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.