2.1.

Photoacoustic Tomography Imaging System

The PAT imaging system used is shown in Fig. 1(b). Here, a Q-switched Nd:YAG laser (Continuum, Surelite Ex) delivers laser pulses of 10 Hz at 532-nm wavelength (5-ns pulse width). Three right-angle uncoated prisms (PS911, Thorlabs) (not shown in the schematic) and one uncoated planoconcave lens L1 (LC1715, Thorlabs) were used to deliver the laser pulses from the Nd:YAG laser to the sample. The laser energy density was maintained at $\sim 9\mathrm{mJ}/{\mathrm{cm}}^2$ on the sample surface, which is much lower than the ANSI safety limit³² of $20\mathrm{mJ}/{\mathrm{cm}}^2$ at 400 to 700 nm. For point source and hair phantoms, a 532-nm wavelength laser was used. In biological tissue, the light penetration is greater in the near-infrared (NIR) wavelength region, so an optical parametric oscillator (OPO) laser (Continuum, Surelite) pumped by the Q-switched (532 nm) laser was used as the light excitation source for “N” shaped blood vessel mimicking phantom and in vivo rat brain imaging. The OPO laser was set to deliver laser pulses of 680-nm wavelength to the sample. Three right-angle coated prisms P1, P2, and P3 (PS908L-B, Thorlabs) and one uncoated planoconcave lens L1 [LC1715, Thorlabs; Fig. 1(b)] were used to deliver the laser pulses from OPO to the sample. The laser energy density was maintained at $\sim 6\mathrm{mJ}/{\mathrm{cm}}^2$ on the sample surface. A flat UST (Olympus NDT, V306-SU), a flat USTR by attaching an acoustic reflector (F102, Olympus NDT) with the flat UST, a focused UST (Olympus NDT, V306-SU-NK, $\mathrm{CF}=1.90$ in.), and a focused USTR by attaching the same acoustic reflector to the focused UST were used to acquire the PA signal. The UST had a 2.25-MHz central frequency with $\sim 70\%$ nominal bandwidth and 13-mm diameter detector surface area. Figure 1(a) shows the flat UST, the acoustic reflector, and the acoustic reflector attached with the flat UST. Each UST acquires the PA data by moving around the sample in full $2\pi$ radians in circular geometry. In all the experiments, the PA signals were collected continuously for 480 s with UST at a rotational speed of $0.75\mathrm{deg}/\mathrm{s}$. The acquired PA signals were regrouped into 800 A-lines. The acquired PA signals were first amplified and filtered using a pulse amplifier (Olympus-NDT, 5072PR) and then recorded using a data acquisition (DAQ) card (GaGe, compuscope 4227) inside a desktop (Intel xeon 3.7 GHz 64-bit processor, 16 GB RAM, running Windows 10 operating system). All PA data were acquired with a 25-MHz sampling rate. The data acquisition was synchronized with the laser illumination with a TTL sync signal from the laser.

2.2.

Phantom Experiments

Figures 1(c)–1(e) show the phantoms used for the experiments. The first phantom was a point source object made using five pencil leads of 0.5 mm diameter held using a pipette and adhered to an acrylic slab [Fig. 1(c)]. One pencil lead was placed at the scanning center, and four other pencil leads were placed at a distance of $\sim 1\mathrm{cm}$ from the scanning center. Figure 1(d) shows the second phantom made of horse hair of $150\mu \mathrm{m}$ thickness in triangular shape glued to the tips of pipettes and adhered to an acrylic slab. Each side of the triangle is $\sim 1\mathrm{cm}$. The third phantom was the blood vessel phantom made by LDPE tubes of 0.38 mm diameter filled with rat blood. These tubes were embedded inside a layer of chicken breast tissue in the shape of “N” [Fig. 1(e)]. Another chicken breast tissue layer of 5 mm thickness was placed on top [Fig. 1(e) inset]. All these phantoms were placed inside a water bath for better acoustic coupling.

2.3.

In Vivo Experiments

In vivo animal experiments were conducted using healthy female rats weighing $\sim 120\mathrm{g}$. The rats were procured from InVivos Pte. Ltd., Singapore. Animal experiments were performed according to the approved guidelines and regulations by the institutional Animal Care and Use committee of Nanyang Technological University, Singapore (Animal Protocol Number ARF-SBS/NIE-A0263). The animals were anesthetized using a cocktail of Ketamine ($120\mathrm{mg}/\mathrm{kg}$) and Xylazine ($16\mathrm{mg}/\mathrm{kg}$) injected intraperitoneally (dosage of $0.2\mathrm{ml}/100\mathrm{g}$). Before the animals were placed in the PAT scanner, the hair on the head was trimmed and then epilated using hair removal cream. A breathing mask covering the mouth and nose of the animal was used to deliver an anesthesia mixture of isoflurane and ${\mathrm{O}}_2$ during PAT imaging. The animal was placed in a sitting position on its abdomen using a custom-made animal holder. Surgical tapes were used to hold the animal in its position. The animal along with the animal holder was mounted on a translational stage during the experiment. The translational stage was used to adjust the height of the animal to adjust the scan plane of the brain. After the data acquisition, animals were euthanized by intraperitoneal injection of Valabarb (sodium pentobarbitone $300\mathrm{mg}/\mathrm{ml}$).

2.4.

Comparison of Reconstructed Images

To study the quality of the images obtained using an acoustic reflector, a signal-to-noise ratio (SNR) was calculated for both flat and focused transducers and compared with SNR levels of the images obtained using unfocused and focused transducers without an acoustic reflector. The SNR was calculated from the reconstructed PAT images as the ratio of PA amplitude to the standard deviation of the background noise, i.e., SNR (in dB) $=20\log (V/n)$, where $V$ is the PA amplitude on the object and $n$ is the standard deviation of the background noise. For point source phantom, PA amplitude was calculated for the point 3 in Fig. 1(c), and, in all the other cases, PA amplitude was calculated as the average of the PA amplitude of the whole target object. The noise was taken from the background where there was no object located.

3.1.

Phantom Experiments

Figure 2 shows the reconstructed PA images of point objects (pencil leads located one at center and four at $\sim 1\mathrm{cm}$ distance from the center). Figure 2(a) shows the reconstructed PAT images of the point targets using a flat UST, and Fig. 2(b) shows the reconstructed PA images using a flat USTR. Figures 2(c) and 2(d) show the reconstructed PA images obtained using a cylindrically focused UST and a cylindrically focused USTR, respectively. To study the quality of the images, the SNR was calculated (for point target 3). The SNR value with the flat UST was 54.9 dB and with the flat USTR was 50.56 dB. There was an $\sim 8\%$ reduction in the SNR with the flat USTR. Corresponding SNR values for the focused UST and the focused USTR were calculated to be 48.18 and 47.76 dB, respectively. There was an $\sim 0.8\%$ degradation in the SNR with the focused USTR.

Fig. 2

Cross-sectional reconstructed PAT images of the point source phantom. (a) Reconstructed PAT image obtained using flat UST, (b) reconstructed PAT image obtained using flat USTR, (c) reconstructed PAT image obtained using focused UST, (d) reconstructed PAT image obtained using focused USTR. Corresponding color bars are shown for images of flat and focused transducers. Scale bar is shown in (a) for all the images.

Next, a study was conducted using a horse hair triangular phantom. The hair was $150\mu \mathrm{m}$ thick. Each side was $\sim 1\mathrm{cm}$ in length. Figure 3(a) shows the reconstructed PAT image of the hair phantom using a flat UST, and Fig. 3(b) shows the reconstructed PA image using a flat USTR. Corresponding SNR values for these images were calculated to be 47.44 and 47.56 dB, respectively. SNR levels were maintained for flat USTR as well as those of flat UST. Figures 3(c) and 3(d) show the reconstructed PA images using focused transducers without and with an acoustic reflector, respectively. For these images, SNR values were computed to be 48.8 and 45.85 dB, respectively. The SNR was $\sim 6\%$ lower using a cylindrically focused transducer with an acoustic reflector compared to its counterpart.

Fig. 3

Cross-sectional reconstructed PAT images of triangular shaped hair phantom. (a) Reconstructed PAT image for flat UST, (b) reconstructed PAT image for flat USTR, (c, d) reconstructed PAT images for focused UST and focused USTR, respectively. Corresponding color bars are shown for flat and focused transducers. Scale bar is shown in (a) for all the images.

Next, an “N” shaped blood vessel phantom (LDPE tubes of 0.38 mm diameter) of $\sim 1\mathrm{cm}\times 1\mathrm{cm}$ dimensions embedded inside a chicken breast tissue layer was studied. A layer of 5-mm thick chicken breast tissue was placed on top of this layer. The reconstructed PAT images for unfocused UST and unfocused USTR are shown in Figs. 4(a) and 4(b), respectively. The corresponding SNR levels for these images were computed to be 24.61 and 24.32 dB, resepctively. Figures 4(c) and 4(d) show cross-sectional reconstructed PA images obtained through a cylindrically focused transducer without and with acoustic reflector, respectively. The SNR values were calculated to be 30.56 and 31.01 dB, respectively. The SNR was $\sim 1.5\%$ higher using a focused USTR than a focused UST.

Fig. 4

Cross-sectional reconstructed PAT images of “N” shaped blood vessel phantom embedded inside chicken breast tissue. (a) Reconstructed PAT image for flat UST, (b) reconstructed PAT image obtained using flat USTR, (c) cross-sectional reconstructed PAT image for focused UST, (d) cross-sectional reconstructed PAT image obtained using focused USTR. Corresponding color bars are shown for the images of flat and cylindrically focused transducers. Scale bar is shown in (a) for all the images.

3.2.

In Vivo Experiments

Next, a study was conducted on small animals to demonstrate the feasibility of using an acoustic reflector augmented to a conventional UST to obtain similar quality images to that of a conventional UST. As described earlier, anesthetized healthy female rats were used for this study. Figure 5 shows the cross-sectional reconstructed PAT images of the animal brain. The images obtained for flat UST without and with acoustic reflector are shown in Figs. 5(a) and 5(b), respectively. Figure 5(c) shows the cross-sectional reconstructed PA image of the focused UST, and Fig. 5(d) shows the cross-sectional reconstructed PA image of the focused USTR. In all four images, superior sagittal sinus (SS) and transverse sinuses (TS) were clearly visible (yellow arrows). Figure 5(e) shows the image of the animal head before taking PAT images, and Fig. 5(f) shows the image of the animal brain after cutting open the top layer of skin on the head after the image acquisition was completed. To study the quality of the images, the SNR was calculated. The SNR was 22.76 dB (flat UST) and 18.66 dB (flat USTR). There was an $\sim 18\%$ reduction in the SNR levels with the usage of an acoustic reflector. The SNR for the images obtained with the focused UST was 26.61 dB and that for the focused USTR was 31.63 dB. There was a $\sim 19\%$ enhancement in the SNR levels using the focused transducer with an acoustic reflector compared to that of the images from the focused transducer without an acoustic reflector.

Fig. 5

Reconstructed cross-sectional PAT images of rat brain. (a) Reconstructed PAT image for flat UST, (b) reconstructed cross-sectional PAT image for flat USTR, (c) cross-sectional reconstructed PAT image obtained using focused UST, (d) reconstructed cross-sectional PAT images obtained using focused USTR. SS: superior sagittal sinus, TS: transverse sinus. Yellow arrows point to the SS, TS. Corresponding color bars are shown for unfocused and cylindrically focused transducers. Scale bar is shown in (a) for all the images. (e) Rat brain after trimming and removal of hair on the head region before PAT imaging. (f) Rat brain after cutting open the top skin layer of the head region after the PAT imaging is completed.

The ultrasound waves generated from the sample fall at 45 deg to the water and acoustic reflector (stainless steel) interface. At this incidence angle, due to the total internal reflection phenomenon, the ultrasound waves are entirely reflected back into the water medium. These reflected waves are detected by the UST. As discussed above, the SNR was maintained in many cases, but there was an $\sim (1\text{to}19)\%$ enhancement or degradation in the SNR levels for a few cases. This was due to the small difference ($\sim 2$ to 3 mm) in the distance at which the UST and USTR were placed from the scanning center. The amplitude of the ultrasound signal detected by the UST is inversely proportional to the square of the distance from the source (target object) to the detector surface, so the small difference in the distances results in SNR variations. If the scanning radii were kept exactly the same for the UST and USTR, then SNR levels in these cases would also have been similar. Visually, the quality of the PAT images obtained using an acoustic reflector augmented to the conventional USTs was nearly similar to that of the PAT images obtained using conventional transducers of both flat and focused UST.

For better understanding of the quality of the PAT images using an acoustic reflector, the resolution of the PAT system was compared for all four transducers. The resolution was calculated using full-width at half-maximum from the point spread function of the hair phantom images. For flat UST and flat USTR, the resolution was 301 and $281\mu \mathrm{m}$, respectively. For focused UST and focused USTR, it was computed to be 294 and $269\mu \mathrm{m}$, respectively. This resolution matches well with the theoretically expected resolution of $250\mu \mathrm{m}$ for a 2.25 MHz UST, so the use of acoustic reflector did not affect the spatial resolution of the PAT system. The frequency response of the transducers with and without the reflector was also compared. As expected, we did not see any degradation of the transducer bandwidth with or without the reflector. The frequency response for all four transducers was calculated by taking Fourier transforms of the Aline PA signal (averaged six times) from the hair phantom. The bandwidth was calculated from the frequency response. The bandwidth for flat UST, flat USTR, focused UST, and focused USTR was found to be 2.29, 2.24, 2.46, and 2.44 MHz, respectively.

Hence, by using the acoustic reflector augmented to the conventional UST surface at 45 deg, we can use the transducer in the vertical plane instead of the horizontal plane. Therefore, the transducer rotates in a smaller circle around the sample. This vertical configuration will lead to a size reduction of the water tank used for housing the transducer as well as the target object (the length and width of the tank will be reduced by $\sim 11.5\mathrm{cm}$: this includes the length of the UST and partial length of the connecting cable). This reduces the volume of the PAT system. Also, the load on the motor that rotates the UST decreases as the torque required to rotate the UST decreases. Hence, the overall PAT system size can be minimized. Together with a PLD-based PAT system,³⁰ this configuration of UST will make the entire PAT system quite compact and hopefully translation to clinical applications will be easier. As the use of the acoustic reflector did not affect the SNR levels or the spatial resolution (as well as the bandwidth of the ultrasound detectors) of the PAT system, this acoustic reflector can be used with any PAT system in which single-element USTs with circular scanning geometry are used. This will reduce the scanning radius and will make the PAT imaging system more compact in size. The USTR-based PAT system can find applications in small animal brain imaging, breast cancer imaging, etc.