The authors would like to thank the Ontario Institute for Cancer Research, Imaging Translation Program and the Natural Sciences and Engineering Research Council of Canada for funding this research. We also like to acknowledge useful discussions with Albert Chen, GE Healthcare, Toronto, Canada, Gianni Ferrante, Stelar s.r.l., Italy, and William Mander, Oxford Instruments, UK.

1. Sample Preparation
NOTE: Steps 1.1-1.8 are performed just once
<ol>
	<li>Prepare 1 mL of stock 13C-enriched pyruvic acid solution, widely used for in vivo research1,2,5,6, consisting of 15-mmol/L of triarylmethyl radical dissolved in [1-13C]pyruvic acid (see Table of Materials). Aliquots from this stock solution will be used for the samples that will be individually polarized and subsequently undergo relaxometry at different magnetic fields. A representation of the [1-13C]pyruvic acid molecule is shown in Figure 1.</li>
	<li>On the dynamic nuclear polarizer software interface (see Table of Materials), click on the Cooldown button to lower the temperature of the variable temperature insert (VTI) to 1.4 K.</li>
	<li>Once the DNP has reached the desired temperature, load 10 &#181;L of the stock solution in a sample cup, open the turret doors and insert the cup into the VTI using an insertion wand specifically designed for this task.</li>
	<li>After that, quickly extract the wand and make sure the cup is released. Then close the turret doors and continue with the following steps while the temperature of the VTI goes back to 1.4 K.</li>
	<li>Prepare the DNP to run a microwave sweep in order to find the optimal RF frequency for hyperpolarization of the stock solution.
	<ol>
		<li>On the computer controlling the spectrometer (part of the DNP system), establish the communication between the spectrometer and the DNP control software by double-clicking on HyperTerminal icon, previously configured with the appropriate serial communication parameters.</li>
		<li>Once the communication is established, launch the RINMR software, type in its command line&#160;.HYPERSENSENMR, and then press enter.</li>
		<li>After that, a new window will be shown on the screen and on it type the number one (1) in the Configuration Number field. Then, click on the Select Configuration button.</li>
		<li>Click on the button Do microwave sweep. A small window with a descending counter of seconds will be launched indicating that the spectrometer is ready and it will be waiting for periodic trigger signals, coming from the DNP control software, to sample the polarization.</li>
		<li>On the DNP control software, select the Calibrate tab and click on the Generate button.</li>
		<li>Using the calibration setup window, enter the following information: Start Frequency = 94.117 GHz, End Frequency = 94.137 GHz, Step Size = 1 MHz, Step Duration = 300 s, Power = 50 mW, Liquid Helium Level = 65%, and Temperature = 1.4 K.</li>
		<li>Click on the button Generate, which will close the setup window and return to the Calibrate tab that will display the number of steps and the time required to perform the desired microwave sweep.</li>
		<li>Once the desired VTI temperature is achieved, click the Enable button and then Start to initialize the microwave sweep process.&#160;&#160;&#160;&#160;&#160;</li>
	</ol>
	</li>
	<li>At the end of the microwave sweep, recover the sample and record the optimal frequency where the maximum polarization is achieved. This optimal frequency is defined as the polarization frequency that provides the maximum polarization as shown in Figure 2. This frequency will be used for hyperpolarizing all the aliquots obtained from that specific stock solution of pyruvic acid.</li>
	<li>Prepare 250 mL of stock dissolution medium using a solution of 40-mmol/L Tris base, 50 mmol/L of sodium chloride, and 80-mmol/L sodium hydroxide in de-ionized water. Add ethylenediaminetetraacetic acid (EDTA) at a concentration of 100 mg/L to sequester any metal ion contamination. Similarly to the pyruvic acid stock solution, this dissolution medium will be used for all the different samples that will be polarized. Refer to the Table of Materials for more specific details regarding the chemicals used.</li>
	<li>Also, prepare 500 mL of stock cleaning solution consisting of 100 mg/L EDTA dissolved in deionized water. Approximately 10 mL of this cleaning solution is used after each polarization to clean the dissolution path of the DNP. 
	NOTE: Steps 1.9-1.27 are performed for each individual sample.</li>
	<li>Cool the DNP apparatus to 1.4 K in preparation of hyperpolarizing a [1-13C]pyruvic acid sample by pressing the Cooldown button in the DNP main window.</li>
	<li>If the software used for the spectrometer is already active with configuration 1 selected, proceed with the following steps. Otherwise, perform steps 1.5.1 to 1.5.3 and then continue with the following steps.</li>
	<li>After verifying that configuration 1 is selected in the window controlling the DNP&#39;s spectrometer, click on the Solid Build Up button.</li>
	<li>Enter the file name SSBuilupXXX, where &#34;XXX&#34;&#160;is a number in the sequence of files stored with build-up data. This number is automatically incremented by the software. Then click OK. Similarly to the microwave sweep case, a small window with a descending counter of seconds will be launched indicating that the spectrometer is ready and it will be waiting for periodic trigger signals, coming from the DNP control software, to sample the polarization.</li>
	<li>Using the pyruvic acid - OX063 stock solution prepared in step 1.1, weigh out 30 mg in a sample cup.</li>
	<li>When the desired VTI temperature is achieved (1.4 K) click on Insert Sample, then select Normal Sample and then click on Next. Following the safety precautions displayed on the screen, insert the cup in the cold DNP apparatus, using a long wand specifically designed for this task.</li>
	<li>Once the cup is inserted, the wand removed, and the DNP doors closed, click Next and then Finish. At that point the hyperpolarizer system lowers the sample cup to the irradiation chamber partially filled (65%) with liquid helium.</li>
	<li>Wait until the temperature has returned to 1.4K and then click on the Polarize Sample button.</li>
	<li>In the new pop up window, set the frequency value to that obtained from the microwave sweep in step 1.6. In the same window, also set the power to 50 mW and the sampling time to 300 s. Click on Next, check the Enable Build-up Monitoring box, and then click on Finish. 
	NOTE: Once the polarization is started, the DNP control software generates trigger signals every 300 s to instruct the spectrometer to sample the polarization using a small tip angle. That way, the spectrometer software adds a sample point to a solid-state magnetization curve, now displayed in both the spectrometer software and in the DNP control software under the tab Polarization Build-Up. After the 4th sample and every sample after that, the spectrometer software fits the curve to an exponential growth function of the form: 
	 
	S = A *exp(-t/Tp) + y0 
	 
	where A is the polarization amplitude, in arbitrary units, t is the sampling time, Tp is the polarization time constant (both in seconds), and y0 is an offset. Based on the fitted parameters, the software also calculates the percentage polarization achieved up to that point in time, which is also displayed in the DNP&#39;s Polarization Status tab.</li>
	<li>Polarize until the build-up of the solid-state magnetization reaches at least 95% of maximum (approximately one hour).</li>
	<li>While the sample is polarizing, prepare the Fast-Field-Cycling Relaxometer as explained in Section 2 below.</li>
	<li>When the desired polarization is achieved, click on Run Dissolution and under Method, select Pyruvic Acid Test. Then, click on Next.</li>
	<li>Following the instructions on the screen, open the DNP turret doors and load the heating and pressurizing chamber at the top of the apparatus with ~ 4.55 mL of the dissolution medium prepared in section 1.5 to produce a concentration of 80-mmol/L pyruvate upon dissolution at a pH of ~7.75 and temperature of ~37 &#176;C.</li>
	<li>Position the recovering wand in the right position, close the turret doors, and at the computer click on Next and then on Finish. At that point the dissolution media will be superheated until the pressure reaches 10 bar.</li>
	<li>Once the 10 bar pressure is attained, the frozen and hyperpolarized pyruvate is automatically lifted from the liquid helium bath, quickly mixed, and thawed with the superheated dissolution media and ejected through a capillary tubing into a pear-shaped flask. While the hyperpolarized pyruvate/dissolution media mixture is ejected, constantly swirl the flask to ensure a homogeneous mixture.</li>
	<li>When all the mixture has been ejected, quickly draw 1.1 mL of the liquid into a syringe, transfer to a pre-warmed (37 &#176;C) 10-mm-diameter NMR tube, and rapidly transport to the field-cycling relaxometer (See step 2.2.12).</li>
	<li>Dispense the remaining aliquot of every pyruvate dissolution into a 0.55-T benchtop NMR spectrometer (see Table of Materials) to check for possible systematic experimental effects.</li>
	<li>Immediately clean DNP fluid path using clean dissolution medium followed by ethanol. Blow helium gas through fluid path to remove remaining cleaning fluids and purge path of oxygen. Clean all glassware.</li>
	<li>After each measurement, record the pH of samples from both the bench top spectrometer and the field-cycling relaxometer. 
	NOTE: Each T1 measurement is a separate hyperpolarized dissolution from the DNP apparatus, so care is required to assure measurement-to-measurement reproducibility of the sample composition. This is accomplished by weighing all agents and solvents with a precision of 0.1 mg to assure accurate and reproducible preparation of the final hyperpolarized solutions.</li>
</ol>
2. Relaxometry
NOTE Please refer to Table 1 for a better understanding of the selection and use of the different parameters described in the following steps. Prior to dissolution, the relaxometer flip angle must be calculate and the relaxometer must be setup and ready for measurement of the hyperpolarized solution (see below).
<ol>
	<li>Flip-angle calibration 
	<ol>
		<li>Prepare 1 mL of neat [1-13C]pyruvic acid in an NMR tube and add a gadolinium contrast agent to reduce the T1 of the 13C nuclei to a value of less than 200 ms but more than 50 ms.</li>
		<li>Seal the NMR tube so it can be used multiple times as a calibration standard.</li>
		<li>Using the depth gauge of the relaxometer, set the depth of insertion of the NMR tube to the proper height to ensure the sample will be located at the center of the relaxometer RF coil.</li>
		<li>Mark the insertion depth of the 13C pyruvate calibration standard with adhesive tape to ensure repeatability.</li>
		<li>Place the depth stopper on the NMR tube to the position indicated by the tape and insert this calibration standard into the bore of the field-cycling relaxometer. Use a weight to keep the NMR tube in position.</li>
		<li>Open the instrument air valve and from the relaxometer front panel set the temperature controller to 37 &#176;C. That will maintain the temperature of the sample at 37&#176;C (&#177; 0.5&#176;C) using heated air during the experiment.</li>
		<li>Setup the field-cycling relaxometer hardware to acquire 13C nuclei signals. That includes installing and energizing the external shim coil (see Table of Materials), tuning and matching the RF coil to 8 MHz (~0.75 T for 13C nuclei), and using the appropriate &#955;/4 cable.</li>
		<li>In the instrument software, perform the following steps:
		<ol>
			<li>Select the Main par tab</li>
			<li>Click on the cell next to the label Experiment and scroll down in the pop-up window to select the pulse sequence &#34;13CANGLE.FFC &#34;.</li>
			<li>Set the following acquisition parameters: RFA = 5; SWT = 0.005, RD = 0.5, BPOL = 30 MHz, TPOL = 0.5.</li>
			<li>Select the Acq. par tab and then select the Basic sub-tab.</li>
			<li>Click on the cell next to the label Nucleus and scroll down in the pop-up window to select 13C.</li>
			<li>Then, set the following parameters: SF = 8 MHz, SW = 1000000, BS = 652, FLTR = 100000, MS = 32.</li>
			<li>Select the Conf sub-tab.</li>
			<li>Set the following parameters: RINH = 25, ACQD = 25.</li>
			<li>Select the nDim sub-tab</li>
			<li>Set NBLK = 32, BINI = 2, BEND = 62.</li>
			<li>Select the Evaluation tab and then the Parameters sub-tab.</li>
			<li>Set the following parameters: EWIP = 10, EWEP = 128, EWIB = 1, EWEB = 32.</li>
			<li>Then, click the Start Acquisition icon to run the pulse sequence.</li>
		</ol>
		</li>
		<li>Once the acquisition is finished, save the data, select the Evaluation dialog icon and from the analysis menu select WAM Window: Absolute Magnitude. Then select Report Sheet, Graphs and Export File and finally click on Execute.</li>
		<li>In the Report window find the RF pulse width that provides the maximum amplitude and fine-tune the value with the help of the cursor in the displayed graph, which is similar to the plots shown at the bottom row of Figure 3. This pulse width will be used for the parameter PW90 of the following experiments.</li>
		<li>Click the F1 icon to adjust the frequency shift of the relaxometer. 
		NOTE: WAM Window: Absolute Magnitude is a procedure to integrate the magnitude of a single or a sequence of free-induction decay acquisitions (FIDs) from the point defined by EWIP to the point specified by EWEP and from the block defined by EWIB to the block specified by EWEB.</li>
	</ol>
	</li>
	<li>T1-Measurements 
	<ol>
		<li>Make sure the external shim coil is installed and energized.</li>
		<li>In the instrument software perform the following steps:
		<ol>
			<li>Select the Main par tab</li>
			<li>Click on the cell next to the label Experiment and scroll down in the pop-up window to select the pulse sequence HPUB/S, which is shown in Figure 4.</li>
			<li>Set the following acquisition parameters: RFA = 25, T1MX = values between 3 and 5; SWT = 0.2, RD = 0, BRLX = Desired relaxation field in MHz (proton Larmor frequency).</li>
			<li>Select the Acq. par tab and then select the Basic sub-tab.</li>
			<li>Click on the cell next to the label Nucleus and scroll down in the pop-up window to select 13C.</li>
			<li>Then, set the following parameters: SF = 8 MHz, SW = 1000000, BS = 652, FLTR = 50000.</li>
			<li>Select the Conf sub-tab.</li>
			<li>Set the following parameters: PW90 equal to the value found in step 2.1.10, RINH = 25, ACQD = 25.</li>
			<li>Select the Puls sub-tab and set PW = 5.</li>
			<li>Select the nDim sub-tab and set NBLK = 100.</li>
			<li>Wait and get ready to receive the hyperpolarized solution to initiate the data acquisition.</li>
			<li>Immediately before inserting the sample into the relaxometer, manually start the pulse sequence from the console, to avoid inserting the sample into a null magnetic field. For this reason, it is important to ignore the first Free Induction Decay (FID) during the data analysis.</li>
			<li>Once the acquisition is done, save the data by clicking the Save button.</li>
		</ol>
		</li>
		<li>Using the analysis software, integrate the magnitude of each FID signal to produce a data series comprised of sample magnetization as a function of time.</li>
		<li>Extract the spin-lattice relaxation time from a three-parameter exponential model using a standard non-linear least-squares fitting algorithm implemented in a commercial analytical software (see Table of Materials) assuming even weighting for all data: 
		<img alt="Equation 1" src="/files/ftp_upload/59399/59399eq1.jpg" /> 
		where A is the initial signal amplitude (y-intercept), T1 is the spin-lattice relaxation time, TR is the repetition time, which is a known value, y0 is the signal offset, and cos(n-1)(&#945;) is a correction for loss of longitudinal magnetization at the nth measurement for a flip angle, &#945;.</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Spin dynamics: basics of nuclear magnetic resonance. , (2001).</li><li>Mieville, P., Jannin, S., Bodenhausen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxometry+of+insensitive+nuclei:+optimizing+dissolution+dynamic+nuclear+polarization.">Relaxometry of insensitive nuclei: optimizing dissolution dynamic nuclear polarization.</a> Journal of Magnetic Resonance. 210 (1), 137-140 (2011).</li><li>Redfield, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shuttling+device+for+high-resolution+measurements+of+relaxation+and+related+phenomena+in+solution+at+low+field,+using+a+shared+commercial+500+MHz+NMR+instrument.">Shuttling device for high-resolution measurements of relaxation and related phenomena in solution at low field, using a shared commercial 500 MHz NMR instrument.</a> Magnetic Resonance in Chemistry. 41 (10), 753-768 (2003).</li><li>Grosse, S., Gubaydullin, F., Scheelken, H., Vieth, H. -. M., Yurkovskaya, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+cycling+by+fast+NMR+probe+transfer:+Design+and+application+in+field-dependent+CIDNP+experiments.">Field cycling by fast NMR probe transfer: Design and application in field-dependent CIDNP experiments.</a> Applied Magnetic Resonance. 17 (2), 211-225 (1999).</li><li>Kimmich, R., Anoardo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field-cycling+NMR+relaxometry.">Field-cycling NMR relaxometry.</a> Progress in Nuclear Magnetic Resonance Spectroscopy. 44 (3-4), 257-320 (2004).</li><li>Gu&#240;j&#243;nsd&#243;ttir, M., Belton, P., Webb, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Magnetic Resonance in Food Science: Challenges in a Changing World. , 65-72 (2009).</li><li>Anoardo, E., Galli, G., Ferrante, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast-field-cycling+NMR:+Applications+and+instrumentation.">Fast-field-cycling NMR: Applications and instrumentation.</a> Applied Magnetic Resonance. 20 (3), 365-404 (2001).</li><li>Chattergoon, N., Martinez-Santiesteban, F., Handler, W. B., Ardenkjaer-Larsen, J. H., Scholl, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+dependence+of+T1+for+hyperpolarized+[1-13C]pyruvate.">Field dependence of T1 for hyperpolarized [1-13C]pyruvate.</a> Contrast Media &amp; Molecular Imaging. 8 (1), 57-62 (2013).</li><li>Mart&#237;nez-Santiesteban, F. M., Dang, T. P., Lim, H., Chen, A. P., Scholl, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T1+nuclear+magnetic+relaxation+dispersion+of+hyperpolarized+sodium+and+cesium+hydrogencarbonate-13C.">T1 nuclear magnetic relaxation dispersion of hyperpolarized sodium and cesium hydrogencarbonate-13C.</a> NMR in Biomedicine. 30 (9), 3749 (2017).</li></ol>

The authors have no disclosures.

The use of DNP to enhance signal acquisition is a technical solution to insufficient magnetic resonance signal available from 13C nuclei at limited concentrations, as those used in animal injections, but presents other experimental challenges. Each relaxation measurement shown in Figure 7 represents a measurement of a uniquely prepared sample because it cannot be repolarized after dissolution for remeasurement. This inevitably leads to experimental variability due to minor differe...

Magnetic resonance spectroscopic imaging (MRSI) can produce spatial maps of metabolites detected by spectroscopic imaging, but its practical use is often limited by its relatively low sensitivity. This low sensitivity of in vivo magnetic resonance imaging and spectroscopy methods stems from the small degree of nuclear magnetization achievable at body temperatures and reasonable magnetic field strengths. However, this limitation can be overcome by the use of dynamic nuclear polarization (DNP) to greatly enhance the in vitro magnetization of liquid substrates, which are subsequently injected to probe in vivo metabolism using MRSI1,2,3,4. DNP is capable of enhancing the magnetization of most nuclei with non-zero nuclear spin and has been used to increase in vivo MRSI sensitivity of 13C-enriched compounds such as pyruvate5,6, bicarbonate7,8, fumarate9, lactate10, glutamine11, and others by more than four orders of magnitude12. Its applications include imaging of vascular disease13,14,15, organ perfusion13,16,17,18, cancer detection1,19,20,21,22, tumor staging23,24, and quantification of therapeutic response2,6,23,24,25,26.
Slow spin-lattice relaxation is essential for in vivo detection with MRSI. Spin-lattice relaxation times (T1s) on the order of tens of seconds are possible for nuclei with low gyromagnetic ratios within small molecules in solution. Several physical factors influence the transfer of energy between a nuclear spin transition and its environment (lattice) leading to relaxation, including the magnetic field strength, temperature, and molecular conformation27. Dipolar relaxation is reduced in molecules for carbon positions with no protons directly attached, and deuteration of dissolution media can further reduce intermolecular dipolar relaxation. Unfortunately, deuterated solvents have limited abilities to extend in vivo relaxation. Increased relaxation of carbonyls or carboxylic acids (such as pyruvate) can occur at high magnetic field strengths due to chemical shift anisotropy. The presence of paramagnetic impurities from the fluid path during dissolution after polarization can cause rapid relaxation and need to be avoided or eliminated using chelators.
Very little data exist for the relaxation of 13C-containing compounds at low fields, where spin-lattice relaxation could be significantly faster. However, it is important to measure T1 at low fields to understand relaxation during preparation of the agent used for in vivo imaging, since the hyperpolarized contrast agents are usually dispensed from the DNP apparatus near or at the earth&#8217;s field. Additional physical factors such as 13C-enriched substrate concentration, solution pH, buffers and temperature also influence relaxation, and consequently have an effect on the formulation of the agent. All these factors are essential in the determination of key parameters in optimizing the DNP dissolution process, and the calculation of the magnitude of signal loss that occurs in transportation of the sample from the DNP apparatus to the imaging magnet.
Nuclear magnetic resonance dispersion (NMRD) measurements, i.e., T1 measurements, as a function of magnetic field are typically acquired using an NMR spectrometer. To acquire these measurements, a shuttling method could be used where the sample is first shuttled out of the spectrometer to relax at some field determined by its position in the fringe field of the magnet28,29,30 and then rapidly transferred back into the NMR magnet to measure its remaining magnetization. By repeating this process at the same point in the magnetic field but with increasing periods of relaxation, a relaxation curve can be obtained, which can then be analyzed to estimate T1.
We use an alternative technique known as fast field-cycling relaxometry31,32,33 to acquire our NMRD data. We have modified a commercial field-cycling relaxometer (see Table of Materials), for T1 measurements of solutions containing hyperpolarized 13C nuclei. Compared with the shuttle method, field-cycling enables this relaxometer to systematically acquire NMRD data over a smaller range of magnetic fields (0.25 mT to 1 T). This is accomplished by rapidly changing the magnetic field itself, not the sample location in the magnetic field. Therefore, a sample can be magnetized at a high field strength, &#34;relaxed&#34;&#160;at a lower field strength, and then measured by acquisition of a free-induction-decay at a fixed field (and Larmor frequency) to maximize signal. This means that the sample temperature can be controlled during the measurement, and the NMR probe does not need to be tuned at each relaxation field promoting automatic acquisition over the entire magnetic field range.
Focusing our efforts to the effects of dispensing and transporting the hyperpolarized solutions at low magnetic fields, this work presents a detailed methodology to measure the spin-lattice relaxation time of hyperpolarized 13C-pyruvate using fast field-cycling relaxometry for magnetic fields in the range of 0.237 mT to 0.705 T. The main results of using this methodology have been previously presented for [1-13C]pyruvate34 and 13C-enriched sodium and cesium bicarbonate35 where other factors such as radical concentration and dissolution pH have also been studied.

<table border="0" cellpadding="0" cellspacing="0" style="width:825px;" width="824">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:263px;">[1-13C]Pyruvic Acid</td>
			<td style="width:244px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:79px;">677175</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">10mm NMR Tube</td>
			<td style="width:244px;">Norell, Inc., Morganton NC, USA</td>
			<td style="width:79px;">1001-8</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">De-ionized water</td>
			<td style="width:244px;"></td>
			<td style="width:79px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td style="width:244px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:79px;">E5134</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="294">
			<td height="294" style="height:294px;width:263px;">HyperSense Dynamic Nuclear Polarizer</td>
			<td style="width:244px;">Oxford Instruments, Abingdon, UK</td>
			<td style="width:79px;"></td>
			<td style="width:240px;">Includes the following: &#34;DNP-NMR Polarizer&#34; software used to control and monitor the whole DNP polarizer; &#34;RINMR&#34; used to monitor the solid state polarization levels; &#34;HyperTerminal&#34; used to communicate the DNP software with the RINMR software that monitors the solid state polarization level. Also includes the MQC bench top spectrometer to monitor the liquid state polarization in conjunction with it own RINMR software</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:263px;">MATLAB R2017b</td>
			<td>MathWorks, Natick, MA</td>
			<td style="width:79px;"></td>
			<td style="width:240px;">Include scripts for non-linear fitting of magnetization decay over time and T1 NMRD analysis of hyperpolarized pyruvic acid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">OX063 Triarylmethyl radical</td>
			<td style="width:244px;">Oxford Instruments, Abingdon, UK</td>
			<td style="width:79px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">pH meter - SympHony</td>
			<td style="width:244px;">VWR International, Mississauga, ON., Canada</td>
			<td style="width:79px;">SB70P</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">ProHance</td>
			<td style="width:244px;">Bracco Diagnostics Inc.</td>
			<td style="width:79px;"></td>
			<td style="width:240px;">Gadoteridol, Gd-HP-DO3A</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Pure Ethanol (100% pure)</td>
			<td style="width:244px;">Commercial Alcohols, Toronto, ON, Canada</td>
			<td style="width:79px;">P016EAAN</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Shim Coil</td>
			<td style="width:244px;"></td>
			<td style="width:79px;"></td>
			<td style="width:240px;">Developed in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sodium Chloride</td>
			<td style="width:244px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:79px;">S7653</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sodium Hydroxide</td>
			<td style="width:244px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:79px;">S8045&#160;</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:263px;">SpinMaster FFC2000 1T C/DC</td>
			<td style="width:244px;">Stelar s.r.l., Mede (PV) Italy</td>
			<td style="width:79px;"></td>
			<td style="width:240px;">Includes the software &#34;AcqNMR&#34; that is used to set experimental parameters, monitor the tuning and matching of the RF coil, loading different pulse sequences, calibrate flip angle, data acquisition and curve fitting, among other functions. Also includes a depth gauge, some weights and a depth stopper.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Trizma Pre-Set Crystals (pH 7.6)</td>
			<td style="width:244px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:79px;">T7943</td>
			<td style="width:240px;"></td>
		</tr>
	</tbody>
</table>

measuring the spin-lattice relaxation magnetic field dependence of hyperpolarized [1-13c]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Figure 2 presents an example of a high-resolution full-range microwave sweep for pyruvic acid. For the presented case, that optimal microwave frequency corresponds to 94.128 GHz, highlighted in the figure insert. Our DNP system can normally work in the range of 93.750 GHz to 94.241 GHz with step size of 1 MHz, polarization time of up to 600 s, and power of up to 100 mW. A full range of frequencies is investigated only for novel substrates. However, based on previous experience with 13</...

Watch this Scientific Journal Video about Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate at JoVE.com

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

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6.5K Views.  Western University. We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

Video: Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...