Name: Anisotropic Polyvinyl Alcohol Phantom Fabrication for Ultrasound Elastography: Procedure and Quality Controls (Video) | JoVE Journal
Uploaded: 2026-06-05T16:50:00.000+00:00
Duration: 9 min 59 s
Description: Learn to fabricate anisotropic polyvinyl alcohol phantoms for ultrasound elastography validation using controlled freeze-thaw cycles and precise stretching techniques. This protocol ensures reproducible tissue-mimicking models with direction-dependent mechanical properties.

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5 minutes

This study was supported by the Beijing Natural Science Foundation (No. 1254038). The authors would like to thank Prof. Wei-Ning Lee from the University of Hong Kong for her help with the ultrasound shear wave imaging test.

NOTE: The following steps are illustrated in Figure 1.
1. 3D printing of phantom mold
<ol>
	<li>Using 3D modeling software, design the phantom geometry to incorporate two holding rings (one at each end) for maintaining stretch, with an effective central region of 35 mm &#215; 60 mm &#215; 5 mm (length &#215; width &#215; height) to ensure uniform anisotropy induction.</li>
	<li>Print the mold using a 3D printer. 
	NOTE: The holding rings prevent slippage during stretching, ensuring consistent strain distribution. All 3D printing files (.stl) are provided in Supplemental File 1.</li>
</ol>
2. Preparation of PVA solution
<ol>
	<li>Weigh PVA powder (10 wt%), potassium sorbate (1 wt%), and graphite particles (1 wt%, ~6.5 &#181;m diameter), and measure the corresponding volume of pure water (deionized water is recommended) to prepare a batch volume of ~200 mL.</li>
	<li>Combine PVA powder and pure water in a beaker, place the beaker on a magnetic stirrer equipped with a stir bar, cover the beaker with aluminum foil, and stir at ~600 rpm at room temperature for 10 min until PVA is fully dispersed.</li>
	<li>Transfer the beaker to an 80 &#176;C water bath and continue heating until PVA is completely dissolved (evidenced by a clear, particle-free solution). 
	NOTE: Wear heat-resistant gloves and eye protection gear during heating to prevent thermal burns. Ensure no undissolved PVA particles remain, as they will compromise phantom homogeneity.</li>
</ol>
3. Addition of additives and casting
<ol>
	<li>Cool the solution to ~40 &#176;C while continuing to stir at ~600 rpm on the magnetic stirrer.</li>
	<li>Remove the aluminum foil, add potassium sorbate and graphite particles, re-cover with foil, and stir at ~600 rpm until the additives are uniformly dispersed (visually confirm homogeneous distribution).</li>
	<li>(Optional) Degas the PVA solution to remove residual air bubbles. Place the beaker containing the mixed solution in a vacuum chamber and apply a vacuum for approximately 30 min to remove air bubbles from the PVA solution.</li>
	<li>Cast the solution into the 3D-printed mold and assemble the mold using screws. 
	NOTE: Avoid introducing air bubbles during casting. Gently tap the mold if necessary to eliminate trapped air bubbles inside the solution.</li>
</ol>
4. Initial FTC for PVA solidification
<ol>
	<li>Place the mold in a &#8722;20 &#176;C freezer for 12 h.</li>
	<li>Remove the mold from the freezer and thaw at room temperature (20 &#176;C) for 12 h.</li>
	<li>Carefully demold the phantom to prevent damage. 
	NOTE: This step forms an isotropic base phantom, providing a stable foundation for subsequent anisotropy induction.</li>
</ol>
5. Inducing anisotropy via stretched FTCs
<ol>
	<li>Place the phantom on the custom-made stretching device, gradually stretch it to 180% of its effective length (80% tensile strain), measure the stretching distance manually, and lock the device to prevent retraction.</li>
	<li>Transfer the entire stretching device (with the phantom secured) into a &#8722;20 &#176;C freezer for 12 h, followed by thawing at room temperature (20 &#176;C) for 12 h.</li>
	<li>Repeat this FTC cycle once more under sustained stretching. 
	NOTE: The custom-made stretching device is made of a commercially available bidirectional, opposite-thread linear stage assembled with two 3D-printed phantom-holding mounts, for which the STL files are provided in Supplemental File 1. Wear insulated cold-protective gloves to avoid frostbite when removing the stretching device from the &#8722;20 &#176;C freezer. Sustained stretching during FTCs aligns PVA molecular chains unidirectionally. Total FTCs employed in this study: one stretch-free cycle + two stretched cycles (80% tensile strain).</li>
</ol>
6. Final release and storage
<ol>
	<li>Following the final FTC thawing step, gently release the stretching device to prevent recoil-induced damage.</li>
	<li>Remove the phantom from the stretching device. The anisotropic PVA phantom is now complete.</li>
	<li>Store the phantom immersed in water inside a sealed plastic container at 4 &#176;C.</li>
</ol>

<ol>
	<li>Fung, Y. C. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomechanics: mechanical properties of living tissues. , (2013).</li><li>Sherwood, L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamentals of human physiology. , (2011).</li><li>Stiver, M. L., Mirjalili, S. A., Agur, A. M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+shear+wave+velocity+in+adult+skeletal+muscle+with+ultrasound+2-D+shear+wave+elastography:+a+scoping+review.">Measuring shear wave velocity in adult skeletal muscle with ultrasound 2-D shear wave elastography: a scoping review.</a> Ultrasound Med Biol. 49 (6), 1353-1362 (2023).</li><li>Bercoff, J., Tanter, M., Fink, M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supersonic+shear+imaging:+a+new+technique+for+soft+tissue+elasticity+mapping.">Supersonic shear imaging: a new technique for soft tissue elasticity mapping.</a> IEEE Trans Ultrason Ferroelectr Freq Control. 51 (4), 396-409 (2004).</li><li>Wang, M., Byram, B., Palmeri, M., Rouze, N., Nightingale, K. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+transverse+isotropic+properties+of+muscle+by+monitoring+acoustic+radiation+force+induced+shear+waves+using+a+2-D+matrix+ultrasound+array.">Imaging transverse isotropic properties of muscle by monitoring acoustic radiation force induced shear waves using a 2-D matrix ultrasound array.</a> IEEE Trans Med Imaging. 32 (9), 1671-1684 (2013).</li><li>Gennisson, J. L., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+and+anisotropic+mechanical+properties+of+in+vivo+muscle+tissue+assessed+by+supersonic+shear+imaging.">Viscoelastic and anisotropic mechanical properties of in vivo muscle tissue assessed by supersonic shear imaging.</a> Ultrasound Med Biol. 36 (5), 789-801 (2010).</li><li>Chatelin, S., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+polyvinyl+alcohol+hydrogel+phantom+for+shear+wave+elastography+in+fibrous+biological+soft+tissue:+a+multimodality+characterization.">Anisotropic polyvinyl alcohol hydrogel phantom for shear wave elastography in fibrous biological soft tissue: a multimodality characterization.</a> Phys Med Biol. 59 (22), 6923 (2014).</li><li>Millon, L., Mohammadi, H., Wan, W. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+polyvinyl+alcohol+hydrogel+for+cardiovascular+applications.">Anisotropic polyvinyl alcohol hydrogel for cardiovascular applications.</a> J Biomed Mater Res B Appl Biomater. 79 (2), 305-311 (2006).</li><li>Dong, J., Lee, W. N. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+numerical-model-based+optimization+strategy+for+design+and+fabrication+of+transversely+isotropic+tissue-mimicking+phantoms+[poster].">A numerical-model-based optimization strategy for design and fabrication of transversely isotropic tissue-mimicking phantoms [poster].</a> , (2022).</li><li>Montaldo, G., Tanter, M., Bercoff, J., Benech, N., Fink, M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+plane-wave+compounding+for+very+high+frame+rate+ultrasonography+and+transient+elastography.">Coherent plane-wave compounding for very high frame rate ultrasonography and transient elastography.</a> IEEE Trans Ultrason Ferroelectr Freq Control. 56 (3), 489-506 (2009).</li><li>Nitta, N., Yamakawa, M., Hachiya, H., Shiina, T. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+physical+and+engineering+factors+potentially+affecting+shear+wave+elastography.">A review of physical and engineering factors potentially affecting shear wave elastography.</a> J Med Ultrason. 48 (4), 403-414 (2021).</li></ol>

The authors have no conflicts of interest to declare.

The success of this protocol relies on achieving complete, uniform PVA dissolution and maintaining sustained stretching during FTCs—two critical steps for inducing stable, homogeneous mechanical anisotropy. The recipe used herein (i.e., PVA concentration, FTC number, and applied tensile strain) was selected to fabricate anisotropic PVA phantoms with stiffness within the physiological range of muscle tissue. Notably, the stiffness and anisotropy ratio of the PVA phantom can be readily tuned by adjusting these recipe param...

Certain biological soft tissues (e.g., skeletal muscle) exhibit macroscopic mechanical anisotropy, which originates from their aligned microstructural architecture1. Specifically, connective tissues organize muscle fibers into a hierarchical structure: endomysium surrounds individual fibers, perimysium bundles fibers into fascicles, and epimysium encapsulates the entire muscle, supporting force transmission and nutrient supply2. Ultrasound-based elastography techniques, such as shear wave imaging (SWI), are widely used for muscle assessment3,4. However, the anisotropic mechanical properties of muscle theoretically result in direction-dependent shear wave propagation5, which complicates the quantification of muscle stiffness6. Mechanically anisotropic tissue-mimicking phantoms are essential for advancing ultrasound elastography in muscle assessment.
Polyvinyl alcohol (PVA) is an ideal material for such phantoms, as maintaining mechanical stretch during freeze/thaw cycles (FTCs) aligns PVA molecular chains, thereby inducing mechanical anisotropy7,8. Conventional approaches that use simple PVA cuboids under stretching often lead to inhomogeneous strain due to surface slippage, thereby compromising reproducibility. This limitation can be addressed by employing an optimized phantom geometry with end holding rings, which ensures reliable stretching and uniform strain distribution9&#8212;markedly enhancing the quality of anisotropic PVA phantoms. In this study, we provide a detailed visual demonstration of the fabrication of such anisotropic PVA phantoms and validate their mechanical anisotropy via both SWI and uniaxial tensile testing.

<table><tbody><tr><td>Analytical balance</td><td>OHAUS</td><td>AX224</td><td>Analytical balance with internal calibration, capacity 220 g, readability 0.1 mg</td></tr><tr><td>Bidirectional, opposite-thread linear stage</td><td>Taobao</td><td>N/A</td><td>Travel range 250 mm for each thread, used for constructing the custom-made stretching device</td></tr><tr><td>FDM 3D Printer</td><td>Bambu Lab</td><td>X1 Carbon</td><td>Used for printing the phantom mold, as well as the phantom?holding mounts for the custom-made stretching device</td></tr><tr><td>FreeCAD</td><td>FreeCAD Open-Source Community</td><td>N/A</td><td>Version 0.22.0, used for designing the phantom mold and the phantom-holding mounts of the custom-made stretching device</td></tr><tr><td>Graphite particles</td><td>MREDA</td><td>M151649</td><td>Graphite particles, 99.95% purity, 2000 mesh(~6.5 um), used as acoustic scatterers, 1% by weight</td></tr><tr><td>Magnetic stir bar</td><td>Corning</td><td>401435</td><td>PTFE-coated magnetic stir bar, 1 x 3/8 inch, for stirring PVA solution</td></tr><tr><td>Magnetic stirrer</td><td>IKA</td><td>25005927</td><td>RCT basic safety control magnetic stirrer with heating function</td></tr><tr><td>Polyvinyl alcohol</td><td>MACKLIN</td><td>P81584</td><td>Polyvinyl alcohol, Mw 89000-98000, 95% hydrolyzed, used as phantom matrix, 10% by weight</td></tr><tr><td>Potassium sorbate</td><td>Sigma-Aldrich</td><td>85520</td><td>Potassium sorbate,&amp;ge;99.0% purity, used as preservative, 1% by weight</td></tr><tr><td>Rotation stage</td><td>Newport</td><td>URS50BPP</td><td>Used for rotating the ultrasound probe in shear wave imaging test for multipe probe angles (0&amp;deg;:2&amp;deg;:90&amp;deg;)</td></tr><tr><td>Single column universal testing machine</td><td>Instron</td><td>Model 5944</td><td>Used for uniaxial tensile testing, 50 N force sensor</td></tr><tr><td>Research Ultrasound System</td><td>Verasonics</td><td>Vantage 256&amp;nbsp;</td><td>Used for conducting shear wave imaging test, L7-4 probe (center frequency: 5.2 MHz)</td></tr><tr><td>Water bath</td><td>LICHEN</td><td>HH-2</td><td>Digital display constant temperature water bath, 2 holes single row, for heating and dissolving PVA solution</td></tr></tbody></table>

anisotropic polyvinyl alcohol phantom fabrication for ultrasound elastography: procedure and quality controls

Anisotropic Polyvinyl Alcohol Phantom Fabrication for Ultrasound Elastography: Procedure and Quality Controls

Polyvinyl alcohol (PVA) phantoms are typically isotropic and are widely used to validate ultrasound elastography techniques for soft-tissue evaluation. However, some biological soft tissues (e.g., skeletal muscle) exhibit distinct mechanical anisotropy, which necessitates the use of anisotropic PVA phantoms for the rigorous testing of elastography methods targeting these tissues. While prior studies have noted that in-house anisotropic PVA phantoms can be fabricated via stretch-integrated freeze/thaw cycles (FTCs), critical technical details (e.g., fabrication process, quality control) remain insufficiently documented. This work presents a visually detailed, reproducible protocol for fabricating anisotropic PVA phantoms, focusing on key materials, stepwise processes, and quality controls to induce stable, uniform anisotropy. Key materials include PVA as the phantom matrix, potassium sorbate as a preservative, graphite particles as acoustic scatterers, and pure water (or deionized water) as the solvent. The fabrication process comprises three core stages: 1) Preparation of a homogeneous PVA-based solution through controlled thermal conditions to ensure complete PVA dissolution; 2) Solidification via FTCs: the cooled solution is poured into 3D-printed molds, followed by stretch-free FTCs to form a preliminary structure; 3) Inducing anisotropy via stretched FTCs: additional FTCs are performed under controlled stretching to induce directional anisotropy. Quality-control measures (e.g., avoiding air bubbles during PVA dissolution) are described in detail. After fabrication, ultrasound shear wave imaging (SWI) and uniaxial tensile testing are employed to confirm the phantom&#39;s mechanical anisotropy. This paper provides a standardized approach for fabricating anisotropic tissue-mimicking phantoms to validate ultrasound elastography techniques with enhanced accuracy and consistency.

Figure 2A presents a schematic of the complete fabrication process, including PVA solution preparation, molding, initial stretch-free FTC, demolding, and subsequent stretched FTCs. Using the proposed protocol, we fabricated two anisotropic PVA phantoms for SWI and uniaxial tensile testing, respectively. SWI was performed using a research ultrasound system equipped with an L7-4 probe (center frequency: 5.2 MHz), employing supersonic mode (3 foci, 100 µs push)4 and cohe...

This protocol describes a robust, reproducible method for fabricating anisotropic polyvinyl alcohol (PVA) phantoms&#8212;essential tools for validating ultrasound elastography techniques targeting mechanically anisotropic soft tissues (e.g., skeletal muscle).

Polyvinyl alcohol (PVA) phantoms are typically isotropic and are widely used to validate ultrasound elastography techniques for soft-tissue evaluation. ...

Ultrasound elastography is a powerful and increasingly vital tool for non-invasively assessing the mechanical properties of soft tissues crucial for diagnosing various medical conditions. For accurate validation and development of these techniques, reliable tissue-mimicking phantoms are indispensable. Traditionally, Polyvinyl Alcohol or PVA phantoms have been widely used due to their tissue-like acoustic and mechanical properties.Most PVA phantoms are inherently isotropic. Meaning their mechanical properties are uniform in all directions. However, many crucial biological tissues, such as skeletal muscle exhibit distinct mechanical anisotropy with varying stiffness along different orientations.Using isotropic phantoms to validate elastography methods for these anisotropic tissues can lead to misinterpretations and limit clinical applicability. To overcome this, there's a significant need for standardized anisotropic tissue-mimicking tools. While previous studies have shown the potential for fabricating anisotropic PVA phantoms, critical technical details, particularly regarding their reproducible fabrication and quality control have often been underspecified.In this video, we present a visually detailed and highly reproducible protocol for fabricating robust inotropic PVA Phantoms. Let's begin. Before starting, ensure you have all necessary materials and equipment ready, and always prioritize safety by wearing appropriate personal protective equipment like lab coats, gloves, and safety glasses.For the phantom matrix, we use Polyvinyl Alcohol powder, specifically a high molecular weight grade to ensure appropriate stiffness. As a preservative, we use potassium sorbate to prevent microbial growth. Graphite particles are incorporated as acoustic scatterers, making the phantoms visible and trackable under ultrasound.And finally, pure water serves as our solvent. Critical to inducing anisotropy are the custom-designed 3D-printed molds. These are specifically designed to hold the phantoms during our fabrication process.And this custom-built stretching apparatus allows us to apply precise and controlled strain during specific freeze-thaw cycles. First, accurately weigh your dry ingredients, 20 grams of PVA powder, 2 grams of potassium sorbate, and 2 grams of graphite particles. Precision here is key for consistent phantom properties.Next, measure 176 milliliters of pure water. Add the pure water to a clean beaker. Place the beaker on a magnetic stirrer, and then carefully add the weighed PVA powder to the pure water.Ensure the stirring bar is added. After the solution is stirred uniformly seal the beaker with aluminum foil. The aluminum foil cover minimizes water evaporation, thereby preventing changes in solution concentration.And then place the beaker into the water bath. Set the temperature of the water bath to 100 degrees Celsius. This is to enable the water bath to heat up more rapidly with our target temperature set at 80 degrees Celsius.After sufficient heating, the PVA will have completely dissolved in the solution. At this point, remove the beaker and place it back on the magnetic stirrer to continue stirring slowly, allowing it to cool down to about 40 degrees Celsius. This process takes approximately 15 minutes.Next, add potassium sorbate and graphite particles to the beaker. Seal the beaker with aluminum foil and keep stirring slowly. After the solution has cooled, carefully pour it into the 3D-printed mold.Pour slowly and keep the pouring spout close to the mold surface to prevent the formation of new air bubbles, ensuring the mold is evenly filled. Place the mold into a freezer set at minus 20 degrees Celsius and freeze it for 12 hours. This initiates the first freezing step.After the freezing process is complete, transfer the mold to room temperature and allow it to thaw completely. This process takes approximately 12 hours. Once the initial stretch-free cycles are complete, carefully demold the base phantom.At this stage, they are still largely isotropic, but possess a stable structure. Now, mount the phantom securely into the stretching apparatus, then apply a precise and controllable uniaxial strain to the phantom, stretching it to 180%of its original length. Uniform stretching along a specific axis is crucial for inducing directional anisotropy as the polymer chains will realign along the stretching direction.While maintaining this precise stretch, transfer the entire apparatus with the stretched phantom back into the freezer for 12 hours. Following this, move it to the thawing environment for another 12 hours still under continuous tension. The freezing locks in the molecular realignment induced by stretching.Repeat these stretched freeze-thaw cycles another times. Each cycle further enhances the anisotropic properties resulting in a robust, directionally dependent phantom. After the final stretched cycle, carefully release the tension from the apparatus, then gently remove the fully formed anisotropic PVA phantom.You will notice that compared to its original preliminary form, there is no obvious change in the phantom's appearance. Yet in reality, significant and reproducible changes have occurred in its microstructure and mechanical properties at this point. After fabrication, it's crucial to validate the induced anisotropy.We employ two primary methods:ultrasound shear wave imaging and uniaxial tensile testing. For ultrasound shear wave imaging, we position the phantom and acquire shear wave speed measurements. When the shear waves are propagated parallel to the direction of induced anisotropy, we observe a higher shear wave speed, indicating greater stiffness.Conversely, when the waves are propagated in the perpendicular direction, we measure a significantly lower sheer wave speed, confirming the directional dependence of the phantom's mechanical properties. This quantifiable difference visually demonstrates the successful anisotropy. Also, similar shear wave speed distribution patterns along the depth confirm the phantom's homogeneity.Uniaxial tensile testing provides a more direct mechanical assessment. We prepare phantom samples cut both parallel and perpendicular to the induced anisotropic axis. As we perform the tensile test, the stress-strain curves clearly illustrate the difference.The phantom sample in the parallel direction to the anisotropy exhibit a higher Young's modulus, indicating greater stiffness and resistance to deformation in that direction. These results unequivocally confirm the successful fabrication of anisotropic PVA phantoms. In summary, we have presented a detailed, reproducible protocol for fabricating anisotropic Polyvinyl Alcohol phantoms.By carefully controlling material preparation, freeze-thaw cycles, and crucial stretching steps, researchers can create high quality tissue-mimicking tools with tunable anisotropic properties. These phantoms address a critical gap in elastography research, offering a more physiologically relevant model for validating techniques aimed at anisotropic biological tissues. Thank you for watching.

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Learn to fabricate anisotropic polyvinyl alcohol phantoms for ultrasound elastography validation using controlled freeze-thaw cycles and precise stretching techniques. This protocol ensures reproducible tissue-mimicking models with direction-dependent mechanical properties.

Anisotropic Polyvinyl Alcohol Phantom Fabrication for Ultrasound Elastography: Procedure and Quality Controls

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