
		<li>Load the experiment from 7.2.4 into the secondary viewport of the spectrometer software.</li>
		<li>Go to Processing&#62;Transformations&#62;Deconvolution, select Slice: all and click apply to perform a deconvolution.</li>
		<li>Save the deconvolved data to disk.</li>
		<li>Use the freely available image reconstruction software20 with the following command: reconstruct --input result_from_8_1_4.DSC --output reconstructed_image.DSC --steps 100 --size 256</li>
		<li>Load the result from 8.1.5 into the spectrometer software for later reference.</li>
	</ol>
	</li>
	<li>Analyze the Recorded Diffusion Experiment
	<ol>
		<li>Start the data analysis software and go to the &#34;Load&#34; tab of the software shown in Figure 3. Load the diffusion experiment from step 6.3 under &#34;diffusion experiment&#34;. Load the corresponding point spread function from step 7.1.2 under &#34;diffusion experiment w/o gradient&#34;. Load the result from step 7.3.2 under &#34;resonator profile experiment&#34; and the result from step 7.3.3 under &#34;resonator profile exp w/o gradient&#34;.</li>
		<li>Go to the resonator sensitivity tab shown in Figure 4 to perform a deconvolution of the data from experiment 7.3.2 using 7.3.3 as point spread function. Decrease the noise power value until the result is noisy, then raise it until the noise just disappears.</li>
		<li>Go to the 1d spin density profile tab shown in Figure 5 in order to deconvolve each field sweep recorded in 6.2 using the experiment from 7.1 as point spread function. Decrease the noise power value until the result is noisy, then raise it until the noise just disappears.</li>
		<li>Switch to the crop area tab shown in Figure 6. Select an area of the diffusion heat map that lies completely inside of the aerogel and where spin probe is just about to enter from above at the first time step of that area. If in doubt, load the reconstructed image from 8.1.6 in the spectrometer software to help identify the exact position of the aerogel.</li>
		<li>Increase the area from step 8.2 in the downward direction of the sample so that no spin probe reaches the lower boundary of the area within the time of the experiment. See Figure 6 for reference.</li>
		<li>Switch to the influx tab shown in Figure 7 and press fit. The left hand panel shows the integral of the cropped region from 8.2.5 along the position axis.</li>
		<li>Verify that the curve shown in the middle panel starts at zero and immediately starts to rise. If that is not the case, go back to 8.2.5.</li>
		<li>Verify that the red line shown in the middle panel follows the black data points.</li>
	</ol>
	</li>
	<li>Simulate the 1d Spin Concentration Over Time and Fit the Diffusion Coefficient
	<ol>
		<li>Switch to the diffusion coefficient tab and press fit.</li>
		<li>Wait for the results of the calculation.</li>
		<li>Verify that the experimental data shown on the left matches the numerical data shown on the right.</li>
		<li>Read the value of the macroscopic translational diffusion coefficient Dmacro that is displayed on the screen.</li>
	</ol>
	</li>
</ol>

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C. 119 (30), 17474-17479 (2015).</li><li>Kruczala, K., Schlick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Diffusion+Coefficients+of+Nitroxide+Radicals+in+Heterophasic+Propylene&#8722;Ethylene+Copolymers+by+Electron+Spin+Resonance+Imaging.">Measuring Diffusion Coefficients of Nitroxide Radicals in Heterophasic Propylene&#8722;Ethylene Copolymers by Electron Spin Resonance Imaging.</a> Macromolecules. 44 (2), 325-333 (2011).</li><li>Wessig, M., Drescher, M., Polarz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Functional+Group+Specific+Surface+Interactions+in+Porous+Solids+Using+ESR+Spectroscopy+as+a+Sensitive+and+Quantitative+Tool.">Probing Functional Group Specific Surface Interactions in Porous Solids Using ESR Spectroscopy as a Sensitive and Quantitative Tool.</a> The J. Phys. Chem. C. 117 (6), 2805-2816 (2013).</li><li>Kuschel, A., Polarz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organosilica+Materials+with+Bridging+Phenyl+Derivatives+Incorporated+into+the+Surfaces+of+Mesoporous+Solids.">Organosilica Materials with Bridging Phenyl Derivatives Incorporated into the Surfaces of Mesoporous Solids.</a> Adv. Funct. Mater. 18 (8), 1272-1280 (2008).</li><li>Spitzbarth, M., Drescher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+iterative+reconstruction+technique+software+for+spectral-spatial+EPR+imaging.">Simultaneous iterative reconstruction technique software for spectral-spatial EPR imaging.</a> J. Magn. Reson. 257, 79-88 (2015).</li><li>Stoll, S., Schweiger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EasySpin,+a+comprehensive+software+package+for+spectral+simulation+and+analysis+in+EPR.">EasySpin, a comprehensive software package for spectral simulation and analysis in EPR.</a> J. Magn. Reson. 178 (1), 42-55 (2006).</li></ol>

The authors have nothing to disclose.

The protocol allows monitoring of the diffusion of paramagnetic guest molecules. A 1d imaging approach has been chosen because it allows for a higher time resolution compared to 2d or 3d imaging. The 1d approach requires a constant cross sectional area of the sample because the intensity of the obtained 1d image depends not only on the concentration but also on the cross sectional area of the sample. The method also requires that the EPR spectra of the spin probes within the samples only change in intensity but not in sh...

Porous materials play a major role in practical applications such as catalysis and chromatography1. By adding surface groups and adjusting the pore size and surface properties, the materials can be tailored to the desired application2,3. The functionality of the porous material crucially depends on the diffusion properties of the guest molecules inside the pores. In porous materials, a distinction must be made between the microscopic translational diffusion constant D&#8203;micro, which describes diffusion on a molecular length scale on one hand and the macroscopic translational diffusion constant Dmacro on the other hand, which is influenced by the diffusion through multiple pores, grain boundaries, tortuosity and inhomogeneity of the material.
There are several magnetic resonance methods available to study diffusion, each suitable for a particular length scale. On the millimeter scale, nuclear magnetic resonance (NMR) imaging4 and electron paramagnetic resonance (EPR) imaging (as presented in this protocol) can be used. Smaller scales become accessible by the use of pulsed field gradients in NMR as well as EPR experiments5,6. On the nanometer scale, EPR spectroscopy can be used by observing changes of the Heisenberg exchange interaction between spin probes7,8. Studies of translational diffusion using EPR imaging range from industrial catalyst supports, e.g., aluminum oxide9, to anisotropic fluids10,11, drug release systems made of polymer gels12-14 and model membranes15.
This protocol presents an in situ approach using EPR imaging to monitor macroscopic translational diffusion of spin probes in cylindrical, porous media. It is demonstrated for a host-guest system consisting of the nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent. This protocol has successfully been used previously16 to compare D&#8203;macro as determined with EPR imaging with D&#8203;micro for the host materials UKON1-GEL and SILICA-GEL and guest species IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl), see Figure 1.
In other methods based on continuous wave (CW) EPR imaging17, diffusion takes place outside of the spectrometer. In contrast, the method presented here uses an in situ approach. A series of snapshots of the 1d spin density distribution &#961;&#8203;1d (t, &#947;) is recorded over a period of several hours. During this time, one snapshot is taken after the other and delivers a real-time diffusion pattern with a time resolution of approximately 5 min.
UKON1-GEL and SILICA-GEL have been synthesized in sample tubes with an inner diameter of 3 mm as described in literature.16,18,19 The UKON1-GEL and SILICA-GEL synthesis leads to a shrinking of the sample. The samples are placed inside a heat-shrink tube to prevent guest molecules from moving between the aerogel and the wall of the sample tube. This additional step is not necessary for samples that can be synthesized directly in the sample tube without changing their size. The aerogel samples collapse when they dry out, so they must be submerged in solvent at all times. The temperature that is needed for the heat shrink tubing is higher than the boiling point of ethanol at ambient pressure. Therefore the protocol describes the use of a pressure cooker to raise the boiling point of ethanol.
The protocol covers the sample preparation of UKON1-GEL synthesized beforehand for the EPR imaging experiment and the spectrometer settings that are used to monitor diffusion of IPSL spin probe. For data analysis, locally written software is provided and its use is described. The raw data from the spectrometer can be directly loaded. The software calculates the spatial 1d spin density distribution &#961;&#8203;1d (t, &#947;) and takes into account the resonator sensitivity profile. The user can select a region of the aerogel and a time window, over which the diffusion constant is to be determined. The software then determines the boundary conditions of the diffusion equation based on that selection and solves the diffusion equation. It supports least square fitting to find the value of Dmacro where the numerical solution best matches the experimental data.
The protocol can be used with adjustments for different guest and host materials as long as the cross sectional area of the sample does not change throughout the sample, that is &#961;&#8203;1d (t, &#947;) gives direct access to the concentration and is not influenced by a change in sample cross section. The range of accessible values for Dmacro is estimated16 between 10-12 m2/sec and 7&#183;10-9 m2/sec.

<table><tbody><tr><td>X-Band spectrometer</td><td>Bruker</td><td>E580</td><td></td></tr><tr><td>Spectrometer software</td><td>Bruker</td><td>Xepr 2.6b.108</td><td></td></tr><tr><td>gradient coil system</td><td>Bruker</td><td>E540 GCX2</td><td></td></tr><tr><td>imaging resonator</td><td>Bruker</td><td>TMHS 1007</td><td></td></tr><tr><td>micro-classic pipette controller</td><td>Brand</td><td>25900</td><td></td></tr><tr><td>microcapillary ringcaps 50&amp;nbsp;&amp;micro;l</td><td>Hirschmann</td><td>9600150</td><td>inner diameter 0.5 mm</td></tr><tr><td>EPR sample tube 2 mm inner diameter</td><td>Bruker</td><td>ER 221TUB/2</td><td></td></tr><tr><td>EPR sample tube 4 mm inner diameter</td><td>Bruker</td><td>ER 221TUB/4</td><td></td></tr><tr><td>heat-shrink tubing DERAY-IB</td><td>DSG-Canusa</td><td>2210048952</td><td>4.8 mm/2.4 mm, 2:1, 95&amp;nbsp;&amp;deg;C - 200&amp;nbsp;&amp;deg;C</td></tr><tr><td>heat gun</td><td>Bosch</td><td>PHG 600-3</td><td></td></tr><tr><td>PTFE&amp;nbsp; band</td><td>VWR</td><td>332362S</td><td>width 12 mm</td></tr><tr><td>test tube</td><td></td><td></td><td>length 16 cm, diameter 1.5 cm</td></tr><tr><td>beaker</td><td></td><td></td><td>250 ml, height 9 cm, diameter 7 cm</td></tr><tr><td>capillary tube sealing</td><td>Fisher Scientific</td><td>02-678</td><td></td></tr><tr><td>pressure cooker, 3 L with trivet</td><td>Beem</td><td>Vital-X-Press V2, F1000675</td><td></td></tr><tr><td>magnetic stirrer with heating element</td><td></td><td></td><td></td></tr><tr><td>ethanol (p.a.)</td><td></td><td></td><td></td></tr><tr><td>ethanol (techn.)</td><td></td><td></td><td></td></tr><tr><td>syringe</td><td>Hamilton</td><td>1705</td><td>0.05 ml, custom length: 20 cm</td></tr><tr><td>Pasteur capillary pipette</td><td></td><td></td><td>length 23 cm</td></tr><tr><td>data analysis software</td><td>homemade</td><td></td><td>Available for download at http://www.uni-konstanz.de/drescher/software. Requires Matlab.</td></tr><tr><td>UKON1-GEL</td><td>kindly provided by Prof. Sebastian Polarz, Martin Wessig and Andreas Schachtschneider&amp;nbsp;</td><td></td><td>See references 16, 18, 19 for the synthesis</td></tr></tbody></table>

in situ monitoring of diffusion of guest molecules in porous media using electron paramagnetic resonance imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. 
A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis.
The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

A photo and schematic of an aerogel within the shrinking tube is shown in Figures 2a and 2b. The 2d EPR image in Figure 2c clearly shows the upper edge of the aerogel. The intensity of &#961;1d within the sample tube above the aerogel is lower although the concentration of the spin probe is at least as high as within the aerogel. However, the sample depth perpendicular to the picture plane is much smaller due to the sm...

Watch this Scientific Journal Video about In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging at JoVE.com

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with ...

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Research

JoVE Journal

Chemistry

6.3K Views.  Universität Konstanz. A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

Video: In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Bioengineering

Exploring the Radical Nature of a Carbon Surface by Electron Paramagnetic Resonance and a Calibrated Gas Flow

While the first Electron Paramagnetic Resonance (EPR) studies regarding the effects of oxidation on the structure and stability of carbon radicals date back to the early 1980s the focus of these early papers primarily characterized the changes to the structures under extremely harsh conditions (pH or temperature)1-3. It is also known that paramagnetic molecular oxygen undergoes a Heisenberg spin exchange interaction with stable radicals that extremely broadens the EPR signal4-6. Recently, we reported interesting results where this interaction of molecular oxygen with a certain part of the existing stable radical structure can be reversibly affected simply by flowing a diamagnetic gas through the carbon samples at STP7. As flows of He, CO2, and N2 had a similar effect these interactions occur at the surface area of the macropore system.
This manuscript highlights the experimental techniques, work-up, and analysis towards affecting the existing stable radical nature in the carbon structures. It is hoped that it will help towards further development and understanding of these interactions in the community at large.

Stable radicals that are present in carbon substrates interact with paramagnetic oxygen through a Heisenberg spin exchange. This interaction can be significantly reduced under STP conditions by flowing a diamagnetic gas over the carbon system. This manuscript describes a simple method to characterize the nature of those radicals.

While the first Electron Paramagnetic Resonance (EPR) studies regarding the effects of oxidation on the structure and stability of carbon radicals date ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Cancer Research

Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due to fundamental constraints imposed by the hyperpolarized state, exotic imaging and reconstruction techniques are commonly used. A practical system for characterization of dynamic, multi-spectral imaging methods is critically needed. Such a system must reproducibly recapitulate the relevant chemical dynamics of normal and pathological tissues. The most widely utilized substrate to date is hyperpolarized [1-13C]-pyruvate for assessment of cancer metabolism. We describe an enzyme-based phantom system that mediates the conversion of pyruvate to lactate. The reaction is initiated by injection of the hyperpolarized agent into multiple chambers within the phantom, each of which contains varying concentrations of reagents that control the reaction rate. Multiple compartments are necessary to ensure that imaging sequences faithfully capture the spatial and metabolic heterogeneity of tissue. This system will aid the development and validation of advanced imaging strategies by providing chemical dynamics that are not available from conventional phantoms, as well as control and reproducibility that is not possible in vivo.

A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents.

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due ...

Engineering

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids with adsorbates. Vibrational spectra provide information on the chemical nature of adsorbed species and their structure. Thus, they are very useful for obtaining molecular level understanding of surface species. The IR spectrum of the sample itself gives some direct information about the material. General conclusions can be drawn concerning hydroxyl groups, some stable surface species and impurities. However, the spectrum of the sample is &#34;blind&#34; with respect to the presence of coordinatively unsaturated ions and gives rather poor information about the acidity of surface hydroxyls, species decisive for the adsorption and catalytic properties of the materials. Furthermore, no discrimination between bulk and surface species can be made. These problems are solved by the use of probe molecules, substances that interact specifically with the surface; the alteration of some spectral features of these molecules as a result of adsorption provides valuable information about the nature, properties, location, concentration, etc., of the surface sites.
The experimental protocol for in-situ IR studies of gas/sample interaction includes preparation of a sample pellet, activation of the material, initial spectral characterization through the analysis of the background spectra, characterization by probe molecules, and study of the interaction with a particular set of gas mixtures. In this paper we investigate a zirconium terephthalate metal organic framework, Zr6O4(OH)4(BDC)6 (BDC = benzene-1,4-dicarboxylate), namely UiO-66 (UiO refers to University of Oslo). The acid sites of the UiO-66 sample are determined by using CO and CD3CN as molecular probes. Furthermore, we have demonstrated that CO2 is adsorbed on basic sites exposed on dehydroxylated UiO-66. Introduction of water to the system produces hydroxyl groups acting as additional CO2 adsorption sites. As a result, CO2 adsorption capacity of the sample is strongly enhanced.

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

The use of FTIR spectroscopy for investigation of the surface properties of polycrystalline solids is described. Preparation of sample pellets, activation procedures, characterization with probe molecules and model studies of CO2 adsorption are discussed.

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at length scales down to the atomic level. In situ closed-cell gas reaction (CCGR) studies performed using (scanning) transmission electron microscopy (STEM) can separate and identify localized dynamic reactions, which are extremely challenging to capture using other characterization techniques. For these experiments, we used a CCGR holder that utilizes microelectromechanical systems (MEMS)-based heating microchips (hereafter referred to as &#34;E-chips&#34;). The experimental protocol described here details the method for performing in situ gas reactions in dry and wet gases in an aberration-corrected STEM. This method finds relevance in many different materials systems, such as catalysis and high-temperature oxidation of structural materials at atmospheric pressure and in the presence of various gases with or without water vapor. Here, several sample preparation methods are described for various material form factors. During the reaction, mass spectra obtained with a residual gas analyzer (RGA) system with and without water vapor further validates gas exposure conditions during reactions. Integrating an RGA with an in situ CCGR-STEM system can, therefore, provide critical insight to correlate gas composition with the dynamic surface evolution of materials during reactions. In situ/operando studies using this approach allow&#160;for detailed investigation of the fundamental reaction mechanisms and kinetics that occur at specific environmental conditions (time, temperature, gas, pressure), in real-time, and at high spatial resolution.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at ...

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 ...

Biology

Preclinical Imaging of Tumor Oxygen Levels Using EPRI and Ultrasound

Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 (i.e., hypoxia) is connected to tumor aggressiveness and poor response to therapy. The quantification of tumor pO2 allows the evaluation of treatment effectiveness. Electron Paramagnetic Resonance Imaging (EPRI), particularly Pulse EPRI, has emerged as an advanced three-dimensional (3D) method of assessing tissue oxygenation in vivo. This innovation was enabled by the technological developments in EPR (Electron Paramagnetic Resonance) and the application of the water-soluble oximetric spin probes from the triaryl family, offering fast and sensitive oxygenation data. The relaxation time of the spin probe (T1 and/or T2) provides accurate information about pO2 in selected voxels.
Human glioblastoma LN229 tumors were grown in the interscapular fad pad of BALB/c nude mice. Ultrasound (US) imaging was used as a reference for tumor anatomical information. To image tissue pO2, the animals were placed in a fixed position in the animal bed with fiducials, enabling registration between the imaging modalities. After the OX071 contrast agent was administered, EPRI was performed, followed by US B-mode. Because of the low spin probe toxicity, the procedure can be repeated during tumor growth or treatment. Following imaging, the registration process was carried out using software written in MATLAB. Ultimately, the hypoxic fraction can be calculated for a specific tumor, and the histogram of pO2 tissue distribution can be compared over time. EPRI combined with ultrasound is an excellent tool for oxygen mapping of tumors in the preclinical setting.

Quantitative 3D oxygen maps of murine tumors were imaged noninvasively using Pulse Electron Paramagnetic Resonance. Ultrasound B-mode and Power Doppler were used for anatomy and vascular structure. Images from both modalities were superimposed enabling multi-parametric tumor analysis.

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 ...

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

Summary

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Chapters in this video

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Exploring the Radical Nature of a Carbon Surface by Electron Paramagnetic Resonance and a Calibrated Gas Flow

In Vivo EPR Assessment of pH, pO₂, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO₂ Adsorption in UiO-66 Metal-Organic Framework

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

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

Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance