Funding support for this research was provided the National Institutes of Health (NIH) and National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/NIH R01-DK-105371. We thank Dr. Houchun (Harry) Hu for advice and suggestions on fat water phantom creation.

1. Prepare the Workstation and Materials
<ol>
	<li>Adhere to all laboratory safety rules. Wear eye protection and gloves. Read the material safety data sheet for each of the reagents used and take appropriate precautions. Review the materials and equipment list, chemical handling procedures, and glassware precautions. 
	Caution: This protocol requires the use of a hotplate at high temperatures. Use caution and wear heat resistant gloves when interacting with hot containers and do not touch the surface of the hotpl...

<ol>
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A., 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=Liver+Fat+Quantification+by+Dual-echo+MR+Imaging+Outperforms+Traditional+Histopathological+Analysis.">Liver Fat Quantification by Dual-echo MR Imaging Outperforms Traditional Histopathological Analysis.</a> Acad Radiol. 19 (10), 1208-1214 (2012).</li><li>Hayashi, T., 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=Influence+of+Gd-EOB-DTPA+on+proton+density+fat+fraction+using+the+six-echo+Dixon+method+in+3+Tesla+magnetic+resonance+imaging.">Influence of Gd-EOB-DTPA on proton density fat fraction using the six-echo Dixon method in 3 Tesla magnetic resonance imaging.</a> Radiol Phys Technol. , (2017).</li><li>Hines, C. D. G., Yu, H., Shimakawa, A., McKenzie, C. A., Brittain, J. H., Reeder, S. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Echo+Water-Fat+Separation+and+Simultaneous+R2*+Estimation+with+Multi-Frequency+Fat+Spectrum+Modeling.">Multi-Echo Water-Fat Separation and Simultaneous R2* Estimation with Multi-Frequency Fat Spectrum Modeling.</a> Spectrum. 60 (5), 1122-1134 (2011).</li><li>Peri, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The extra-virgin olive oil handbook. , (2014).</li><li>Kell, G. 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We describe a robust method to create fat water phantoms suitable for validating the medical imaging techniques used to quantify adipose tissue and triglyceride content in vivo. By creating two reservoirs (one for the oil solution and one for the water solution), stable phantoms with a variety of FF values &#8211; including values exceeding 50% &#8211; were constructed without the need for expensive equipment. High FF phantoms (&#62;50%) provide the utility to ensure imaging techniques for adipose quantification...

Interest in quantifying adipose tissue and triglyceride content using imaging modalities, such as magnetic resonance imaging (MRI), extends across many fields. Research areas include the investigation of white and brown adipose tissue depots and ectopic storage of lipid in organs and tissues such as the liver1, pancreas2, and skeletal muscle3. As these novel techniques for adipose quantification are developed, methods are needed to confirm that imaging parameters are valid for research and clinical applications.
Phantoms, experimental replicas of a tissue or organ, provide a low-cost, flexible, and controlled tool to develop and validate imaging techniques4. Specifically, phantoms can be constructed to consist of fat and water in a volume ratio or fat fraction (FF) comparable to that of the tissue of clinical interest. Clinically, FF values in tissues and organs can vary widely: FF in brown adipose tissue falls between 29.7% and 93.9%5; the average liver FF in steatosis patients is 18.1 &#177; 9.0%6; the pancreatic FF in adults at risk for type 2 diabetes ranges between 1.6% and 22.2%7; and in some cases of advance disease, patients with Duchenne muscular dystrophy can have FF values of almost 90% in some muscles8.
Because non-polar molecules such as lipids do not dissolve well in solutions composed of polar molecules such as water, creating stable phantoms with a high target FF remains challenging. For FF up to 50%, many existing methods can be used to create fat water phantoms9,10,11,12. Other methods that achieve higher FFs typically require expensive equipment such as a homogenizer or an ultrasonic cell disruptor13,14. Although these techniques provide a roadmap for high FF phantoms, equipment constraints and varying amounts of experimental details limit the efforts to create reproducible and robust fat water phantoms.
Building upon these previous techniques, we developed a method to construct cost-effective and stable fat water phantoms across a customizable range of FF values. This protocol details the steps needed to make 5x 100 mL of fat phantoms with FF values of 0%, 25%, 50%, 75%, and 100% using a single hotplate. It can easily be adjusted to create various volumes (10 to 200 mL) and fat percentages (0 to 100%). The efficacy of the phantom technique was evaluated in the feasibility study comparing fat-water MRI FF values to the target FF values in the constructed phantoms.

<table>
	<tbody>
		<tr>
			<td>Distilled Water</td>
			<td>Amazon</td>
			<td>B000P9BY38</td>
			<td>Base of water solution</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>A1296-100G</td>
			<td>Gelling agent</td>
		</tr>
		<tr>
			<td>Water-Soluble Surfactant</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>P1379-500ML</td>
			<td>Surfactant/emulsifying agent</td>
		</tr>
		<tr>
			<td>Gadolinium-DTPA Contrast Agent</td>
			<td>Bayer Healthcare</td>
			<td>50419-0188-01</td>
			<td>Magnetic Resonance Imaging Contrast Agent.</td>
		</tr>
		<tr>
			<td>Sodium Benzoate</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>71300-250G</td>
			<td>Preservative</td>
		</tr>
		<tr>
			<td>Peanut Oil</td>
			<td>Amazon</td>
			<td>54782-LOU</td>
			<td>Base of oil solution</td>
		</tr>
		<tr>
			<td>Oil-Soluble Surfactant</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>S6760-250ML</td>
			<td>Surfactant/emulsifying agent</td>
		</tr>
		<tr>
			<td>Hotplate w/ Stirrer</td>
			<td>Fisher Scientific</td>
			<td>07-770-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir bars (Egg-Shaped)</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>Z127116-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>400 mL Beaker</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>CLS1003400-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Erlenmeyer Flask</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>CLS4450250-6EA</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Glass Volumetric Pipette</td>
			<td>Fisher Scientific</td>
			<td>13-650-2P</td>
			<td>Quantity = 2</td>
		</tr>
		<tr>
			<td>50 mL Glass Volumetric Pipette</td>
			<td>Fisher Scientific</td>
			<td>13-650-2S</td>
			<td>Quantity = 2</td>
		</tr>
		<tr>
			<td>75 mL Glass Volumetric Pipette</td>
			<td>Fisher Scientific</td>
			<td>13-650-2T</td>
			<td>Quantity = 2</td>
		</tr>
		<tr>
			<td>3.0 mL Syringe</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>Z248002-1PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 mL Syringe</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>Z230723-1PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>S3897-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale (100g X 0.01g Resolution)</td>
			<td>Amazon</td>
			<td>AWS-100-BLK</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigh Boats</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>Z740499-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>120 mL Glass Jars</td>
			<td>McMaster Carr Supply Co</td>
			<td>3801T73</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Resistant Gloves (pair)</td>
			<td>Amazon</td>
			<td>B075GX43MN</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Needles</td>
			<td>Sigma Aldrich Incorporated</td>
			<td>Z192341-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>18&#34; stir bar retriver</td>
			<td>Fisher Scientific</td>
			<td>14-513-70</td>
			<td></td>
		</tr>
		<tr>
			<td>1 Dram Clear Glass Vial</td>
			<td>Fisher Scientific</td>
			<td>03-339-25B</td>
			<td></td>
		</tr>
	</tbody>
</table>

fat-water phantoms for magnetic resonance imaging validation: a flexible and scalable protocol

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental replicas of a tissue or organ of interest, provide a low cost, flexible solution. However, without access to expensive and specialized equipment, constructing stable phantoms with high fat fractions (e.g., &#62;50% fat fraction levels such as those seen in brown adipose tissue) can be difficult due to the hydrophobic nature of lipids. This work presents a detailed, low cost protocol for creating 5x 100 mL&#160;phantoms with fat fractions of 0%, 25%, 50%, 75%, and 100% using basic lab supplies (hotplate, beakers, etc.) and easily accessible components (distilled water, agar, water-soluble surfactant, sodium benzoate, gadolinium-diethylenetriaminepentacetate (DTPA) contrast agent, peanut oil, and oil-soluble surfactant). The protocol was designed to be flexible; it can be used to create phantoms with different fat fractions and a wide range of volumes. Phantoms created with this technique were evaluated in the feasibility study that compared the fat fraction values from fat-water magnetic resonance imaging to the target values in the constructed phantoms. This study yielded a concordance correlation coefficient of 0.998 (95% confidence interval: 0.972-1.00). In summary, these studies demonstrate the utility of&#160;fat phantoms for validating adipose tissue imaging techniques across a range of clinically relevant tissues and organs.

If the water solution has been prepared correctly, a small amount of the solution should congeal quickly in a test vial (Figure 1, left). If the solution separates (Figure 1, right), the solution should be prepared again (as instructed in step 3.8 of the protocol). If the emulsion separates (examples in Figure 2, left and right), the phantom is not v...

Watch this Scientific Journal Video about Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol at JoVE.com

Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol

The purpose of this work is to describe a protocol for creating a practical fat-water phantom that can be customized to produce phantoms with varying fat percentages and volumes.

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental ...

This method can help to optimize magnetic resonance imaging sequences and sequence parameters for quantitative fat-water imagining of tissues with high fat fractions. The main advantage of this technique is that it can be performed using commonly available lab supplies that can be purchased at a relatively low cost. Generally, individuals new to this method will struggle because creating stable phantoms with fat fractions greater than 50%is difficult due to the hydrophobic nature of the lipids.To prepare the water solution, first place a stir bar into a 400 milliliter beaker. Then, use a 100 or 200 milliliter graduated cylinder to measure 300 milliliters of distilled water, and pour the water into the beaker. Place the beaker on the hot plate and set at 90 degrees Celsius with a stir rate of 100 RPM.Use a calibrated scale to measure 0.30 grams of sodium benzoate into a weigh boat. Add the sodium benzoate to the water solution. Next, use a syringe to measure 0.6 milliliters of the water soluble surfactant.Make sure there are no air bubbles. Hold the needle a few milliliters over the center of the solution, and slowly release the water soluble surfactant to avoid splatter on the walls of the beaker. Using a clean syringe, measure 0.24 milliliters of the gadolinium DTPA contrast agent.Add it to the beaker using the same technique. Then, slowly spoon the 9.0 grams of agar with a spatula into the beaker with water. Once everything has been added to the water solution, increase the hot plate temperature to 350 degrees Celsius and stir bar speed to 1100 RPM for five to 10 minutes to melt the agar.To check if the agar is melted, briefly remove the water solution from the hot plate, stop stirring, and check the color of the solution. Melted agar should be clear and yellow or amber in color. Once the agar is fully melted, remove the water solution beaker from the hot place and pour about 3.5 milliliters of the water solution into a one dram vial.Check the solution to ensure it is clear and homogenous. Leave the water solution on the hot place at 50 degrees Celsius and 100 RPM. Clean the workspace and prepare for the oil solution.Peanut oil is used for the oil solution because it has a similar nuclear magnetic resonance spectrum as compared to triglycerides and human adipose tissue. Place a new stir bar into a clean 400 milliliter beaker. Use a graduated cylinder to measure 300 milliliters of peanut oil, and pour into the beaker.Remove the beaker containing the water solution and place the oil solution beaker on the hot plate. Set the hot plate to 90 degrees Celsius with a stir of 100 RPM for one minute. Do not leave the oil on the hot plate unattended.Measure 3.0 milliliters of the oil soluble surfactant with a clean syringe. Using the same technique as before, add the oil soluble surfactant to the beaker. Set the hot plate to 150 degrees Celsius and 1100 RPM for five minutes to fully mix the oil solution.Take the oil solution off the hot place and clean the workspace in preparation for creating the phantom. To create the phantom emulsion, prepare volumetric pipettes for the oil and water solution. Volumetric pipettes should only be used for the respective solution to prevent cross-contamination.Match the size of the pipette to the volume being used in the protocol. For example, use two 50 milliliter volumetric pipettes to create a 100 milliliter phantom with a target fat fraction of 50%fat. Place the water solution on the hot plate and set the hot plate to 300 degrees Celsius and 1100 RPM.After four to five minutes, turn the stir off. Using a volumetric pipette, check if the water solution is ready for extraction by partially filling the pipette with a small amount of the solution and releasing it back into the beaker. If the water solution can be easily removed and released without excessive remnants in the pipette, move onto the next step.Otherwise, leave it on the hot plate and check again in two to three minutes. Now, carefully add a clean stir bar to a 250 milliliter Erlenmeyer flask. Take the water solution off the hot plate, measure the proper volume, and transfer it to the Erlenmeyer flask.Place the oil solution on the hot plate and set at 90 degrees Celsius and 1100 RPM to ensure the solution is homogenous. After one to two minutes, remove the oil solution from the hot plate and replace it with the Erlenmeyer flask. Measure the proper amount of the oil solution and slowly add to the water solution into the Erlenmeyer flask.Now you'll be able to find if your previous steps in creating the emulsion have worked. Once all oil solution has been added, increase the temperature to 300 degrees Celsius and maintain the stirring at 1100 RPM for four to five minutes. There should be a vortex from the stir bar.The emulsion should be white with a creamy texture. Now, remove the stir bar with a magnetic stir bar retriever. Use heat resistant gloves to carefully pour the mixture in the Erlenmeyer flask into a clean 120 milliliter glass jar.Slowly pour the mixture down the side of the glass jar to prevent bubbles in the mixture as it cools. After cleaning the Erlenmeyer flask and stir bar, repeat these steps, adjusting the amounts of water and oil solutions, until all phantoms are created. Successful phantoms will congeal to form a homogenous mixture, which can be imaged and measured via magnetic resonance imaging, or MRI.Shown here are pictures of slight color differences in the constructed phantoms, with fat fractions of 0%25%50%75%and 100%Proton density fat signal fraction, or FSF maps, obtained from magnetic resonance imaging, reveal a homogenous FSF measurement similar to the target fat content. A high concordance correlation coefficient and the inclusion of the line of identity within the 95%confidence band of the regression line suggests the mean MRI-observed FSF values did not differ significantly from the known FF values in the fat water phantoms. We first had the idea for this method when we attempted to make some of our own high fat fraction phantoms for our studies of human brown adipose tissue, and we found that many of the methods described in the literature were either incompletely described or required expensive equipment.After watching this video, you should have a good idea how to create phantoms with fat fractions greater than 50%While attempting this procedure, it is critical to have accurately measured the amount of oil and water solution that will be used in the emulsion. This is important because the ratio ultimately determines the fat fraction of the phantom.

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11.0K visualizações.  Vanderbilt University Medical Center. This method can help to optimize magnetic resonance imaging sequences and sequence parameters for quantitative fat-water imagining of tissues with high fat fractions. The main advantage of this technique is that it can be performed using commonly available lab supplies that can be purchased at a relatively low cost. Generally, individuals new to this method will struggle because creating stable phantoms with fat fractions greater than 50%is difficult due to the hydrophobic nature of the lipid...