Solid phantom recipe for diffuse optics in biophotonics applications: a step towards anatomically correct 3D tissue phantoms

: We present a tissue mimicking optical phantom recipe to create robust well tested solid phantoms. The recipe consists of black silicone pigment (absorber), silica microspheres (scatterer) and silicone rubber (SiliGlass, bulk material). The phantom recipe was characterized over a broadband spectrum (600-1100 nm) for a wide range of optical properties (absorption 0.1-1 cm − 1 , reduced scattering 5-25 cm − 1 ) that are relevant to human organs. The results of linearity show a proper scaling of optical properties as well as the absence of coupling between the absorber and scatterer at different concentrations. A reproducibility of 4% among different preparations was obtained, with a similar grade of spatial homogeneity. Finally, a 3D non-scattering mock-up phantom of an infant torso made with the same recipe bulk material (SiliGlass) was presented to project the futuristic aspect of our work that is 3D printing human organs of biomedical relevance.

Multiple phantom recipes are thus found in literature, indicating remaining challenges to find a phantom material that is ideal for a wide range of needs in biophotonics. Possible limiting factors for the acceptance of some of the published phantom recipes include that the recipe does not provide a detailed manufacturing procedure or a systematic study of phantom behavior over a wide range of absorber and scatterer concentrations. In addition, liquid phantoms are cumbersome to handle especially for realistic tissue geometries and typically have a short shelf life. An example of a phantom developed for a specific purpose is given by Maughan Jones et al. They proposed an improved method for phantom manufacturing using silica microsphere as a scatterer for OCT applications. This phantom was thereby characterized at a single wavelength only [4]. Similarly, multiple groups have proposed various manufacturing procedures, some proposed complex methods to ensure absorber and scatterer uniformity [14,15], while some simpler recipes are also available [9,13,16]. Yet, all of these methodslack one of the following aspects: i) A detailed protocol for the procedure of phantom manufacturing and the phantom recipe.
ii) A characterization of the behavior of the phantom over a wide range of absorber and scatterer concentrations relevant to the tissue optical properties.
iii) A broadband (600-1100 nm) optical characterization of the phantoms.
iv) Characterization of the phantoms in terms of reproducibility, shelf life, and homogeneity. The first (i) point takes care of robust manufacturing procedure, whereas the second point ensures the phantoms have linear optical behavior over the entire range of absorber and scatterer concentrations. Point (iii) makes sure that point (ii) was tested over a broad wavelength range, while (iv) ensures the phantoms are optically reproducible and homogeneous over the entire phantom volume. In addition, thorough consideration of phantom ingredients is important in order to facilitate the best possible properties. These points form the building block to provide a well-tested, robust solid phantom that could be manufactured by any group interested in optical properties relevant to human organs.
In our opinion there is still a need for a recipe for a good 3D tissue-mimicking phantom that is robust and tested over the wide range of optical properties covering most of the human organs, preferably characterized over the entire therapeutic wavelength range to benefit the wide community in biophotonics. The aim of this work was to fulfill this need. The study was designed to cover optical properties (absorption 0.1-1 cm −1 , reduced scattering 5-25 cm −1 ) relevant to various organs of the human body. The optical characterization was performed using photon time-of-flight diffuse optical spectrometer (pTOFS) which has been validated in various phantom and clinical studies [17]. Time domain measurements can inherently disentangle absorption and scattering properties, providing reliable results of both absorption and scattering properties of the phantoms [18][19][20][21]. To the best of our knowledge, this was the first study that covers all the above-mentioned challenges to provide an integrated recipe solution that could provide 3D solid phantoms in biomedical optics. To emphasize the cause, a realistic anatomically correct 3D phantom of an infant's torso was provided in the results section.

Phantom recipe
A detailed description of the phantom ingredients, the method to make the phantoms, and the concentration of absorber and scatterer for different optical properties are shown in Fig. 1. The bulk material of the phantom was made of SiliGlass which is a transparent silicone rubber procured from MBFibreglass (PlatSil SiliGlass). SiliGlass is a two-component cured room-temperature-vulcanizing (RTV) silicone. It consists of two liquid parts, part A (base material) and part B (hardener). When the two parts are mixed together in a 1:1 ratio, the mixture cures to a solid at room temperature within 1 hour. Compared to other silicone based rubbers, SiliGlass is a clear medium instead of becoming bluish or turbid on curing. The scattering contribution from the silicone matrix needs to be negligible for better control of scattering properties in the produced phantoms. Also, low scattering regions, such as the cerebrospinal fluid (CSF) in the human head can be accurately modeled. The absorber employed was black silicone pigment (Polycraft Black Silicone Pigment) compatible with silicone and silica microspheres (440345, Sigma-Aldrich) were used to obtain the desired reduced scattering. The pure absorber was highly absorbing hence it was diluted in Part A (silicone base) at a ratio of (2272:1), here onwards whenever the absorber is mentioned it is the diluted (2272:1) stock solution of black silicone pigment in Part A.
The phantom preparation includes two parallel processes. One part comprises of mixing part A of the SiliGlass and the absorber (stock solution). The absorption properties of the phantoms were tuned by changing the quantity of absorber (stock solution) and part A with their total sum kept at constant value (75 g). The second process constitutes mixing part B of the SiliGlass with the microspheres. Both processes follow a similar procedure as shown in Fig. 1. Process B includes an extra step where the silica microspheres were disaggregated manually for 15 minutes with a spatula prior to the mixing with a magnetic stirrer (two magnetic beads (25x8 mm), speed-5, VWR VMS-C7). In general, both processes start with magnetic stirring for 15 minutes followed by ultrasonication (power-5, VWR USC500THD, 45 kHz) for 15 minutes. The entire preparation of the phantoms produced in this study was carried out at a controlled temperature (21° C) to avoid temperature-related reactions and extensive bubbling in the vacuum chamber employed for degassing. Following the bubble removal in a vacuum, both parts were mixed together and stirred manually for 5 minutes to ensure uniformity of the phantom mixture. The final step was to pour the mixture in the phantom cast, which for the characterization was a 3D printed cylinder with large diameter (10 cm). Subsequent measurements were performed in transmittance geometry. Therefore, the phantoms were made with large diameter (10 cm) to avoid boundary effects and granting use of a slab model [22]. The thickness of the phantoms was 18 mm. This value was chosen as a good compromise between the validity of the Diffusion equation and the signal level over the entire spectrum. A previous study on the validity of the Diffusion equation for time-resolved measurements at different source detector distances is reported in reference [23]. In total 24 phantoms were created with a matrix combination of 6 absorption values and 4 reduced scattering values as shown in Fig

Instrume
A time-doma phantoms. Th The setup co Pellin Broca wavelength-sc wavelength, th was injected transmittance phantom on th on a silicon p by Politecnico The system h various phant The use of we inherently dis optical proper The measu source-detecto repeated 3 tim (600-1100 nm minimize inst after the meas The acqui semi-infinite [29]. The the 1 [11], [34]. Th pted us to choo a), 3(b)), a tab broadband ran with well char characterized o ttering 5-25 cm is range that w ompute:  Figure 3(c), 3(d) illustrate the dependency of reduced scattering coefficient on absorber concentration and vice versa. The straight lines observed in these plots confirm the absence of any chemical reaction between the scatterer and absorber. The slight coupling of absorption to scatterer concentration observed in Fig. 3(d) (especially for the f-series) relates to the change in total volume of the phantom due to increasing scatterer concentration and may be partly due to no longer fulfilling the assumptions of the diffusion approximation for high absorption (μ a ≈0.8 cm −1 ) and low reduced scattering (μ s ' ≈5 cm −1 ).
A key feature of a robust recipe is to ensure that the outcome is insensitive to irrelevant parameters like the person who made the phantom, the day it was produced, the batch of raw material used, etc. To this end, a reproducibility test was conducted by manufacturing four phantoms (series code: cB) on different days by different people using a different batch of bulk material. Figure 4(a), 4(b) show the absorption and reduced scattering spectra, respectively, of the same phantom (series code: cB) produced under different conditions. Though the CV was found to be slightly higher in the shorter wavelengths, the overall CV of around 4% reaffirms the robustness of the recipe. Finally, a test to confirm the homogeneity of the phantom (series code: cB) was carried out by measuring optical properties at different points on the same phantom surface. Five points on the phantom surface were chosen, one in the middle and four points were around the midpoint of the phantom top surface (Fig. 4(c) and 4(d)). Similar to the reproducibility test, the CV of the homogeneity test was well below 4%, which confirms the homogenous nature of thek sample. A long term stability of the phantom was performed by measuring phantom (cB) 3 times in the last 5 months and the results showed that the phantom were fairly stable with a CV close to 5% with more variation in the short wavelength range.

Conclusio
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