Name: fabrication and characterization of optical tissue phantoms containing macrostructure
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Description: Watch this Scientific Journal Video about Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure at JoVE.com

This work was supported by the National Science Foundation CAREER award no. CBET-1254767 and National Institute of Allergy and Infectious Diseases grant no. R01 AI104960. We gratefully acknowledge Patrick Griffin and Dan Tran for their assistance with characterization measurements and the Texas A&amp;M Cardiovascular Pathology Laboratory for micro-CT imaging.

1. Selection and Verification of Matrix Material Properties
<ol>
	<li>Before starting the phantom fabrication process (Figure 1), find the absorption and reduced scattering coefficients for the biological tissue of interest at the imaging wavelength(s). Preliminary estimates may be found in the references35,36. However, validation of the optical coefficients might be necessary.</li>
	<li>Using the look-up tables for absorption coef...

<ol>
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We have demonstrated a method for creating optical phantoms to represent a murine lung with an internal branching structure to simulate the internal air-tissue interface. The optical properties of murine lung tissue are achieved by incorporating unique concentrations of optically scattering and absorbing particles distributed homogenously within the bulk matrix polymer. These optical properties can be tuned to mimic the physiological values within different spectral ranges of tissues in different states (i.e. he...

Tissue phantoms are used widely for system characterization and calibration of optical imaging and spectroscopy instruments, including multimodality systems incorporating ultrasound or nuclear modalities1,2,3,4. Phantoms provide a controlled optical environment for system characterization and quality control of multiple biological imaging techniques. Tissue-mimicking phantoms are useful tools in predicting system performance and optimizing system design for the physiological task at hand; for example, to predict the probing depth of spectroscopic probes for assessing tumor margins5. Optical properties and structural design of the phantoms can be tuned to mimic the specific physiological environment in which the instrument will be used, therefore allowing for both feasibility studies and verification of system performance3,6,7. Verification of imaging system performance with realistic optical phantoms prior to entering pre-clinical or clinical trials reduces the risk of malfunction or acquisition of unusable data during in vivo studies. The reproducibility and stability of optical phantoms make them customizable calibration standards for optical techniques to monitor intra- and inter-instrument variability, particularly in multicenter clinical trials with different instruments, operators, and environmental conditions8,9.
Tissue-mimicking phantoms also serve as tunable and reproducible physical models for validation of theoretical optical models. Simulations aid in the design and optimization of in vivo optical instruments, while reducing the need for animal experiments10,11. The development and validation of optical simulations to accurately represent the in vivo environment can be encumbered by the complexity of the tissue structure, the biochemical content, and the location of the target or tissue within the body. Variability between subjects makes validation of theoretical models challenging using animal or human measurements. Polymer optical tissue phantoms allow for validation of theoretical models by supplying a known and reproducible optical environment in which to study photon migration12,13,14,15.
For the purpose of system calibration, solid optical phantoms may consist of a single homogeneous slab of cured polymer with the optical scattering, absorption, or fluorescence tuned for the wavelengths of interest. Layered polymer phantoms are frequently used to mimic the depth variance of the tissue optical properties in epithelial tissue models16,17. These phantom structures are sufficient for epithelial imaging and modeling, because the tissue structure is fairly homogeneous through each layer. However, larger scale and more complex structures affect radiative transport in other organs. Methods to create more complex phantoms have been developed to simulate the optical environment of subcutaneous blood vessels18,19 and even whole organs, such as the bladder20. Modeling light transport in the lungs provides a unique problem due to the branching structure of the air-tissue interface; a solid phantom would not likely replicate radiative transport in the organ accurately21. To describe a method for incorporating complex structure into an optical phantom, we describe a method to create an internal, reproducible fractal tree void that represents the three-dimensional (3D) macroscopic structure of the airway (Figure 1).
In the past few decades, 3D printing has become a predominant method for rapid prototyping of medical devices and models22, and optical tissue phantoms are no exception. 3D printing has been used as an additive manufacturing tool for fabricating optical phantoms with channels23, blood vessel networks24, and whole-body small animal models25. These methods use one or two printing materials with unique optical properties. Methods have also been developed to tune the optical properties of the printing material to mimic general, turbid biological tissue25,26. However, the range of achievable optical properties are limited by the printing material, usually a polymer such as acrylonitrile butadiene styrene (ABS)26, so this method is not suitable for all biological tissues. Polydimethylsiloxane (PDMS) is an optically clear polymer that can be readily mixed with scattering and absorbing particles with a higher level of tunability27,28. PDMS has also been used to mold phantoms with aneurysm models for deployment of embolic devices29,30. These phantoms also utilize a dissolvable 3D printed part, but remain optically clear for visualizing device deployment. Here, we combine this method with tunability of the optical properties of PDMS with scattering and absorbing particles to fabricate a preliminary model of the tissue and airways of the murine lung.
While the phantom presented here is specific to the lungs, the process can be applied to a variety of other organs. 3D printing of the internal structure of the phantom allows the design to be customizable for any purpose and printable scale, whether it be a blood or lymph vessel network, bone marrow, or even the four chambered structure of the heart31. Because we are interested in optical imaging and modeling of the lung32,33,34, we have opted to use a four-generation fractal tree as the internal structure to replicate within the polymer phantom. This structure was designed to approximate the branching structure of the airway and have break-away support material for the 3D printing process. A more anatomically correct airway could be printed if break-away support material is not necessary. Although this particular model represents an airway, the internal structure of the phantom does not have to remain a material void. Once the surrounding polymer is cured and the 3D printed part is dissolved, the internal structure can be used as a flow pathway or as a secondary mold for a material with its own unique absorption and scattering characteristics. For example, if the internal structure from this protocol was designed as a digital bone rather than an airway, the bone structure could be 3D printed, molded with PDMS with optical properties of the finger, and then dissolved out of the phantom. The void could then be filled with a PDMS mixture with different optical properties. Additionally, each mold is not limited to a single dissolvable part. A phantom of the finger could be created to include bone, veins, arteries, and a general soft tissue layer, each with its own unique optical properties.

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			<td height="42" style="height:42px;width:228px;">Dow Corning Sylgard 184 Silicone Encapsulant Clear 0.5 kg Kit</td>
			<td style="width:195px;">Ellsworth Adhesives</td>
			<td style="width:187px;">184 SIL ELAST KIT 0.5KG</td>
			<td style="width:259px;">Polydimethylsiloxane: polymer base for optical phantoms</td>
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			<td height="42" style="height:42px;width:228px;">White Rutile Titanium Dioxide powder</td>
			<td style="width:195px;">Atlantic Equipment Engineers</td>
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			<td style="width:259px;">Scattering particles for optical phantoms</td>
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			<td height="42" style="height:42px;width:228px;">Higgins Fountain Pen India Ink</td>
			<td style="width:195px;">Michaels Craft Stores</td>
			<td style="width:187px;">&#160;10015483</td>
			<td style="width:259px;">Absorbing particles for optical phantom</td>
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			<td height="21" style="height:21px;width:228px;">Heat Resistant tape</td>
			<td style="width:195px;">Uline</td>
			<td style="width:187px;">S-7595</td>
			<td style="width:259px;">Heat resistant tape for polymer molds</td>
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			<td height="42" style="height:42px;width:228px;">Fortus 360mc 3D printer</td>
			<td style="width:195px;">Stratasys</td>
			<td style="width:187px;">N/A</td>
			<td style="width:259px;">Able to switch build and support material with this model printer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:228px;">ABS Ivory Model Material</td>
			<td style="width:195px;">Stratasys</td>
			<td style="width:187px;">SDS-000001</td>
			<td style="width:259px;">Material for printing mold parts and/or using as support for printing internal structure&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">SR-30 Soluble Support</td>
			<td style="width:195px;">Stratasys</td>
			<td style="width:187px;">400638-0001</td>
			<td style="width:259px;">Base soluble support material for printing internal structure</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Flacktek Speedmixer</td>
			<td style="width:195px;">Flacktek Inc.</td>
			<td style="width:187px;">DAC 150.1 FV</td>
			<td style="width:259px;">For efficient mixing of polymer and particles&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Integrating sphere</td>
			<td style="width:195px;">Edmund Optics</td>
			<td style="width:187px;">58-585</td>
			<td style="width:259px;">For measuring optical properties</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Polycarbonate build plates (1 mm)</td>
			<td style="width:195px;">Stratasys</td>
			<td style="width:187px;">N/A</td>
			<td style="width:259px;">Used polycarbonate build plates from Stratasys printer can also be used</td>
		</tr>
	</tbody>
</table>

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.

To demonstrate the phantom fabrication technique, mouse lung tissue phantoms were fabricated to simulate measured optical properties of excised healthy and inflamed murine lung tissue at 535 nm (Table 5). This wavelength of interest is the excitation wavelength for tdTomato fluorescent protein used in recombinant reporter strains of mycobacteria in previous studies33. Optical measurements of mouse lung tissue were obtained with the same methods des...

Watch this Scientific Journal Video about Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure at JoVE.com

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

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 ...

The overall goal of this Phantom Fabrication process is to model the mouse airway within an optically tuned polymer. Since the internal structure is 3D printed, this molding process can be applied to a variety of other anatomies. This method can help answer key questions in the field of tissue optics such as Health three dimensional anatomy can affect light transport in tissue.The main advantage of this technique is that it is customizable for any structure that can be resolved by a 3D printer. Although this method can provide insight as a physical model for light transport in the lung, it can also be applied to other systems such as close systems or 3D models with multiple materials. Begin with fabrication of a polydimethylsiloxane or PDMS slab of the selective recepie for confirmation of optical properties.Using a 10:1 ratio in weight of PDMS resin curing agent pour PDMS resin, titanium dioxide, India ink, and PDMS curing agent into the mixing cup in that order. Thorough mixing of materials will allow for a modernist material with an intenant absorption and scattering properties. This step is integral for reproducible optical properties.Mix the ingredients in a speed mixer for 60 seconds. If titanium dioxide particles stick to the mixing cup, mix by hand to remove the particles from the base of the cup before mixing in the mixer for another 30 seconds. Next pour the mixture into Ross or Petri dishes to make thin slabs of the mixture.Degas the slabs for 10 minutes by placing them in an airtight negative pressure chamber. Then place the slabs in a preheated oven at 80 degree celsius for 30 to 60 minutes. After removing the slabs from the oven and letting them cool, remove the cooled polymer slab from its container.Then trim of the edges to leave a flat uniform slab. Measure the thickness of the slab using calibers. Next, measure the transmittance and reflectance of the slabs using an integrating sphere.Turn on the light source and spectrometer of the integrating sphere setup. Check the alignment of the system to ensure a small collimated beam is centered on the entry and exit ports of the integrating sphere. To calibrate the integrating sphere system, turn off the source and cap the exit port of the integrating sphere.Record three dark spectra. Turn the source back on to obtain the transmission reference with the exit port capped and the entrance port empty. Now record three spectra.Next obtain reflectance reference measurements using reflectance standards. Place each standard at the exit port of the sphere. Record three spectra for each reflectance standard.Next, measure the transmittance of the slab with the cap on the exit port. Place the slab on the entry port of the integrating sphere for the transmission measurement. Record three spectra.To measure the reflectance of the slab, remove the exit port cap and place the slab on the exit port for the reflectance measurement. Record three spectra. Proceed to determine optical properties as described in the text protocol.Select a dissolvable material for printing such as polyvinyl alcohol or high impact polystyrene. And print the solid model in this dissolvable material. When the print is complete, break, dissolve or machine the support material off of the printed part.File or sand off any large in perfections. Vapor polishing is important to control surface roughness. The axial resolution of the 3D printer will give a rough internal surface causing diffused reflectance off of that surface.To vapor polish the printed part, first drill a through hole with clearance for a thin steel or nitinol wire in the base of the printed part while it is secured in a vise. Next, spread a stainless steel or nitinol wire through the hole. Bend the ends of the wire and hook them together.This will allow for the part to be fully immersed in acetone vapor within the beaker. Then set the wire and part aside. Working in a fume hood to prevent inhalation of acetone vapor fill a large beaker roughly 10%full of acetone and place it on a hot plate.Then heat to 100 degree celsius. When acetone vapor condensation reaches about half way up the wall of the beaker hand the looped wire with the marked airway on a second wire and suspend it in the acetone vapor for 15 to 30 seconds. Ensure printed parts do not touch the beaker walls or each other.Remove the printed part and suspend it over the empty beaker or container. Let the part dry for at least 4 hours. Pour 9.1 g of PDMS resin into a plastic mixing cup.Add 20 mg of rutile titanium dioxide followed by 35 micro liters of India ink. Finally add 0.91 g of the curing agent to the top of the mixture. Pour the final polymer mixture into the heat resistant mold.Then pour a small amount of the mixture into a separate container to create a polymer slab for confirmation of material optical properties. Place both the marked airway mold in the separate slab into a bell jar for degassing. Begin the vacuum process.If the polymer in the marked airway mold starts to rise, let the air back into the bell jar to burst the surface bubbles. Then begin to pull air again. Repeat this process until the polymer does not rise significantly.This will take between 5 to 10 minutes depending on how much air was trapped when pouring the mixture into the mold. Once the PDMS no longer rises, continue to degas for another 15 minutes. After degassing, slowly let the air back into the chamber.Remove both the marked airway phantom and the polymer slab and place them in a level oven at 80 degree celsius for two hours. Remove the phantom and slab from the oven and let them cool for 20 minutes. Then disassemble the polymer mold with the scalpel without cutting the cured polymer.Snap the base plate half of the marked airway base. Next, place the phantom in the heated sodium hydroxide base bath until the internal part is fully dissolved. And optically cleared referenced phantom may help to determine the dissolving time for the internal component.Once the internal structure is dissolved, take the phantom out of the bath and let it fully dry before taking any optical measurements. After verifying phantom geometry as described in the text protocol, verify the optical properties of the phantom using the polymer slab and the integrating sphere. Representative results of phantom imaging and verification of the internal structure of a 3D lung phantom are shown.Internal illumination with green light of an optically cleared phantom shows the diffused reflection off of the internal surface when the 3D printed part was not vapor polished. Conversely a phantom made with vapor polished internal structure has minimal diffused reflectance at the internal surface. Phantoms illuminated internally with green light and imaged with external detection show a different surface of radiance when different concentrations of optical particles are used.The phantom produced in this video shows spatially diffused surface radiance. However, a phantom with a same internal structure but different concentrations of titanium dioxide and India ink has a much higher absorption and a different radiance profile as absorbed at the surface. Because this phantom includes titanium dioxide and India ink, it is optically opaque, therefore a verification of the internal structure with micro computer tomography is shown.Once mastered, this technique can be done in two days if it is performed properly. After watching this video, you should have a good understanding how to fabricate optical phantoms with a 3D printed structure. This technique of phantom fabrication is used to explore light transport in the mouse lung for optimization of bacteria sensing imaging systems.

Research

JoVE Journal

Bioengineering

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