*2.3. Tissue-Mimicking Phantoms*

The performance of the three different illumination methods was evaluated with regards to the PA imaging depth using a phantom made of polyvinyl chloride (PVC) (M-F Manufacturing Super Soft, Fort Worth, TX, USA), with 0.2% fine ground silica (US Silica MIN-U-SIL5, Stow, OH, USA) used as an optical diffuser. To create a PVC phantom [43], the plastisol was first mixed with the ground silica and then heated in a microwave to 170 ◦C. In a mold, three graphite rods with a 2 mm diameter were placed horizontally, and the PVC was poured to create the three layers as shown in Figure 3. After cooling, the phantom was removed from the mold and used for the described experiments. Graphite was chosen as an absorber due to its broadband absorption characteristics and ease of placement inside the phantom.

**Figure 3.** (**a**) Graph illustrates the polyvinyl chloride (PVC) phantom and graphite inclusions and their dimensions. (**b**) A photograph of the experimental setup including the ring US transducer. A motorized translational stage was used to adjust the position of the phantom to acquired images at multiple cross-sections.

#### *2.4. UST and PAT Image Reconstruction*

In all instance, the waveform method was used to reconstruct the UST images [44], while filtered back-projection was utilized to reconstruct the PAT images [45]. For PAT back-projection reconstruction, the RF (radio frequency) values for each cross-section were averaged 10 times to increase the overall SNR. In UST mode, a 20 dB linear gain TGC was used for acquiring the US images, while in PAT mode, as previously described, a linear 10 dB TGC was used for data acquisition. For US imaging, the used TGC was optimized to minimize the transducer saturation, which resulted in cleaner images. This was done empirically. For PAT imaging, the value chosen was designed to reduce the signal emanating from other cross-sections from appearing in the cross-section of interest.

#### **3. Results and Discussion**

#### *3.1. A Comparison of the Three Di*ff*erent Illumination Methods*

The results discussed in this section focus on analyzing the PAT images and PA signal amplitudes from progressively deeper phantom cross-sections using the discussed illumination methodologies. Figure 4 shows the UST and PAT images for the first and third cross-sections, which are separated by 60 mm. The PAT images are masked based on the region of interest (ROI) as determined by the UST image. For each illumination method, the PA amplitude is normalized to the highest value for the method across all cross-sections. For example, for the full-ring illumination method, images were normalized to the highest amplitude of PA detected within the three slices, which are separated by 30 mm each. This allows for visualization of the effect of depth on the PA signal amplitudes for each illumination method. As can be seen in the PAT images, the graphite absorber is visible in the first and third layers (i.e., larger vertical depth) of the full-ring illumination method, which is not the case for the diffuse and point-source methods.

To further quantify the results shown in Figure 4, the PA signal amplitude across the graphite absorber for each illumination method, and for each cross-section, is plotted in Figure 5. For the full-ring illumination method, the PA values are nearly constant across the three cross-sections (Figure 5a). However, the peak amplitude value of the PA signal decreases by 25 times for point-source illumination and 15 times for the diffused-beam illumination between the first and third cross-sections. As seen by the uniformity in the amplitudes between three cross-sections for the full-ring illumination method, this imaging technique can provide a consistent image regardless of vertical depth. On the other hand, the diffused and point illumination methodologies show variance in PA amplitude signals. This finding demonstrates that the full-ring illumination is capable of providing sufficient fluences at lower vertical depths, which results in detectable and reliable PAT images across cross-sections.

**Figure 5.** PA amplitude across the graphite absorber for three different cross-sections for: (**a**) full-ring, (**b**) diffused-beam, and (**c**) point illumination.

To further compare the performance of the three illumination strategies, the SNR and contrastto-noise ratio (CNR) of the PAT images were measured. In the SNR, the value is calculated by:

$$\text{SNR} = 20 \times \log 10 \left(\frac{M\_S}{M\_B}\right)^2$$

where *MS* refers to the mean of the PA signal, as marked by the US image region of interest, while *MB* refers to the mean of the phantom background. The CNR is determined by:

$$\text{CNR} = 20 \times \log 10 \left( \frac{M\_S - M\_B}{\sigma\_B} \right)^2$$

where *MS* refers to the mean of the PA signal, *MB* refers to the mean of the phantom background, and σ*B* is the standard deviation of the phantom background. For the full-ring illumination method, the used PA amplitude values are from irradiating the target cross-section 15 mm below the cross-section of interest. As can be seen in Figure 6a,b, the SNR and CNR are nearly constant for the full-ring illumination across the three cross-sections, which is not the case for the diffuse and point illuminations. Figure 6c also plots the amplitudes across the graphite object at the third cross-section for all illumination methods. Based on the laser beam diameter of 8 mm; optical losses in the system; and 100 mJ input energy, the diffuse illumination method has a fluence of 9.3 mJ/cm2, compared to 175 mJ/cm<sup>2</sup> for point illumination, and 7 mJ/cm<sup>2</sup> for the full ring illumination. Full-ring calculations use a beam height of 4 mm circumferentially on a 9 cm diameter phantom, and diffused beam calculations use a beam diameter of 30 mm at the phantom. Even though point illumination has about 25 times the fluence of the full-ring illumination method, its amplitude is much smaller at this cross-sectional depth.

**Figure 6.** The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the PA amplitudes at three different cross-sections are plotted in (**<sup>a</sup>**,**b**), respectively. For the full-ring illumination, the values were determined based on the illumination at 15 mm below the cross-section of interest. (**c**) Plots the PA amplitude for the top cross-section (cross-section 3) for full-ring, diffuse, and point illumination.

When compared to the diffused-beam and point illuminations, full-ring illumination has a higher PA amplitude at Cross-section 3. It has a near constant SNR, CNR, and PA signal amplitude across all cross-sections, making it an effective illumination method for breast imaging. Given that imaging breast regions close to the chest wall (i.e., large vertical depth) are clinically important, the full-ring illumination shows promise in accessing these regions and thus provides a means for reliable whole breast PAT imaging with a ring US transducer.

#### *3.2. The PA Amplitude of the Targeted Cross-Section as a Function of Illumination Position*

The optimum position for the full-ring beam was investigated by evaluating the effect of the distance between the ring-shaped beam and the targeted cross-section. The distance between the targeted cross-section and the full-ring beam was changed within a range of 0–20 mm. Zero millimeters represents the case where the ring beam is illuminating the targeted cross-section at the graphite rod, while the 20 mm case is when the ring beam is 20 mm below the graphite rod (Figure 7a). In this study, a selected cross-section was imaged while changing the illumination location from 0 to 20 mm. The five different positions chosen to illuminate the targeted cross-section of the graphite rod were 0, 5, 10, 15, and 20 mm. A 532 nm laser with 100 mJ per pulse energy was used for this experiment. The location of the full-ring illumination was adjusted by translating the phantom in steps of 5 mm in the vertical direction.

**Figure 7.** (**a**) The image shows the different positions of the ring beam based on the targeted cross-section (Cross-section 1). The targeted cross-section is located in the central field of view of the US elements. (**b**) PA amplitude at Cross-section 1 plotted as a function of illumination depth below the cross-section.

In Figure 7b, the PA amplitude was measured by drawing a line across the targeted graphite for all five positions. The overall shape of the PA amplitude is constant while the maximum increases as one moves further below the desired imaging cross-section. This increased visibility could be due to the fact that the incidence angle of the beam illuminates the central part of the object more directly as it moves below the cross-section. A possible reason for the stronger PA signal, when illuminating 20 mm below the targeted cross-section, could the incident angle of the light diffusion within the tissue. In the current prototype, the ring mirror is illuminating the object cross-section at an angle of 39 degrees with respect to the object surface. The optimum illumination will occur if the ring beam falls normal to the surface. The 0 and 5 mm cross-sections use the US image as a mask to crop out the large PA peaks generated at the surface of the phantom. The graphite absorber was embedded within the PVC background with a margin from the surface. Here, we only evaluated the signal arising from the absorber. In cases where the illumination was coincident with the center of the transducer, a strong PA signal from the surface was observed (not shown in these masked images). Hence, imaging below the transducer can help to better visualize more central parts of the object due to elimination of a large PA signal arising from the light-entering surface.

The large PA signal at the surface is primarily due to the large fluence at the surface, which can affect the visualization of deeper regions due to a limited dynamic range in PA acquisition. This is not the case where the illumination was adjusted to 10 to 20 mm with respect to the center of the US detectors (i.e., illuminating below the imaged plane). The averaged PA amplitude from all five illumination methods (shown in red in Figure 7b) is similar to the 10 mm illumination results. It is worth mentioning that these results are not necessarily general for all ring illumination systems and are dependent on the incident angle of the beam with respect to the object (39 degrees in our case). The results might vary in other ring illumination systems if the incident angle is changed.

These findings are important because they help to define the illumination scenario based on the characteristics and size of the given object. For example, for a large diameter phantom one might need to acquire PAT images using more than one position of full-ring illumination to cover the regions closer to the outer surface and deeper regions (with illumination offset from the imaging plane). In addition, illuminating below the targeted cross-section (below the central line of the US elements) could help to limit the high PA signals that are generated from the outer surface of the scanned object. The used illumination method has a significant effect on the imaging's vertical depth, which is significant when visualizing anatomy such as the breast.
