The work was supported by the National Natural Science Foundation of China (Grant Nos. 11002139 and 81327803) and the Fundamental Research Funds for the Central Universities. We thank Zachary J. Smith of the University of Science and Technology for providing the audio voiceover.

1. Preparing materials for 3D printing
NOTE: Our optical phantom production line uses a variety of printing materials to simulate the structural and functional heterogeneities of biological tissue. The selection of the printing materials also depends on the manufacturing processes.
<ol>
	<li>Material preparation for spin coating printing
	<ol>
		<li>Add 100 mg of titanium dioxide (TiO2) powder into a beaker containing 100 mL of stereolithography (SLA) photopolymer resin.</li>
		<li>Stir the mixture in the beaker for 30 min on a magnetic stirrer.</li>
		<li>Seal the beaker with tinfoil and sonicate it in an ultrasonic machine for 15 min.</li>
		<li>Vacuum the material for 10 min and load it into the storage syringe of the device.</li>
	</ol>
	</li>
	<li>Material preparation for polyjet printing
	<ol>
		<li>Add 17.56 g of 2-hydroxy-2-methylpropiophenone (1-hydroxycyclohexyl phenyl ketone) into the beaker containing 80 g of triethylene glycol dimethacrylate to get 18% (w/w) material.</li>
		<li>Seal the beaker with tinfoil and sonicate it in an ultrasonic machine for 15 min.</li>
		<li>Take out 20 mL of the mixture and add 5 mg of the oil-soluble Chinese red dye into it. Repeat step 1.2.2.</li>
		<li>Vacuum all the materials, load the solution with dye into the cartridges for the Y (Yellow) channel, and load the pure solution into the cartridges for the K (Black) channel.</li>
	</ol>
	</li>
	<li>Material preparation for FDM printing
	<ol>
		<li>Load 200 g of gel wax into each of three beakers and then heat them to 60 &#176;C on a magnetic stirrer.</li>
		<li>Add 600 mg TiO2 powder into the first beaker. Add 80 mg of graphite powder into the second one.</li>
		<li>Stir the gel wax mixed with TiO2 and gel wax mixed with graphite powder in different beakers for 30 min on the magnetic stirrer.</li>
		<li>Vacuum the three different materials for 2 min and load them into the extruder of the hybrid-three-nozzle module before solidification.</li>
	</ol>
	</li>
</ol>
2. Preparing computer models for multimodal 3D printing
NOTE: The heterogeneous skin tissue is simplified into three layers: epidermis, dermis, and subcutaneous tissue. The epidermis layer is produced by spin coating using the material introduced in step 1.1. The dermis layer is produced by polyjet printing using the photosensitive polymer introduced in step 1.2. The subcutaneous tissue layer is produced by FDM using the material introduced in step 1.3. A prototype computer aided design (CAD) file of different printing parameters is generated to guide the aforementioned fabrication processes.
<ol>
	<li>Design of a digital optical phantom for skin
	<ol>
		<li>Design the skin phantom with the following three layers: an epidermis layer of 100 &#181;m thick, a dermis layer of 400 &#181;m thick, and a subcutaneous tissue of 1 cm thick.</li>
		<li>Draw a tumor model using a 3D modeling software package (e.g., Solidworks) (Figure 5A).</li>
	</ol>
	</li>
	<li>Parameter setting for spin coating
	<ol>
		<li>Set the parameters of rotating speed and duration in the control software of the printing device. The first-stage spin coating speed used in this demonstration is 200 revolutions per min (rpm), the spin coating time is 20 s, the speed in the second-stage spin coating is 1,000 rpm, and the spin coating time is 40 s.</li>
		<li>Set the amount of spin coating material as 3 mL and the time of light-curing as 180 s in the control software.</li>
	</ol>
	</li>
	<li>Preparation of the source file for polyjet printing
	<ol>
		<li>Import the blood vessel image to be printed into the AcroRIP Color software package and set the parameters (printing position and inkjet amount) according to the relationship between the optical parameters of the printed phantoms and the image properties. In this printed blood vessel picture, the K channel is loaded with a transparent photocurable material, and the Y channel is loaded with a photocurable material mixed with Chinese red dye.</li>
		<li>Generate a &#34;.prn&#34; file with parameters defined for 3D printing.</li>
	</ol>
	</li>
	<li>Preparation of G code for FDM printing
	<ol>
		<li>Draw a frustum model with a 3D mapping software package (e.g., Solidworks) to simulate a tumor.</li>
		<li>Import the &#34;.stl&#34; file of the tumor model into a Cura software package installed with an all-in-one nozzle slicing script.</li>
		<li>Slice the model to generate the G code required for printing.</li>
	</ol>
	</li>
	<li>Loading of the documents to the printing control software
	<ol>
		<li>Click the &#34;File&#34; menu item in the menu bar, select the &#34;Import UV Print File&#34; submenu item, and load the UV printing &#34;.prn&#34; files introduced in step 2.3.</li>
		<li>Load the G code generated in step 2.4 into the print control software as in step 2.5.1.</li>
		<li>Click the Start Printing button to start the automated 3D printing procedure.</li>
	</ol>
	</li>
</ol>
3. Printing the skin epidermis layer phantom component by spin coating
NOTE: The spin coating module is mainly comprised of three parts: 1) a spin coater; 2) a glue dispenser; and 3) a UV lamp.
<ol>
	<li>Move the substrate on the loading station to the sample stage of the spin coater with a mechanical hand. Start the vacuum pump to fix the substrate by adsorption.</li>
	<li>The glue dispenser controls the syringe to drip the material introduced in step 2.2.2 at the center of the substrate.</li>
	<li>The spin coater starts to work following the set speed and time parameters.</li>
	<li>Put down the UV lamp (wavelength: 395 nm) and turn it on for 180 s.</li>
	<li>Lift the UV lamp, turn off the spin-coater, and print the skin epidermis layer.</li>
</ol>
4. Printing the skin dermis layer phantom component by polyjetting
NOTE: The polyjet printing module consists of a piezoelectric inkjet nozzle, a three-dimensional mobile platform, a control panel, and a UV lamp (mercury lamp). The solvent-based photocurable material, absorption material, and scattering material are used as a matrix. Different optical parameters are obtained by spraying materials in different proportions in different regions. Finally, the dermis layer phantom is printed and cured layer-by-layer.
<ol>
	<li>Move the substrate to the 3D mobile platform and open the suction valve to adsorb the substrate on the platform.</li>
	<li>The 3D mobile platform holds the substrate to the initial position of the UV printer.</li>
	<li>Push the inkjet printer to the working position by the cylinder, and the inkjet printer works the time specified in the &#34;.prn&#34; file sent by the host computer. Here, the paper feed signal of the inkjet printer is used to drive the movement of the Y-axis mobile platform.</li>
	<li>The inkjet printer prints the layer designed in step 2.5.1 and the cylinder pushes the inkjet printer back to the original position. The Y-axis of the 3D moving platform placed with the substrate is initialized by moving to its initial position.</li>
	<li>The substrate moves 50 mm in the positive direction of the Y-axis. The UV lamp is pushed down by the cylinder (10 mm above the substrate).</li>
	<li>Turn on the UV lamp for 180 s according to the curing time setting.</li>
	<li>Push the UV lamp to the initial position with the cylinder. The Y-axis of the 3D mobile platform placed with the substrate is initialized and returned to its initial position.</li>
	<li>Move the 3D mobile platform placed with substrate down by 0.1 mm along the Z-axis.</li>
	<li>Repeat steps 4.1&#8211;4.8 to print the next layer until the multilayer printing is completed.</li>
</ol>
5. Printing the subcutaneous tissue phantom component by FDM
NOTE: The FDM module is comprised of a hybrid-three-head module, a single-head module, and a 3D mobile platform. The gel wax, the absorbing material, and the scattering material are used as raw materials to prepare a phantom simulating subcutaneous tissue/tumor. The gel wax is heated and melted in the feeder. Uniformly stirred by the extrusion head, it is extruded to print the final phantoms with the desired optical parameters.
<ol>
	<li>Turn on the heating power of the nozzle module and set the temperature to 60 &#176;C.</li>
	<li>Move the mixing nozzle to the working position by pushing the cylinder.</li>
	<li>The FDM module receives the G code commands sent by the host computer, and the mixing nozzle is heated up to 68 &#176;C.</li>
	<li>Turn on the agitation motor and mix the materials well.</li>
	<li>Initialize the 3D mobile platform and the XYZ axes move to the initial position.</li>
	<li>The printing process is executed following the G code commands. In a layer-by-layer printing procedure, the materials are extruded in proportion to the mixing ratio that determine the optical parameters of the phantom in each layer. The printing continues until the subcutaneous tissue portion or the tumor portion is fully printed.</li>
	<li>Move the mixing nozzle module to the initial position by pushing the cylinder. 
	CAUTION: Because graphite powder has strong light absorption, it needs to be mixed as uniformly as possible to avoid changes in the optical parameters induced by aggregation. TiO2 powder of large particle size easily precipitates and affects material placement accuracy, so it is necessary to fully mix it. TiO2 should be replaced if stored for a long time.</li>
</ol>
6. Moving the substrate back to the loading station
<ol>
	<li>Initialize the 3D mobile platform and move the XYZ axis to the initial position. Move the 3D mobile platform to the handover location.</li>
	<li>Move the mechanical hand to the position above the substrate by pushing the cylinder.</li>
	<li>Pick up the substrate and move it over the loading station with the mechanical hand. Place the substrate on the loading station and complete the automated printing.</li>
</ol>
7. Casting the subcutaneous tissue layer phantom component by molding
NOTE: If the tumor model for the phantom is designed, it will be necessary to cast the entire phantom by pouring the polydimethylsiloxane (PDMS) outside the tumor. Steps 7.1&#8211;7.3 are not required for the FDM module to print subcutaneous tissue layer without a tumor.
<ol>
	<li>Press on a substrate with a 3D printed rectangular mold.</li>
	<li>Pour liquid PDMS into the mold.</li>
	<li>Place the substrate in an incubator and store at 60 &#176;C for 2 h.</li>
	<li>Remove the phantom from the substrate.</li>
</ol>

<ol>
	<li>Lu, G., Fei, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+hyperspectral+imaging:+a+review.">Medical hyperspectral imaging: a review.</a> Journal of Biomedical Optics. 19 (1), 010901 (2014).</li><li>Wang, K., 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+non-uniform+discrete+Fourier+transform+based+high+speed+spectral+domain+optical+coherence+tomography+system.">Development of a non-uniform discrete Fourier transform based high speed spectral domain optical coherence tomography system.</a> Optics Express. 17 (14), 12121-12131 (2009).</li><li>Zhao, H., Gao, F., Tanikawa, Y., Homma, K., Yamada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-resolved+diffuse+optical+tomographic+imaging+for+the+provision+of+both+anatomical+and+functional+information+about+biological+tissue.">Time-resolved diffuse optical tomographic imaging for the provision of both anatomical and functional information about biological tissue.</a> Applied Optics. 44 (10), 1905-1916 (2005).</li><li>Ding, Z., Ren, H., Zhao, Y., Nelson, J. S., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+optical+coherence+tomography+over+a+large+depth+range+with+an+axicon+lens.">High-resolution optical coherence tomography over a large depth range with an axicon lens.</a> Optics Letters. 27 (4), 243-245 (2002).</li><li>Iida, H., 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=Three-dimensional+brain+phantom+containing+bone+and+grey+matter+structures+with+a+realistic+head+contour.">Three-dimensional brain phantom containing bone and grey matter structures with a realistic head contour.</a> Annals of Nuclear Medicine. 27 (1), 25-36 (2013).</li><li>Mobashsher, A. T., Abbosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+human+head+phantom+with+realistic+electrical+properties+and+anatomy.">Three-dimensional human head phantom with realistic electrical properties and anatomy.</a> IEEE Antennas and Wireless Propagation Letters. 13, 1401-1404 (2014).</li><li>Li, J. B., 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+new+head+phantom+with+realistic+shape+and+spatially+varying+skull+resistivity+distribution.">A new head phantom with realistic shape and spatially varying skull resistivity distribution.</a> IEEE Transactions on Biomedical Engineering. 61 (2), 254-263 (2013).</li><li>Bykov, 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=Multilayer+tissue+phantoms+with+embedded+capillary+system+for+OCT+and+DOCT+imaging.">Multilayer tissue phantoms with embedded capillary system for OCT and DOCT imaging.</a> Life Sciences. (International Society for Optics and Photonics). , 73760 (2011).</li><li>Bykov, A. V., Popov, A. P., Priezzhev, A. V., Myllyl&#228;, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+phantoms+with+realistic+vessel+structure+for+OCT+measurements+in+Laser+Applications.">Skin phantoms with realistic vessel structure for OCT measurements in Laser Applications.</a> European Conference on Biomedical Optics. , 80911 (2010).</li><li>Park, J., 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=Fabrication+of+double+layer+optical+tissue+phantom+by+spin+coating+method:+mimicking+epidermal+and+dermal+layer.">Fabrication of double layer optical tissue phantom by spin coating method: mimicking epidermal and dermal layer.</a> Design and Performance Validation of Phantoms Used in Conjunction with Optical Measurement of Tissue V. , 85830 (2013).</li><li>Wr&#243;bel, M. S., 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=Use+of+optical+skin+phantoms+for+preclinical+evaluation+of+laser+efficiency+for+skin+lesion+therapy.">Use of optical skin phantoms for preclinical evaluation of laser efficiency for skin lesion therapy.</a> Journal of Biomedical Optics. 20 (8), 085003 (2015).</li><li>Sheng, S., Wu, Q., Han, Y., Dong, E., Xu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabricating+optical+phantoms+to+simulate+skin+tissue+properties+and+microvasculature.">Fabricating optical phantoms to simulate skin tissue properties and microvasculature.</a> Design and Performance Validation of Phantoms Used in Conjunction with Optical Measurement of Tissue Vii. , 932507 (2015).</li><li>Lurie, K. L., Smith, G. T., Khan, S. A., Liao, J. C., Ellerbee, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional,+distendable+bladder+phantom+for+optical+coherence+tomography+and+white+light+cystoscopy.">Three-dimensional, distendable bladder phantom for optical coherence tomography and white light cystoscopy.</a> Journal of Biomedical Optics. 19 (3), 36009 (2014).</li><li>Hahn, C., Noghanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+breast+phantom+development+for+microwave+imaging+using+regression+models.">Heterogeneous breast phantom development for microwave imaging using regression models.</a> Journal of Biomedical Imaging. 2012, 6 (2012).</li><li>Ansari, M. A., Mohajerani, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+optical+abnormalities+in+breast+phantom+by+diffuse+equation.">Estimation of optical abnormalities in breast phantom by diffuse equation.</a> Optik-International Journal for Light and Electron Optics. 125 (20), 5978-5981 (2014).</li><li>Roman, M., Gonzalez, J., Carrasquilla, J., Erickson, S. J., Godavarty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Gen-2+Hand-Held+Optical+Imager:+Phantom+and+Preliminary+in-vivo+Breast+Imaging+Studies.">A Gen-2 Hand-Held Optical Imager: Phantom and Preliminary in-vivo Breast Imaging Studies.</a> 29th Southern Biomedical Engineering Conference. , 103-104 (2013).</li><li>Michaelsen, K. E., 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=Anthropomorphic+breast+phantoms+with+physiological+water,+lipid,+and+hemoglobin+content+for+near-infrared+spectral+tomography.">Anthropomorphic breast phantoms with physiological water, lipid, and hemoglobin content for near-infrared spectral tomography.</a> Journal of Biomedical Optics. 19 (2), 026012 (2014).</li><li>Park, J., 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=Optical+tissue+phantoms+based+on+spin+coating+method.">Optical tissue phantoms based on spin coating method.</a> Design and Performance Validation of Phantoms Used in Conjunction with Optical Measurement of Tissue VII. , 93250 (2015).</li><li>Mustari, 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=Agarose-based+tissue+mimicking+optical+phantoms+for+diffuse+reflectance+spectroscopy.">Agarose-based tissue mimicking optical phantoms for diffuse reflectance spectroscopy.</a> Journal of Visualized Experiments. (138), e57578 (2018).</li><li>Luciano, N. J., 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=Utilizing+3D+printing+technology+to+merge+MRI+with+histology:+A+protocol+for+brain+sectioning.">Utilizing 3D printing technology to merge MRI with histology: A protocol for brain sectioning.</a> Journal of Visualized Experiments. (118), e54780 (2016).</li><li>Dong, E., 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=Three-dimensional+fuse+deposition+modeling+of+tissue-simulating+phantom+for+biomedical+optical+imaging.">Three-dimensional fuse deposition modeling of tissue-simulating phantom for biomedical optical imaging.</a> Journal of Biomedical Optics. 20 (12), 121311 (2015).</li><li>Beltrame, E. D. V., 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=3D+Printing+of+Biomolecular+Models+for+Research+and+Pedagogy.">3D Printing of Biomolecular Models for Research and Pedagogy.</a> Journal of Visualized Experiments. (121), e55427 (2017).</li><li>Bentz, B. Z., Chavan, A. V., Lin, D., Tsai, E. H., Webb, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+and+application+of+heterogeneous+printed+mouse+phantoms+for+whole+animal+optical+imaging.">Fabrication and application of heterogeneous printed mouse phantoms for whole animal optical imaging.</a> Applied Optics. 55 (2), 280-287 (2016).</li><li>Liu, G., 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=Fabrication+of+a+multilayer+tissue-mimicking+phantom+with+tunable+optical+properties+to+simulate+vascular+oxygenation+and+perfusion+for+optical+imaging+technology.">Fabrication of a multilayer tissue-mimicking phantom with tunable optical properties to simulate vascular oxygenation and perfusion for optical imaging technology.</a> Applied Optics. 57 (23), 6772-6780 (2018).</li></ol>

The authors have nothing to disclose.

In the fabrication of the multilayered phantom, the material used for spin coating is a kind of light-curable material instead of PDMS. The intermediate layer is printed with the polyjet printing method, which uses the light-curable resin as raw material. Although thin PDMS phantoms can be made by spin coating after adding tert-butyl alcohol, a PDMS layer cannot effectively bind to the light-curable material during polyjet printing. Therefore, we chose the light-curable resin for spin coating.
<p class="jove_content"...

Biomedical optical imaging represents a family of medical imaging tools that detect diseases and tissue anomalies based on light interactions with biological tissue. In comparison with other imaging modalities, such as magnetic resonance imaging (MRI) and computed tomography (CT), biomedical optical imaging takes the advantage of noninvasive measurement of tissue structural, functional, and molecular characteristics using low-cost and portable devices1,2,3,4. However, despite its superiority in cost and portability, optical imaging has not been widely accepted for clinical diagnosis and therapeutic guidance, partially due to its poor reproducibility and lack of quantitative mapping between optical and biological parameters. The main reason for this limitation is the lack of traceable standards for quantitative calibration and validation of biomedical optical imaging devices.
In the past, a variety of tissue-simulating phantoms were developed for biomedical optical imaging research in various tissue types, such as brain5,6,7, skin8,9,10,11,12, bladder13, and breast tissues14,15,16,17. These phantoms are primarily produced by one of the following fabrication processes: 1) spin coating10,18 (for simulating homogenous and thin-layered tissue); 2) molding19 (for simulating bulky tissue with geometric features); and 3) three-dimensional (3D) printing20,21,22 (for simulating multilayered heterogeneous tissue). Skin phantoms produced by molding are able to mimic the bulk optical properties of skin tissue but cannot simulate the lateral optical heterogeneities19. Bentz et al. used a two-channel FDM 3D printing method to mimic different optical properties of biological tissue23. However, using two materials cannot sufficiently simulate tissue optical heterogeneity and anisotropy. Lurie et al. created a bladder phantom for optical coherence tomography (OCT) and cystoscopy by combining 3D printing and spin coating13. However, heterogeneous features of the phantom, such as blood vessels, had to be hand painted.
Among the above phantom fabrication processes, 3D printing provides the most flexibility for simulating the structural and functional heterogeneities of biological tissue. However, many biological tissue types, such as skin tissue, consist of multilayered and multiscaled components that cannot be effectively duplicated by a single 3D printing process. Therefore, integration of multiple manufacturing processes is necessary. We propose a 3D printing production line that integrates multiple manufacturing processes for automatic production of multilayered and multiscaled tissue simulating phantoms as a traceable standard for biomedical optical imaging (Figure 1). Although spin coating, polyjet printing, and FDM are automated in our 3D printing production line, each modality retains the same functional characteristics as the established processes. Therefore, this paper provides a general guideline for producing multiscaled, multilayered, and heterogeneous tissue-simulation phantoms without the need for physical integration of multiple processes in a single apparatus.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60563/60563fig1.jpg" /> 
Figure 1: The CAD diagram of the 3D printing production line. (A) The 3D printing production line with the top shell removed. (B) The schematic of the spin coating module and the mechanical hand module. (C) The schematic of the polyjet printing module. (D) The schematic of the FDM printing module (the UV lamp belongs to the polyjet printing module). <a href="https://www.jove.com/files/ftp_upload/60563/60563fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>2-Hydroxy-2-methylpropiophenone</td><td>aladdin</td><td>H110280-500g</td><td>Light initiator&lt;br/&gt; &lt;a href=&quot;http://www.aladdin-e.com/&quot; target=&quot;_blank&quot;&gt;http://www.aladdin-e.com/&lt;/a&gt;</td></tr><tr><td>3D printing control system</td><td>USTC</td><td>USTC-3DPrinter_control1.0</td><td>custom-made&lt;br/&gt; github:&lt;br/&gt; &lt;a href=&quot;https://github.com/macanzhen/3D_printing_control_system_of_USTC&quot; target=&quot;_blank&quot;&gt;https://github.com/macanzhen/&lt;/a&gt;</td></tr><tr><td>3D printing system</td><td>USTC</td><td>USTC-3DPrinter1.0</td><td>custom-made</td></tr><tr><td>AcroRip color</td><td>Human Plus</td><td>AcroRip v8.2.6</td><td></td></tr><tr><td>All-in-one nozzle slicing script</td><td>Shenzhen CBD Technology Co.,Ltd.</td><td></td><td>github:&lt;br/&gt; &lt;a href=&quot;https://github.com/macanzhen/3D_printing_Cura_all-in-one-nozzle-slicing-script&quot; target=&quot;_blank&quot;&gt;https://github.com/macanzhen/&lt;/a&gt;</td></tr><tr><td>Chinese Red Dye</td><td>Juents</td><td></td><td>Oil-soluble</td></tr><tr><td>Cura</td><td>Ultimaker</td><td>Cura_15.04.6</td><td></td></tr><tr><td>Gel Wax</td><td>Shanghai Lida Industry Co.,ltd.</td><td>LP</td><td>melting point: 56 &amp;deg;C</td></tr><tr><td>Graphite</td><td>aladdin</td><td>G103922-100g</td><td>Change object optical absorption parameters&lt;br/&gt; &lt;a href=&quot;http://www.aladdin-e.com/&quot; target=&quot;_blank&quot;&gt;http://www.aladdin-e.com/&lt;/a&gt;</td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>184</td><td></td></tr><tr><td>Titanium dioxide</td><td>ALDRICH</td><td>24858-100G</td><td>347 nm</td></tr><tr><td>Triethylene glycol dimethacrylate</td><td>aladdin</td><td>T101642-250ml</td><td>Photocured monomer&lt;br/&gt; &lt;a href=&quot;http://www.aladdin-e.com/&quot; target=&quot;_blank&quot;&gt;http://www.aladdin-e.com/&lt;/a&gt;</td></tr><tr><td>UV ink SLA Photopolymer Resin</td><td>time80s</td><td>RESIN-A</td><td>&lt;a href=&quot;http://www.time80s.com/zlxz&quot; target=&quot;_blank&quot;&gt;http://www.time80s.com/zlxz&lt;/a&gt;</td></tr></tbody></table>

multimodal 3d printing of phantoms to simulate biological tissue

Biomedical optical imaging is playing an important role in diagnosis and treatment of various diseases. However, the accuracy and the reproducibility of an optical imaging device are greatly affected by the performance characteristics of its components, the test environment, and the operations. Therefore, it is necessary to calibrate these devices by traceable phantom standards. However, most of the currently available phantoms are homogeneous phantoms that cannot simulate multimodal and dynamic characteristics of biological tissue. Here, we show the fabrication of heterogeneous tissue-simulating phantoms using a production line integrating a spin coating module, a polyjet module, a fused deposition modeling (FDM) module, and an automatic control framework. The structural information and the optical parameters of a &#34;digital optical phantom&#34; are defined in a prototype file, imported to the production line, and fabricated layer-by-layer with sequential switch between different printing modalities. Technical capability of such a production line is exemplified by the automatic printing of skin-simulating phantoms that comprise the epidermis, dermis, subcutaneous tissue, and an embedded tumor.

Phantom fabricated by spin coating 
The spin coating evenly distributes the droplets on the substrate by rotating the turntable, and a single layer of the original body is fabricated after curing. The rotational speed of the substrate and the time of rotation not only affect the surface quality of the phantom, but also determine the thickness of each layer of the phantom. Phantoms of different thicknesses can be fabricated by repetitive spin coating layer-by-layer. The optical parameters of the phan...

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Multimodal 3D Printing of Phantoms to Simulate Biological Tissue

Spin coating, polyjet printing, and fused deposition modeling are integrated to produce multilayered heterogeneous phantoms that simulate structural and functional properties of biological tissue.

Biomedical optical imaging is playing an important role in diagnosis and treatment of various diseases. However, the accuracy and the reproducibility of ...

multimodal-3d-printing-of-phantoms-to-simulate-biological-tissue

Research

JoVE Journal

Engineering

7.4K Views.  University of Science and Technology of China. Spincoating, polyjetprinting en gesmolten depositiemodellering zijn geïntegreerd om meerlaagse heterogene fantooms te produceren die structurele en functionele eigenschappen van biologisch weefsel simuleren. Gebrek aan traceerbare fantoomnormen die structurele en functionele heterogeniteit van biologisch weefsel simuleren, is het knelpunt geworden voor de ontwikkeling en validatie van biomedische optische apparaten. Deze video toont laag voor laag fabricage van heterogene weefsel simuleren f...

Video: Multimodal 3D Printing of Phantoms to Simulate Biological Tissue

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

Bioengineering

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...

Voxel Printing Anatomy: Design and Fabrication of Realistic, Presurgical Planning Models through Bitmap Printing

Most applications of 3-dimensional (3D) printing for presurgical planning have been limited to bony structures and simple morphological descriptions of complex organs due to the fundamental limitations in accuracy, quality, and efficiency of the current modeling paradigm. This has largely ignored the soft tissue critical to most surgical specialties where the interior of an object matters and anatomical boundaries transition gradually. Therefore, the needs of the biomedical industry to replicate human tissue, which displays multiple scales of organization and varying material distributions, necessitate new forms of representation. 
Presented here is a novel technique to create 3D models directly from medical images, which are superior in spatial and contrast resolution to current 3D modeling methods and contain previously unachievable spatial fidelity and soft tissue differentiation. Also presented are empirical measurements of novel, additively manufactured composites that span the gamut of material stiffnesses seen in soft biological tissues from MRI and CT. These unique volumetric design and printing methods allow for deterministic and continuous adjustment of material stiffness and color. This capability enables an entirely new application of additive manufacturing to presurgical planning: mechanical realism. As a natural complement to existing models that provide appearance matching, these new models also allow medical professionals to "feel" the spatially varying material properties of a tissue simulant-a critical addition to a field in which tactile sensation plays a key role.

This method demonstrates a voxel-based 3D printing workflow, which prints directly from medical images with exact spatial fidelity and spatial/contrast resolution. This enables the precise, graduated control of material distributions through morphologically complex, graduated materials correlated to radiodensity without loss or alteration of data.

Most applications of 3-dimensional (3D) printing for presurgical planning have been limited to bony structures and simple morphological descriptions of ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Biology

Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. The model can be used for planning and simulating critical steps of a surgical approach. Therefore, it is important that anatomical structures such as blood vessels inside a tumor can be printed to be colored not only on their surface, but throughout their whole volume. During simulation this allows for the removal of certain parts (e.g., with a high-speed drill) and revealing internally located structures of a different color. Thus, diagnostic information from various imaging modalities (e.g., CT, MRI) can be combined in a single compact and tangible object.
However, preparation and printing of such a fully colored anatomical model remains a difficult task. Therefore, a step-by-step guide is provided, demonstrating the fusion of different cross-sectional imaging data sets, segmentation of anatomical structures, and creation of a virtual model. In a second step the virtual model is printed with volumetrically colored anatomical structures using a plaster-based color 3D binder jetting technique. This method allows highly accurate reproduction of patient-specific anatomy as shown in a series of 3D-printed petrous apex chondrosarcomas. Furthermore, the models created can be cut and drilled, revealing internal structures that allow for simulation of surgical procedures.

The protocol describes the fabrication of fully colored three-dimensional prints of patient-specific, anatomical skull models to be used for surgical simulation. The crucial steps of combining different imaging modalities, image segmentation, three-dimensional model extraction, and production of the prints are explained.

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. ...

Medicine

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training physicians&#39; wire-skills as well as improving planning of interventional procedures. The aim of this study was to develop a manufacturing process of patient-specific 3D-printed models for cardiovascular interventions.
To create a 3D-printed elastic phantom, different 3D-printing materials were compared to porcine biological tissues (i.e., aortic tissue) in terms of mechanical characteristics. A fitting material was selected based on comparative tensile tests and specific material thicknesses were defined. Anonymized contrast-enhanced CT-datasets were collected retrospectively. Patient-specific volumetric models were extracted from these datasets and subsequently 3D-printed. A pulsatile flow loop was constructed to simulate the intraluminal blood flow during interventions. Models&#39; suitability for clinical imaging was assessed by x-ray imaging, CT, 4D-MRI and (Doppler) ultrasonography. Contrast medium was used to enhance visibility in x-ray-based imaging. Different catheterization techniques were applied to evaluate the 3D-printed phantoms in physicians&#39; training as well as for pre-interventional therapy planning.
Printed models showed a high printing resolution (~30 &#181;m) and mechanical properties of the chosen material were comparable to physiological biomechanics. Physical and digital models showed high anatomical accuracy when compared to the underlying radiological dataset. Printed models were suitable for ultrasonic imaging as well as standard x-rays. Doppler ultrasonography and 4D-MRI displayed flow patterns and landmark characteristics (i.e., turbulence, wall shear stress) matching native data. In a catheter-based laboratory setting, patient-specific phantoms were easy to catheterize. Therapy planning and training of interventional procedures on challenging anatomies (e.g., congenital heart disease (CHD)) was possible.
Flexible patient-specific cardiovascular phantoms were 3D-printed, and the application of common clinical imaging techniques was possible. This new process is ideal as a training tool for catheter-based (electrophysiological) interventions and can be used in patient-specific therapy planning.

Here we present development of a mock circulation setup for multimodal therapy evaluation, pre-interventional planning, and physician-training on cardiovascular anatomies. With the application of patient-specific tomographic scans, this setup is ideal for therapeutic approaches, training, and education in individualized medicine.

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training ...

A Personalized 3D-Printed Model for Preoperative Evaluation in Thyroid Surgery

The anatomic structure of the surgical area of thyroid cancer is complex. It is very important to comprehensively and carefully evaluate the tumor location and its relation with the capsule, trachea, esophagus, nerves, and blood vessels before operation. This paper introduces an innovative 3D-printed model establishment method based on computerized tomography (CT) DICOM images. We established a personalized 3D-printed model of the cervical thyroid surgery field for each patient who needed thyroid surgery to help clinicians evaluate the key points and difficulties of the surgery and select the operation methods of key parts as a basis. The results showed that this model is conducive to preoperative discussion and the formulation of operation strategies. In particular, as a result of the clear display of the recurrent laryngeal nerve and parathyroid gland locations in the thyroid operation field, injury to them can be avoided during surgery, the difficulty of thyroid surgery reduced, and the incidence of postoperative hypoparathyroidism and complications related to recurrent laryngeal nerve injury reduced too. Moreover, this 3D-printed model is intuitive and aids communication for the signing of informed consent by patients before surgery.

Here, a new method of establishing a personalized 3D-printed model for preoperative evaluation of thyroid surgery is proposed. It is conducive to preoperative discussion, reducing the difficulty of thyroid surgery.

Multimodal 3D Printing of Phantoms to Simulate Biological Tissue

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