Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.

This missile can help address key issues in the Biomedical Optics field such as the development of optical missiles based on diffuse reflector spectroscopy. The main advantage of this technique is that diffuse reflector specter help living, biological tissues invisible to near infrared ray brains region can be represented using easily available materials. The creation of monolayer gel phantoms requires using molds.

The epidermal phantom mold consists of a one millimeter thick acrylic plate cut into a U-shape. There is also a to millimeter thick acrylic plate size to cover the first plate. This plate forms one side of the mold.

A second two millimeter thick plate forms the other side of the mold. With the components in place, fix them using five clips. Construct the dermal phantom mold similarly with a five millimeter thick U-shaped acrylic plate between two-two millimeter thick plates.

Fix these pieces with clips. Producing the base material requires saline solution and Agarose powder. Put 500 milliliters of saline in a pot.

Then stir while slowly adding five grams of agarose. Add a thermometer and place the pot on a hot plate at 1000 watt setting for five minutes. Once the mixture boils, turn the heat to low heat for three minutes.

Wait for the mixture to cool to a temperature of about 70 degrees Celsius before pouring it into a container. Keep it in a constant temperature bath at 60 degrees Celsius for 30 minutes. The melanin in the epidermis is mimicked by a coffee solution in the epidermal phantom.

Create this by mixing four milliliters of brewed coffee an 16 milliliters of saline in a glass bottle. Next, put five milliliters of lipid emulsion into a transparent plastic cup and add 10 milliliters of the coffee solution. Stir the mixture while adding 35 milliliters of the base material.

Have the epidermal phantom mold ready and aspirate the mixture into a syringe. Inject the mixture slowly into the mold while avoiding bubble formation. This filled mold is ready to be cooled to create the gel.

Take it to be cooled at five degrees Celsius for 20 minutes. When the cooling is over, remove the clips from the mold. Slide one of the acrylic pieces outward to remove it.

Take the one millimeter thick solidified gel phantom from the mold. Use a surgical scalpel to cut the phantom to the desired size. Then, place and hold the gel phantom between two glass slides.

Start with five milliliters of lipid emulsion in a transparent plastic cup. For oxygenated blood, add whole equine blood. Then, stir the mixture while adding base material.

Have the dermal phantom mold ready and aspirate the mixture into a syringe. Slowly inject the mixture into the mold and avoid forming bubbles. Here is the mold after it is been filled and is ready for cooling.

Take the mold to be cooled at five degrees Celsius for 20 minutes. Retrieve the mold after cooling and remove the clips and one outer acrylic piece. Take the exposed five millimeter thick solidified gel phantom from the mold.

Use a surgical scalpel to cut it to the desired size. Place and hold the phantom between two glass slides. For deoxygenated phantom, begin with an oxygenated blood phantom.

Use a syringe to drop sodium dithionite solution onto the phantom to deoxygenate the blood. Place and hold the phantom between two glass slides to prevent it from drying. Obtain and epidermal and a dermal phantom.

Drop 0.1 milliliters saline solution onto the dermal phantom to aid with optical coupling. Then place the epidermal phantom on top of the dermal phantom and saline solution. Stroke the surface to push out any air bubbles between the layers.

Build the two layered phantom between two glass slides to prevent it from drying. Set up the apparatus to measure the reflectance spectra. Key to the measurements is an integrating sphere which has a sample holder on one side.

On the other side are a light trap and an entrance port for the incident light. A spectrometer collects light with an optical fiber from the sphere's detector port. This schematic provides and overview of the setup.

Note that the unused ports are plugged to prevent light from entering the sphere. The light source is 150 watt halogen lamp with a light guide using achromatic lens to focus the light onto the sample. Turn on the halogen lamp.

As it warms, move to the sample port of the integrating sphere. There, place a standard white diffuser on the sample holder and prepare for a measurement. Adjust the spectrometer integration time and store a reference spectrum.

Take one of the phantoms to the sample holder. Place the phantom, still sandwiched between two glass slides, at the sample port and prepare for another measurement. Measure a transmitted spectrum and store the data to a file.

To measure transmittance spectra, alter the setup to be consistent with this schematic. Note that light now enters the integrating sphere through the sample in the sample port. The other ports are plugged.

This is the integrating sphere ready for measuring transmittance spectra. For a measurement, place a phantom sandwiched between two glass slides in the sample holder. Perform the measurement and save the data.

These spectra are from a two layered phantom with oxygenated blood in the dermal layer that has a concentration of 3%The spectra are for samples with two different coffee solution concentrations in the epidermal layer. The diffuse reflectance decreases with increasing coffee solution which mimics melanin. This is particularly noticeable at short wave lengths.

Here are spectra for a constant coffee solution concentration in the epidermal layer of 7%and a varying blood concentration in the dermal layer. Strong light absorption of hemoglobin explains the differences in the spectra in the 500 to 600 nanometer range. The diffuse reflectance spectra differs with oxygenation state of the blood.

For each of these spectra the coffee solution concentration in the epidermal layer is fixed at 7%and the blood concentration in the dermal layer is fixed at 3%The collected data is useful for estimating optical properties of the phantoms via inverse Monte Carlo simulation. These are the examples of the calculated average reduced scattering coefficient spectrum and the absorption coefficient of the epidermal and dermal layers. Once mastered, this technique can be done in six hours if it is programmed properly.

While making the phantoms it's important to remember to maintain the base material in a constant temperature bath at 60 degrees Celsius. Doing this procedure, three or four layer phantoms can be constructed to explore other topics like representations of the previous reflectance spectra for biological tissues with more complicated schematics. After its development, this technique paved the way for researchers in the field of Biomedical Optics to delegate new ways of optical missiles and systems.

After watching this video, you should have a good understanding of how to create an optical phantom that mimics light transport in biological tissues and to characterize its optical properties. Don't forget that working with sodium dithionite can be hazardous and proper equipment such as googles, gloves, and a face mask should be worn while making a dermal phantom containing the oxygenated blood.