This project was supported by grants NIH/NIBIB 1K99EB020075 to Pedro Brugarolas and an Innovation Fund Award from the Chicago Innovation Exchange to Brian Popko and Pedro Brugarolas. Prof. Brian Popko is gratefully acknowledged for his mentorship and financial support to the project. Prof. Chin-Tu Chen and the Integrated Small Animal Imaging Research Resource at the University of Chicago are acknowledged for generously sharing laboratory space and equipment. IBA is acknowledged for sponsoring open-access of this article.

CAUTION: All procedures involving the use of radioactive materials must be approved by the local Office of Radiation Safety. When working with radioactive materials wear a lab coat and personal radiation badges. Use two layers of gloves at all times and check hands with a Geiger counter after each step that involves handling radioactivity. If the gloves are contaminated with radioactivity discard and replace outer gloves. Use appropriate shielding, minimize time in contact with the radiation source and maximize distance.
1. One Week before Experiment: Preparation of Materials
<ol>
	<li>Download [18F]3F4AP sequence: Synthera users can log into the Users Database (http://www.iba-radiopharmasolutions.com/products/chemistry) and download the sequence file for 3F4AP. Users of other synthesizers may need to write their own script based on the sequence of steps. Browse through the annotated sequence to become familiar with the steps involved in the synthesis.</li>
	<li>Ensure that there is enough gas for the synthesis. The synthesizer requires compressed gas, either helium or nitrogen. It also requires &#62;75 psi compressed air. Ensure that the pressures are within the recommended by the manufacturer.</li>
	<li>Prepare HPLC mobile phase: prepare 1 L of 50 mM sodium phosphate and 10 mM triethyl amine. Using a pH meter adjust the pH to 8.0 &#177;0.1 by adding dropwise saturated sodium hydroxide while stirring. Filter the solution through a 0.22 &#181;m bottletop filter and add 5% volume of ethanol.</li>
	<li>Dry glassware in the oven overnight.</li>
</ol>
2. Day of Experiment: Before Arrival of Fluorine-18
<ol>
	<li>Using 1 mL syringes, fill reagent vials with the appropriate reagents. For vials 2 and 3 use oven-dried vials and anhydrous solvents kept under argon. Seal the vials with crimp seals using a crimper.
	<ol>
		<li>Fill Vial 1 (11 mm diameter / 2 mL volume vial) with 400 &#181;L of TBA-HCO3 + 800 &#181;L of acetonitrile (MeCN).</li>
		<li>Fill Vial 2 (13 mm / 4 mL vial) with 50 &#181;L of precursor solution 1.0&#160;mg/mL + 450 &#181;L of MeCN.</li>
		<li>Fill Vial 3 (11 mm / 2 mL vial) with 500 &#181;L of MeCN.</li>
		<li>Fill Vial 4 (13 mm / 4 mL vial) with 4 mL of 0.2% oxalic acid in methanol (MeOH).</li>
	</ol>
	</li>
	<li>Condition the QMA (strong anion exchange) and Alumina-N solid phase extraction cartridges. Using a 10 mL syringe, pass 5 mL of 8.4% NaHCO3 drop-wise through the QMA followed by 5 mL of ultrapure deionized Type I water (18.2 &#924;&#937;&#8226;cm at 25 &#186;C). Pass 5 mL of ultrapure water drop-wise through Alumina-N cartridge followed by 5 mL of MeOH + 0.2% oxalic acid.</li>
	<li>Turn on the HPLC and condition the C-18 column with 4 mL per min of mobile phase for 30 min.</li>
	<li>Load a new catalyst cartridge onto the hydrogenator cartridge holder and start a flow of 0.5 mL/min of 100% MeOH. Set the hydrogen regulator to 50 psi and condition the cartridge for 15 min (Figure 2).</li>
	<li>Assemble the Integrated Fluid Processor (IFP) by introducing vials 1 through 4 in their positions, attaching the cartridges and collection vial as shown in Figure 3. Attach a collection vial with a vent needle to the output line of the hydrogenator.</li>
	<li>Start the synthesizer&#39;s software. Enter login and password. Perform pre-run checks on the synthesizer according to manufacturer instructions.</li>
	<li>Click on &#34;Sequences&#34; and then &#34;Open&#34; to load the 3F4AP sequence.</li>
	<li>Load the IFP by clicking the &#34;Load&#34; button on the screen. Type a file name for the run and start the sequence by clicking &#34;Start&#34;. (The automated synthesizer will automatically pause before the 18F loading step.)</li>
	<li>Watch as the synthesizer goes through the routine self-check steps (part one of the sequence). Look at the screen to ensure that there are no warnings or alarms. Pay attention to sounds as the synthesizer flushes lines and preheats reaction vessel in preparation for the run. The temperature indicator should rise and stay at 65 &#186;C. Wait for the signal (auditory beep) indicating that the synthesizer is ready for the 18F transfer.</li>
</ol>
3. Day of Experiment: 18F Labeling
<ol>
	<li>Remotely transfer desired amount of cyclotron-produced 18F from the cyclotron target to 18F vial. Verify the amount of radioactivity and record it with the time of delivery. 
	NOTE: If not using a direct line for 18F- transfer use a prefilled syringe with a needle attached to transfer the activity to the vial through the septum. The amount of starting radioactivity depends on the limits set by the Office of Radiation Safety and the desired amount of final tracer. Typical amount ranges between 50 and 500 mCi.</li>
	<li>Resume sequence on the synthesizer by pressing &#34;Resume&#34;. This will initiate the transfer of the 18F into the QMA.</li>
	<li>Monitor the progression of the synthesis throughout the automated sequence on the computer screen.
	<ol>
		<li>Watch the transfer the 18F from the vial onto the QMA for 90 s. After trapping 18F- on QMA it elutes with TBA-HCO3 solution (vial 1). (Part two of the sequence)</li>
		<li>Monitor the pressure and temperature traces on the synthesizer while the TBA18F is dried under reduced pressure (5 kPa) and heating (100 &#186;C), followed by additional drying and cool down steps. (Part 3 of the sequence)</li>
		<li>Watch the transfer of anhydrous MeCN (vial 3) and precursor solution (vial 2) to the reactor and how it reacts for 1 min at room temperature. The solution should be colorless or very faint yellow. (Part 4 of the sequence)</li>
		<li>Watch the transfer of oxalic acid solution (vial 4) to the reactor. Watch as the solution is pressure transferred from the reactor through alumina-N cartridge to the final product vial. (Part 5 of the sequence)</li>
	</ol>
	</li>
	<li>At the end of the sequence, print out the report, eject the IFP, shut off gas tanks, and close the software.</li>
	<li>While first establishing the procedure, measure the radioactivity in the alumina-N cartridge and the collection vial by separately introducing the cartridge and vial in the dose calibrator. Record the activity and time of measurement. Place the used cartridge in a leaded waste container. Place the collection vial in a shielded container for transport to the next step.</li>
	<li>Using a 1 mL syringe with a 2&#34; needle attached, manually transfer approximately 100 &#956;L of sample of the intermediate product solution into a standard HPLC vial for in-process quality control. Inject 10 &#956;L of this sample into the HPLC to evaluate the purity and identity of the intermediate compound. 
	NOTE: HPLC conditions: XDB 5 &#181;m, 9.4 x 250 mm C18 column. Flow 4 mL/min. Mobile phase (50 mM Na2HPO4, 10 mM TEA, 5% EtOH). Isocratic 15 min.</li>
</ol>
4. Day of Experiment: Hydrogenation
CAUTION: Injection of the product into the hydrogenator has to be done using proper shielding precautions. Hydrogen gas must be properly handled and vented.
NOTE: the hydrogenation reactor can be connected in place of the HPLC column on the synthesizer and controlled using the synthesizer software.
<ol>
	<li>Set up the hydrogenator flow at 0.5 mL/min by starting the synthesizer HPLC sequence. Manually set up hydrogen pressure to 50 psi.
	<ol>
		<li>After finishing the labeling and quenching steps the synthesizer will transfer the intermediate product solution into hydrogenator/HPLC loop.</li>
	</ol>
	</li>
	<li>When the radioactive peak appears on the HPLC software toggle the collection valve to collect the product. Measure radioactivity of crude product using a dose calibrator. 
	NOTE: The crude product should be injected into an automated HPLC system inside a hot cell. After purification, the final product is then collected and dispensed into an aseptic ISO class 5 laminar air flow hot cell as per USP and FDA regulations.</li>
</ol>
5. Day of Experiment: Purification and Preparation of the Dose
<ol>
	<li>Inject crude product into the HPLC and use the automated fraction collector to collect the fractions corresponding to the final product peak. Each tube contains 0.66 mL of solution. 
	NOTE: HPLC conditions: XDB 5 &#181;m, 9.4 x 250 mm C18 column. Flow 4 mL/min. Mobile phase (50 mM Na2HPO4, 10 mM TEA, 5% EtOH). Isocratic 15 min. Collect 4-15 min.</li>
	<li>Measure the radioactivity of each fraction using a dose calibrator and record it. Combine the fractions with highest amounts of radioactivity (typically tubes 14-18).</li>
	<li>Draw the product solution with a 10 mL syringe and pass the sample through 0.22 &#181;m filter into a sterile vial. Record the amount of radioactivity, end of synthesis time and solution volume on the vial label. This is the final dose for injection. Put aside ~0.8 mL of the solution for quality control tests.</li>
</ol>
6. Day of Experiment: Quality Control (QC) Tests
<ol>
	<li>Before dose release: 
	Inspect the dose through lead-shielded glass. The solution must be clear, colorless and free of particulate matter.</li>
	<li>Radiochemical identity:
	<ol>
		<li>For RadioTLC: spot a drop of the sample on a TLC plate side-by-side with the reference standard. Run TLC plate on a TLC chamber using 95% MeOH: 5% acetic acid. Visualize the reference standard under UV illumination and mark its position with pencil.</li>
		<li>Tape the TLC plate to the stage of the radioTLC scanner and record time of the peak. Rf values of the reference standard and radioactive peak must match within 5%.</li>
		<li>For RadioHPLC: run 10 &#181;L of the dose with and without the reference standard on the HPLC. Retention time of the reference standard and the radioactive peak must match. A single coelution peak must be seen on the spiked sample.</li>
	</ol>
	</li>
	<li>For radiochemical purity: measure the area under curve for the radioHPLC and radioTLC target peaks. The area of the target peak must be &#62;95% of the area for all radioactive peaks combined.</li>
	<li>For the specific radioactivity: calculate the specific radioactivity as the amount of radioactivity in the peak (measured on step 5.2) over the mass amount determined from the area under the curve of the UV HPLC trace using a pre-established calibration curve. The specific radioactivity must be higher than 50 mCi/&#181;mol.</li>
	<li>For the residual solvent analysis: measure the amount of residual solvents (MeCN, MeOH) in the dose using gas chromatography. Solvents levels must be &#60; 0.04% for acetonitrile and &#60;3,000 ppm for methanol. The amount of EtOH must be less than 10% w/v.</li>
	<li>For the sterile filter integrity test (bubble point): connect the filter used in step 5.3 to a nitrogen supply equipped with a pressure regulator and submerge the needle in water. Gradually open the gas valve while watching the pressure gauge. The filter should withstand pressures up to 50 psi without bursting as evidenced by the lack of a stream of bubbles from the needle. Increase the pressure beyond 50 psi until a stream of bubbles comes out of the needle. Record this pressure, it is the burst pressure and it must be &#62;50 psi.</li>
	<li>For the radionuclide half-life: measure the radioactivity of the product at two time points &#8805;10 min apart in a dose calibrator. Calculate the half-life using the equation below. The half-life must match that of 18F to within 5 minutes (109 &#177;5 min): 
	t&#189;calculated = 0.693 t &#247; ln (A1/A2) 
	where t is the interval between measurements and A 1, A 2 the activity measured at each time point.</li>
	<li>For the radionuclidic identity and purity: obtain the &#947;-ray spectrum of a sample of the product using a gamma counter. The spectrum should exhibit one single photo-peak at an energy of 511 keV. There should be no other photo-peaks in the spectrum.</li>
	<li>For the endotoxin analysis: measure the endotoxin levels using a LAL chromogenic endotoxin quantitation test. Endotoxin levels must be &#60;1.75 EU/mL for a 1:10 diluted product with a final product volume of 10 mL.</li>
	<li>Document the results from each QC test. Release the dose for animal studies only if all the tests passed.</li>
	<li>Post-dose release: 
	For the sterility test: add a sample of the dose to both fluid thioglycolate and trypticase soy broth. No growth must be seen on the media after 14 days.</li>
</ol>
7. Day of Experiment: Calculations (Table 1)
<ol>
	<li>For the non-decay corrected radiochemical yield (n.d.c. RCY): calculate the n.d.c. RCY as the amount of radioactivity in the final product over the starting radioactivity.</li>
	<li>For the radiolabeling efficiency: calculate the labeling yield as the ratio of radioactivity in the collection vial over radioactivity in the alumina-N cartridge (unincorporated [18F]F-) and the collection vial.</li>
	<li>For the hydrogenation yield: calculate the hydrogenation yield as the amount of radioactivity in the desired peak over the radioactivity injected into the HPLC.</li>
	<li>For the filtering losses: calculate filtering loses as the radioactivity remaining in the filter and syringe over radioactivity before filtering.</li>
</ol>

<ol>
	<li>Valk, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Positron emission tomography : basic science and clinical practice. , (2003).</li><li>Phelps, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PET: molecular imaging and its biological applications. , (2004).</li><li>Ametamey, S. M., Honer, M., Schubiger, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+with+PET.">Molecular imaging with PET.</a> Chem Rev. 108 (5), 1501-1516 (2008).</li><li>Oriuchi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+role+and+future+prospects+of+positron+emission+tomography+in+clinical+oncology.">Present role and future prospects of positron emission tomography in clinical oncology.</a> Cancer Sci. 97 (12), 1291-1297 (2006).</li><li>Heiss, W. D., Herholz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+receptor+imaging.">Brain receptor imaging.</a> J Nucl Med. 47 (2), 302-312 (2006).</li><li>Brugarolas, P., Freifelder, R., Cheng, S. -. H., DeJesus, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+meta-substituted+[18F]3-fluoro-4-aminopyridine+via+direct+radiofluorination+of+pyridine+N-oxides.">Synthesis of meta-substituted [18F]3-fluoro-4-aminopyridine via direct radiofluorination of pyridine N-oxides.</a> Chemical Communications. , (2016).</li><li>Jones, R. E., Heron, J. R., Foster, D. H., Snelgar, R. S., Mason, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+4-aminopyridine+in+patients+with+multiple+sclerosis.">Effects of 4-aminopyridine in patients with multiple sclerosis.</a> J Neurol Sci. 60 (3), 353-362 (1983).</li><li>Davis, F. A., Stefoski, D., Rush, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orally+administered+4-aminopyridine+improves+clinical+signs+in+multiple+sclerosis.">Orally administered 4-aminopyridine improves clinical signs in multiple sclerosis.</a> Ann Neurol. 27 (2), 186-192 (1990).</li><li>Goodman, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained-release+oral+fampridine+in+multiple+sclerosis:+a+randomised,+double-blind,+controlled+trial.">Sustained-release oral fampridine in multiple sclerosis: a randomised, double-blind, controlled trial.</a> Lancet. 373 (9665), 732-738 (2009).</li><li>Brugarolas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Abstracts Of Papers Of The American Chemical Society. , (2016).</li><li>Brugarolas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+PET+tracer+for+MS.">Development of a PET tracer for MS.</a> J Nucl Med Meeting Abstracts. 55 (1), 1124 (2014).</li><li>Brugarolas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorinated+4-aminopyrdines+as+PET+tracers+for+MS.">Fluorinated 4-aminopyrdines as PET tracers for MS.</a> Journal of Nuclear Medicine. 56, 493 (2015).</li><li>Yoswathananont, N., Nitta, K., Nishiuchi, Y., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+hydrogenation+reactions+in+a+tube+reactor+packed+with+Pd/C.">Continuous hydrogenation reactions in a tube reactor packed with Pd/C.</a> Chem Comm. (1), 40-42 (2005).</li><li>St&#246;cklin, G., Pike, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Radiopharmaceuticals for Positron Emission Tomography-Methodological Aspects. 24, (1993).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+evaluation+of+a+high-affinity+18F-trifluoroborate+octreotate+derivative+for+somatostatin+receptor+imaging.">Preclinical evaluation of a high-affinity 18F-trifluoroborate octreotate derivative for somatostatin receptor imaging.</a> J Nucl Med. 55 (9), 1499-1505 (2014).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18F-trifluoroborate+derivatives+of+[des-arg(10)]kallidin+for+imaging+bradykinin+b1+receptor+expression+with+positron+emission+tomography.">18F-trifluoroborate derivatives of [des-arg(10)]kallidin for imaging bradykinin b1 receptor expression with positron emission tomography.</a> Mol Pharm. 12 (3), 974-982 (2015).</li><li>Scott, P. J. H., Hockley, B. G., Kilbourn, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Radiochemical Syntheses, Volume 1: Radiopharmaceuticals for Positron Emission Tomography. , (2012).</li></ol>

Authors have nothing to disclose.

The preparation of PET tracers requires efficient labeling with minimal user intervention to minimize radiation exposure14. Here, we described the first semi-automated procedure for the radiochemical synthesis of [18F]3F4AP, a PET tracer currently under investigation for imaging demyelination. This semi-automated method produces the radiotracer with high purity and sufficient specific activity for animal studies. Prior methods for the synthesis of this compound relied on manual synthesis6, which significantly limits the amount of radioactive tracer that can be produced. Having an automated method for the synthesis also provides more reproducible yields and makes it easier to transfer the procedure to other laboratories with similar equipment. Future efforts to fully automate the procedure will be instrumental to the production of the tracer in high amounts for studies in large animals or humans.
This procedure uses nucleophilic exchange of 19F for 18F to incorporate the radioisotope into the molecule of interest. The advantages of this reaction are that it is fast and produces almost exclusively the desired product without the need to perform a potentially lengthy purification step to remove excess of precursor. One limitation of using fluoride-exchange labeling reactions such as the one used here is that due to initial mass of cold compound the final specific activity defined as amount of radioactivity in mCi over amount of compound in &#181;mol may be limited. Under our standard conditions, starting with 100-200 mCi of 18F- and 50 &#181;g of precursor, the typical specific activity at the end of synthesis is up to 100-200 mCi/&#181;mol, which appears to be sufficient for preclinical PET imaging studies. Nevertheless, the specific activity may improve by increasing the starting amount for 18F- while keeping the mass amount low. There have been several reports of producing radioligands by fluoride-exchange with high specific activity (1-3 Ci/&#181;mol) by starting with high activity and low precursor amounts15,16.
As with all radiochemical syntheses of PET tracers, it is critical to work quickly in order to minimize radioactive decay. It is also important to minimize the time handling the radioactive materials, use proper shielding and maximize the distance between the radioactive material and the user to minimize radiation exposure. These aspects are particularly important during the second half of the protocol (purification and quality control) in which the user has to manually inject the solution into the HPLC, collect the fractions and filter the final product.
As with all radiochemical syntheses of PET tracers, it is critical to work quickly in order to minimize radioactive decay. It is also important to minimize the time handling the radioactive materials, use proper shielding and maximize the distance between the radioactive material and the user to minimize radiation exposure. These aspects are particularly important during the second half of the protocol (hydrogenation and purification) in which the user has to manually inject the solution into the hydrogenator, collect the fractions, set up the drying procedure, redissolve the product in buffer and filter it. During the filtering step it is easy to lose a large amount of radioactive material in the walls of the vials. Thus, it is important to try to collect all the liquid prior to filtering. Using a greater amount of buffer to dissolve may improve the yield of recovery but its use is discouraged because it will require injecting a larger volume on the HPLC, causing the peak to broaden and increase the volume of the final dose.
In order to troubleshoot and optimize the procedure is important to keep track of the yields of each step. For most steps this is done simply by measuring the amount of radioactivity before and after any step. In the case of the reaction the yields can be calculated through quantification of the HPLC peaks. Table 1 in the Results Section shows the typical yields for each step. Table 2 below lists many of the commonly encountered failures with potential reasons for the failure and how to correct them.
Finally, even though the procedure demonstrated here is specific for the synthesis of [18F]3F4AP, the general workflow and many of the individual steps are common to the synthesis of other compounds17. In this article we also demonstrated the typical QC tests performed on any PET tracer.

The ability to trace a small-molecule drug non-invasively within the human body has great potential towards precision medicine. Among molecular imaging techniques, positron emission tomography (PET) has many favorable characteristics: the high sensitivity of PET detectors allows detection and quantification of very small amounts of radioactive material and the characteristics of the scanners allow accurate spatial mapping of the drug localization1,2,3. For example, PET allows detection and localization of tumors and metastasis based on the level of uptake of a radioactive glucose analog, [18F]FDG4. PET can also provide localization and quantification of specific brain receptors and their occupancy which can be valuable for diagnosing and understanding neurological and psychiatric disorders5. In order to develop a small molecule PET tracer, the compound of interest must be labeled with a positron-emitting isotope, typically 11C or 18F. Between these two radioisotopes, 18F has a longer half-life (109 min vs. 20.3 for 11C), which allows multi-dose and offsite production. Nevertheless, adding 18F to a molecule can be challenging. 18F labeling requires fast reactions compatible with automation relieving the chemist of direct handling of the activity and receiving high-absorbed radiation doses.
We recently described the use of pyridine N-oxides as precursors for the fluorination of pyridines and the use of this chemistry in the radiochemical synthesis of [18F]3F4AP6, a radiofluorinated analog of the FDA-approved drug for multiple sclerosis, 4-aminopyridine (4AP)7,8,9. This novel radiotracer is currently under investigation as a PET tracer for demyelination10,11,12. In this video article, we demonstrate the semi-automated synthesis of this compound using an IBA Synthera Synthesis Unit (henceforth referred to as &#34;the synthesizer&#34;) and an in-house made flow hydrogenation device. The synthesis is based on the reaction shown in Figure 1. Preparation for the procedure takes approximately 1 h, radiolabeling and purification 1.5 h and quality control procedures 0.5 h.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cyclotron produced [18F]fluoride</td>
			<td>House supplied/Zevacor</td>
			<td>IBA Cyclone 18</td>
			<td>100-200 mCi</td>
		</tr>
		<tr>
			<td>Integrated fluid processor for production FLT/FDG</td>
			<td>ABX</td>
			<td>K-2715SYN</td>
			<td>Cassette used for nucleophilic substitution</td>
		</tr>
		<tr>
			<td>Anhydrous acetonitrile</td>
			<td>Janssen</td>
			<td>36431-0010</td>
			<td>Transfer under nitrogen</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Janssen</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>ultrapure water</td>
			<td>house supplied</td>
			<td></td>
			<td>Millipore MilliQ system</td>
		</tr>
		<tr>
			<td>TBA-HCO3</td>
			<td>ABX</td>
			<td>808.0000.6</td>
			<td>abx.de</td>
		</tr>
		<tr>
			<td>QMA</td>
			<td>Waters</td>
			<td>WAT023525</td>
			<td>Quaternary methyl ammonium: Anion exchange solid phase extraction cartridge for trap and release of 18F- from the target water</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>ABX</td>
			<td>K-28XX.03</td>
			<td>Prefilled 5 mL syringes</td>
		</tr>
		<tr>
			<td>Alumina-N</td>
			<td>Waters</td>
			<td>WAT020510</td>
			<td>Alumina-N solid phase extraction cartridge (for trapping unreacted 18F-)</td>
		</tr>
		<tr>
			<td>3-fluoro-4-nitropyridine N-oxide</td>
			<td>Synthonix</td>
			<td>76954-0</td>
			<td>Store in desicator. Precursor</td>
		</tr>
		<tr>
			<td>3-fluoro-4-aminopyridine</td>
			<td>Sigma Aldrich</td>
			<td>704490-1G</td>
			<td>Reference standard</td>
		</tr>
		<tr>
			<td>Oxalic acid</td>
			<td>Sigma Aldrich</td>
			<td>75688-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Fisher Scientific&#160;</td>
			<td>S80191-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethyl amine</td>
			<td>Fisher Scientific&#160;</td>
			<td>04885-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>DSP-MD.43</td>
			<td>USP</td>
		</tr>
		<tr>
			<td>Final product vial</td>
			<td>ABX</td>
			<td>K28XX.04</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex Filter Syringe</td>
			<td>Millex</td>
			<td>SLGVR04NL</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Pd/C cartridge</td>
			<td>Sigma Aldrich</td>
			<td>THS-01111-12EA</td>
			<td></td>
		</tr>
		<tr>
			<td>11 mm vials + crimp seals</td>
			<td>Fisher Scientific&#160;</td>
			<td>03-250-618, 06-451-117, or equivalent</td>
			<td></td>
		</tr>
		<tr>
			<td>13 mm vials + crimp seals</td>
			<td>Fisher Scientific</td>
			<td>06-718-992, 06-718-643, or equivalent</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC vials</td>
			<td>Fisher Scientific</td>
			<td>03-391-16, 03-391-17, or equivalent</td>
			<td></td>
		</tr>
		<tr>
			<td>SEMIPREP C18 column</td>
			<td>Agilent</td>
			<td>990967-202</td>
			<td></td>
		</tr>
		<tr>
			<td>V-vials</td>
			<td>Alltech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes: 1, 3, 10 mL</td>
			<td>Fisher Scientific</td>
			<td>14-829-10D, 14-829-13Q, 14-829-18G, or equivalent</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressed gases: N2, He, H2</td>
			<td>Airgas</td>
			<td>UHP N300, UHP HE300, UHP H300, or equivalent</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC plates</td>
			<td>Sigma Aldrich</td>
			<td>Z193275, or equivalent</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synthera automated synthesizer</td>
			<td>IBA SA, Belgium, iba-worldwide.com</td>
			<td>Synthera, 250.001</td>
			<td>Automatic synthesis unit</td>
		</tr>
		<tr>
			<td>In-house hydrogenator</td>
			<td>See picture</td>
			<td></td>
			<td>See text description</td>
		</tr>
		<tr>
			<td>Hot cells</td>
			<td>Comecer</td>
			<td></td>
			<td>For manipulating radioactive materials</td>
		</tr>
		<tr>
			<td>RadioTLC scanner</td>
			<td>Eckert and Ziegler</td>
			<td></td>
			<td>For handling sterile materials</td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Dionex</td>
			<td>Ultimate 3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dose calibrator</td>
			<td>Capintec</td>
			<td>CRC15</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Gamma counter</td>
			<td>Capintec, 7 Vreeland Road, Florham Park, NJ 07932</td>
			<td>CRC 15, PET-CRC25, or equivalent</td>
			<td>For measuring radioactivity</td>
		</tr>
		<tr>
			<td>Personal dosimeters</td>
			<td>Packard</td>
			<td>Cobra II</td>
			<td>For measuring gamma spectrum</td>
		</tr>
		<tr>
			<td>Personal radiation badges and rings</td>
			<td>Atlantic Nuclear</td>
			<td>Rados Rad-60 Electronic Dosimeter, or equivalent</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotavap + vacuum pump</td>
			<td>Landauer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lead pigs + syringe shields</td>
			<td>Heidolph</td>
			<td></td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Geiger counters</td>
			<td>Pinestar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Ludlum&#160;</td>
			<td>Model 3 + Pancake GM detector, 4801605, 47-1539, or equivalent</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated radiochemical synthesis of [18f]3f4ap: a novel pet tracer for imaging demyelinating diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

The radiochemical synthesis of [18F]3F4AP comprises two steps (Figure 1). The first step is carried out in a fully automated fashion using the synthesis unit (Figure 3). This cassette-based system uses four reagent vials and one reactor vial and has computer-controlled valves that allow transfer and mixing of reagents as well as heating, pressurizing and evacuating the reactor. In addition, it supports standard solid-phase extraction cartridges for separation of reagents. The computer interface allows users to write and modify scripts in order to run their own syntheses. In the case of [18F]3F4AP, the synthesis procedure is comprised of five basic parts. In the first part, the synthesizer performs self-check steps, preheats the reactor and waits for operator&#39;s signal that the 18F is ready. During the second part, the [18F]fluoride is transferred from the 18F vial into the anion exchange cartridge and eluted from the cartridge into the reactor using a solution tetrabutyl ammonium bicarbonate. The third part, the synthesizer azeotropically dries the [18F]fluoride under vacuum to make it reactive towards nucleophilic displacement. In the fourth part, the precursor is automatically added to the reactor where it reacts with the 18F- to generate the labeled compound. Finally, the reaction is quenched by the addition of 0.2% oxalic acid in methanol, which prevents base-promoted decomposition of the product, and the final solution is pressure-transferred to the collection vial after passing through an alumina-N cartridge that traps any unreacted fluoride.
After the labeling step is completed a small sample can be taken for quality control. Running a sample on the HPLC provides confirmation that the labeling step worked and an estimation of the radiochemical purity (Figure 4). Also, from the UV trace on the HPLC the mass amount of product can be calculated using a pre-established calibration curve.
While the in-process quality control HPLC is running, the second reaction step, reduction of the N-oxide and nitro groups, is performed. In order to do this, the labeled product is automatedly injected into an in-house hydrogenation device based on the method published by Yoswathananont et al.13 (Figure 2). This device consists of an HPLC pump and a compressed hydrogen tank connected to the flow hydrogenation device through lines equipped with check valves to prevent back-streaming. The product is pushed by the HPLC pump and mixed with hydrogen in a T-shaped mixer. This mixture is then passed through a small cartridge containing 10% Pd/C catalyst on a solid support. After passing through the catalyst the reduced product is then collected in small fractions.
Following hydrogenation, the crude product is transported and manually injected into the HPLC for purification of the final product (Figure 5). The mobile phase of the HPLC has been selected to be compatible with animal injection. The peaks corresponding to the product are then collected and filtered-sterilized to obtain the final dose.
Prior to releasing the dose for PET imaging studies, quality control tests are performed. These tests are performed to ensure that the tracer is the chemical entity that it is supposed to be and that it is safe for injection. Some of these tests may not be required for injection into animals but it is generally recommended to follow the human use guidelines. Doing so ensures quality of the product, which increases confidence in the results and greatly facilitates future transition to manufacturing the product for human injection.
Table 1 contains the typical synthesis parameters including initial amount radioactivity, initial amount of precursor, yield for each step, specific activity, filtering loses, etc. These parameters are useful troubleshooting occasional failures and future optimization of the procedure.
<img alt="Figure 1" src="/files/ftp_upload/55537/55537fig1.jpg" /> 
Figure 1. Reaction scheme. Radiochemical synthesis consists of labeling by 19F/18F exchange followed by palladium-catalyzed hydrogenation. <a href="http://cloudflare.jove.com/files/ftp_upload/55537/55537fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55537/55537fig2.jpg" /> 
Figure 2. Hydrogenation system. Schematic of the device. This device is based on the publication by Yoswathananont et al. (ref 13).&#160;
<img alt="Figure 3" src="/files/ftp_upload/55537/55537fig3.jpg" /> 
Figure 3. Scheme of synthesizer integrated fluidic processor (IFP) and reagents. IFP contains four reagent vials, a QMA cartridge and one reactor vial. <a href="http://cloudflare.jove.com/files/ftp_upload/55537/55537fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a> 
&#160;
<img alt="Figure 4" src="/files/ftp_upload/55537/55537fig4.jpg" /> 
Figure 4. UV and radioHPLC tracers for intermediate product. 3-fluoro-4-nitropyridine N-oxide has a characteristic absorption at 313 nm. <a href="http://cloudflare.jove.com/files/ftp_upload/55537/55537fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55537/55537fig5.jpg" /> 
Figure 5. UV and radioHPLC tracers for final product. 3-fluoro-4-aminoopyridine absorbs at a 254 nm. <a href="http://cloudflare.jove.com/files/ftp_upload/55537/55537fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Concept</td>
			<td>Mean (n = 4)</td>
			<td>S.D.</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Initial 18F activity (mCi)</td>
			<td>148.0</td>
			<td>44.9</td>
			<td>Start of synthesis</td>
		</tr>
		<tr>
			<td>Precursor amount (&#956;g)</td>
			<td>50</td>
			<td></td>
			<td>Use 50 &#956;L of 1.0 mg/mL stock</td>
		</tr>
		<tr>
			<td>Activity left in QMA (mCi)</td>
			<td>3.0</td>
			<td>1.7</td>
			<td>Measured at the end of labeling step</td>
		</tr>
		<tr>
			<td>Radiolabeling yield</td>
			<td>29.7%</td>
			<td>6.3%</td>
			<td>Act_collection_vial &#247; (Act_collection_vial + Act_AluN)</td>
		</tr>
		<tr>
			<td>Radiochemical purity (HPLC-1)</td>
			<td>&#62; 98%</td>
			<td></td>
			<td>From HPLC-1 QC</td>
		</tr>
		<tr>
			<td>Spec. act. intermediate (mCi/&#956;mol)</td>
			<td>122.9</td>
			<td>29.7</td>
			<td>From HPLC-1 using calibration curve</td>
		</tr>
		<tr>
			<td>Hydrogenation recovery (d.c.)</td>
			<td>74%</td>
			<td>9.0%</td>
			<td>Corrected for decay</td>
		</tr>
		<tr>
			<td>HPLC radiochemical purity (HPLC-2)</td>
			<td>90.7%</td>
			<td>2.9%</td>
			<td>Calculated from HPLC-2</td>
		</tr>
		<tr>
			<td>Drying efficiency</td>
			<td>&#62; 98%</td>
			<td></td>
			<td>Corrected for decay</td>
		</tr>
		<tr>
			<td>Filtering recovery</td>
			<td>93.5%</td>
			<td>1.7%</td>
			<td>Corrected for decay</td>
		</tr>
		<tr>
			<td>Dose volume (mL)</td>
			<td>3.3</td>
			<td></td>
			<td>Collect fractions with highest radioactivity</td>
		</tr>
		<tr>
			<td>Spec. act. final product (mCi/&#181;mol)</td>
			<td>75.5</td>
			<td>30.0</td>
			<td>From HPLC-3 using calibration curve</td>
		</tr>
		<tr>
			<td>Synthesis efficiency</td>
			<td>8.5%</td>
			<td>3.6%</td>
			<td>Non-decay corrected</td>
		</tr>
		<tr>
			<td>Synthesis time (min)</td>
			<td>104</td>
			<td>11.2</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Radiochemical synthesis parameters.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Common problems</td>
			<td>Potential reasons and solutions</td>
		</tr>
		<tr>
			<td rowspan="3">[18F]fluoride is not efficiently eluted from the QMA</td>
			<td>&#183; TBA-HCO3 was not prepared correctly. Ensure the concentration is adequate.</td>
		</tr>
		<tr>
			<td>&#183; There are leaks on the TBA-HCO3 vial. Make sure the crimp seal is tight and the septum is not pierced prior to installing it on the IFP.</td>
		</tr>
		<tr>
			<td>&#183; TBA-HCO3 is not in good condition. Order a fresh batch.</td>
		</tr>
		<tr>
			<td rowspan="2">Labeling yield is low</td>
			<td>&#183; There is moisture in the precursor solution. Dry precursor and solvents.</td>
		</tr>
		<tr>
			<td>&#183; Temperature is too low.</td>
		</tr>
		<tr>
			<td rowspan="3">Reaction solution is yellow</td>
			<td>&#183; The product is decomposing due to base. Use less TBA-HCO3.</td>
		</tr>
		<tr>
			<td>&#183; There is too much precursor. Use less precursor.</td>
		</tr>
		<tr>
			<td>&#183; There is too little solvent for the amount of 18F-. Use more solvent.</td>
		</tr>
		<tr>
			<td>Additional peaks on radioHPLC</td>
			<td>&#183; Nitro group is being substituted: reduce the reaction temperature or shorten reaction time.</td>
		</tr>
		<tr>
			<td rowspan="3">Hydrogenation reaction does not work</td>
			<td>&#183; Catalyst is not good. Use a new cartridge.</td>
		</tr>
		<tr>
			<td>&#183; Flow is too fast and does not allow sufficient contact between catalyst and substrate. Decrease flow.</td>
		</tr>
		<tr>
			<td>&#183; Hydrogen pressure is too low. Increase H2 pressure.</td>
		</tr>
		<tr>
			<td>Hydrogen pressure increases dramatically during procedure</td>
			<td>&#183; Cartridge integrity is compromised and solid support is clogging the lines. Stop the flow and shut off the gas. Let radioactivity decay. Remove catalyst cartridge and flush the system. Put a new cartridge.</td>
		</tr>
		<tr>
			<td>Hydrogenation yield is low</td>
			<td>&#183; Too many impurities competing for the catalyst (MeCN, oxalic acid). Decrease amount of impurities or increase mass of precursor (Warning: increasing precursor amount will reduce specific activity).</td>
		</tr>
		<tr>
			<td rowspan="2">Recovery of radioactivity from hydrogenation step is low</td>
			<td>&#183; There is a leak in the system. Check for leaks and backflush into the hydrogen line.</td>
		</tr>
		<tr>
			<td>&#183; Compound is defluorinating in the reactor. Evaluate different reaction conditions (pressure, temperature, flow, etc.).</td>
		</tr>
		<tr>
			<td rowspan="2">Too much radioactivity is lost during filtration</td>
			<td>&#183; Wet the filter prior to use.</td>
		</tr>
		<tr>
			<td>&#183; Use filter with a lower dead volume.</td>
		</tr>
		<tr>
			<td rowspan="4">The final product peak on the HPLC looks broad</td>
			<td>&#183; Too much volume injected. Inject lower amount. Use column with larger diameter.</td>
		</tr>
		<tr>
			<td>&#183; The column is not well conditioned. Condition the column for at least 30 column volumes.</td>
		</tr>
		<tr>
			<td>&#183; pH of the mobile phase is low. Make sure that the pH &#8805; 8.</td>
		</tr>
		<tr>
			<td>&#183; Column is not in good condition. Replace column. Use column compatible with basic pH.</td>
		</tr>
	</tbody>
</table>
Table 2. Troubleshooting guide.

Watch this Scientific Journal Video about Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases at JoVE.com

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

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9.9K Views.  The University of Chicago. We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

Video: Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases

Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls. 	
DTI data analysis is performed in a variate fashion, i.e. voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies. The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e. differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders. Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels.	
In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis.

Diffusion tensor imaging (DTI) basically serves as an MRI-based tool to identify in vivo the microstructure of the brain and pathological processes due to neurological disorders within the cerebral white matter. DTI-based analyses allow for application to brain diseases both at the group level and in single subject data.

Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo. The present ...

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...