Sample Preparation Protocols

For system characterization, we use tissue-mimicking phantoms rather than biological samples. This allows us better control over the relevant properties of the sample (such as optical

scattering and elastic modulus) as well as ensuring that the samples will last for a long time so that repeated imaging over the course of many months will yield comparable results. For MMOCT, the relevant metrics for a tissue phantom are the optical scattering and the elastic properties of the phantom. Following the sample preparation protocol given in Ref [5], we first made samples using silicone because the elasticity is easy to manipulate by diluting cross-linking PDMS with non-cross linking silicone oil. Oldenburg et al reported that a ratio of 90.4% silicone oil (50 cSt viscosity), 8.8% PDMS part A and 0.8% PDMS part B (the curing agent) produced tissue phantoms that qualitatively matched the mechanical properties of soft human tissue [5], with an elastic modulus of approximately 12 kPa [99]. To match the scattering coefficient of biological tissues, TiO2 micro-particles were added at a concentration of 4.1 mg/g. This

concentration was found by comparing the peak OCT signal and OCT signal attenuation in depth to that of 2% intralipid, which is representative of human skin (as reported in Ref [100]).

Using this protocol as a starting point, we found that a similar ratio worked well for us. We combine 89.1% silicone oil (pure PDMS, 50 cSt viscosity, Clearco Products), 9.9% Sylgard 185 Silicone Elastomer Kit part A, (the base), and 0.99% of the elastomer kit part B (the curing agent). We add TiO2 micro-particles (Sigma Aldrich rutile powder, 224227, mean diameter 1m) at a concentration of 4.11 mg/g, and we add varying concentrations of Fe3O4 nanopowder (Sigma Aldrich, 637106, mean diameter 50-100 nm) to make samples with homogeneous distributions of paramagnetic nanoparticles for characterizing the MMOCT system. The TiO2 nanopowder is highly electrostatic and tends to settle out of the silicone polymer matrix and stick to the sides of the glass or plastic sample holder during the ~36 hours that it takes the silicone to cross-link. The protocol that we ultimately found to produce the most homogenous distribution of both the TiO2 and the Fe3O4 (with the least settling out of the nanopowders) is as follows. To make an entire

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array of silicone samples with varying concentrations of Fe3O4, make three large batches of the silicone mixture, one with no Fe3O4, one with a high concentration, and one with an intermediate concentration. These three are then be combined in varying ratios to produce individual samples with the desired concentration of Fe3O4. Working in large batches this way minimizes systematic errors from measuring small amounts of the Fe3O4 nanopowder. For each batch, pour all the components (silicone oil, PDMS parts A and B, TiO2 and Fe3O4) into a 200 mL glass beaker. Manually stir the contents for five minutes. Then place in a sonicating bath and leave for three hours. Manually stir each batch right before combining them in the desired ratio into each individual sample mold. Cure the samples overnight at 70°C and then cure at room temperature for 24 hours.

The benefits of the silicone samples are that silicone stays cross-linked for many years. This means that the same samples can be used to measure the Fe sensitivity of an MMOCT system over long time scales, ensuring that the results are always comparable with previous measurements. While silicone is the best choice for longevity, it presents problems for LF- MMOCT samples. As shown in Figure 3-7, the magnet in the LF-MMOCT system is placed below the sample because with our large line illumination, it would be difficult to image through the magnet bore. Because the magnetic field gradient drops off steeply with distance, we need the magnet to be as close as possible to the portion of the sample being imaged. This limits the thickness of the sample we can use in LF-MMOCT because we can only image ~ 0.5 mm deep into the tissue and we need the magnetic field gradient not to have fallen off too much at this distance from the magnet. As described in section 3.2.2, the magnetic field delivery system was designed to be used with samples ~2 mm thick. Making such thin samples proved challenging because the TiO2 and Fe3O4 settles out and clings to the bottom of the sample mold when such a

thin layer of the silicone mixture is poured. The optical scattering of the 2mm-thick silicone samples was greatly reduced compared to ~8mm-thick samples made for a point-scanning

MMOCT system (in which the magnet is placed above the sample so sample can be much larger). We tried using gelatin rather than silicone to make thin samples for the LF-MMOCT system, but the melting point of the gelatin (37°C) was so low that the gels melted under illumination from the high-powered SC source in the LF-OCT system. Ultimately, the best protocol we found for making 2 mm-thick samples for the LF-MMOCT system was to use agarose powder (Sigma Aldrich A0169). The agarose has a melting point of 87°C and has never shown any sign of melting after many minutes of continuous exposure to the line illumination in the LF-OCT system. Evidence of melting can be seen by taking an image stack of the agarose or gelatin sample over time. If the processed B-mode images are converted to a video, the surface of the sample lowers in the image over time if the sample is melting. Additionally, a hollow with approximately the same dimensions as the line illumination will appear on the surface of the gel. For all the samples made in this dissertation, we use an agarose concentration of 0.4% by weight (e.g. for a typical sample size, combine 10 mL of distilled water and 42 mg of agarose powder). From Ref [101], we can estimate the elastic modulus of this agarose concentration as ~13 kPa. This is a biologically relevant elasticity because the range of the Young’s modulus of soft tissues varies from ~10-1 kPa – 101 kPa [102]. In fact, we are at the upper end of the biologically relevant range of Young’s moduli meaning that the displacements we measure will be smaller (and

therefore harder to detect) in these samples compared to soft tissues with smaller Young’s moduli. (From equation 3-6, the vibration amplitude of an MNP mechanically coupled to an elastic

medium is inversely proportional to Young’s modulus, so smaller Young’s moduli will produce larger displacements.) If we can detect magnetic motion in these agarose samples, we can expect

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to expect similar or even larger displacements in soft tissues.

The agarose sample preparation is as follows. Heat water to at least 90°C and pour into a 50mL centrifuge tube, and then add the agarose powder, the TiO2, and the Fe3O4. (Note that using a smaller and narrower centrifuge tube results in the TiO2 and Fe3O4 getting stuck in the tip of the centrifuge tube.) Immediately mix using a vortexer, and then place in a 90°C water bath. Keep the agarose mixture in the hot water bath for 20 minutes, vortexing periodically. After 20 minutes, remove the water bath from its heat source and allow it to begin cooling. Continue to vortex the sample periodically. Once the agarose mixture has cooled to 55°C, pour into the sample molds, cover, and refrigerate for 20 minutes. If the agarose samples are kept hydrated and in an air-tight container in the fridge, they will last for many months.