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 ...

Research

JoVE Journal

Bioengineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

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Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

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Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

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Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...