2.1.

Dual-Wavelength PA Technique

In this study, we examined the simplest case where the PA signals were generated from locations close to tissue surface. In this case, Eq. (1) can be simplified into $\mathrm{\Delta }P(z=0)=\mathrm{\Gamma }{\mu }_a{F}_0$. In most former studies,^{12–16,18,20} the temperature dependency of tissue optical properties was not considered because major tissue absorbers, such as hemoglobin, have been reported to exhibit minor changes in optical absorption ($<3.5\%$) over the temperature range of 20 to 40°C.³⁰ It was also found that, in the temperature range below coagulation threshold, the optical absorption coefficient ${\mu }_a$ in both human skin³¹ and canine prostate³² did not change much with increased temperature. Although in these studies negligible changes in optical absorption with temperature increase were observed only at low temperature ranges (up to 40°C) since it is not feasible to measure the optical properties at high temperatures without inducing changes in tissue composition, we assume that the temperature dependency of tissue optical properties would still be negligible through the entire temperature range (up to temperatures above coagulation threshold). On the contrary, ${\mu }_a$ is highly dependent on the tissue type/status. For example, thermal-induced tissue coagulation could lead to significant changes in ${\mu }_a$.^33–37 Therefore, in this work, ${\mu }_a$ is expressed as a function of laser wavelength and tissue type/status. The laser-induced pressure rise can now be expressed as

Upon examination of the pressure rise obtained at different temperatures, $T_0$ and $T_1$, with corresponding tissue statuses $s_0$ and $s_1$, we can write $\mathrm{\Delta }{P}_{(s_0,T_0,\lambda )}={\mathrm{\Gamma }}_{(s_0,T_0)}{\mu }_{a(s_0,\lambda )}{F}_{0(\lambda )}$ and $\mathrm{\Delta }{P}_{(s_1,T_1,\lambda )}={\mathrm{\Gamma }}_{(s_1,T_1)}{\mu }_{a(s_1,\lambda )}{F}_{0(\lambda )}$ based on Eq. (2). The relative signal change from $T_0$ to $T_1$ is therefore expressed as

If the tissue status remains the same from $T_0$ to $T_1$, then $s_1=s_0$ and the relative signal change is simplified to $[{\mathrm{\Gamma }}_{(s_0,T_1)}-{\mathrm{\Gamma }}_{(s_0,T_0)}]/{\mathrm{\Gamma }}_{(s_0,T_0)}$, which is independent of the laser wavelength. Therefore, we can expect the relative signal change to be the same for different wavelengths when there is no change in tissue type/status.

2.2.

HIFU Ablation and Dual-Wavelength PA Sensing

Ex vivo porcine myocardium specimens obtained from the local abattoir were used in this study. To demonstrate the ability of dual-wavelength PA detection technique in monitoring tissue status without being affected by temperature change, one group of tissue specimens was treated with HIFU application to generate thermal lesions prior to PA measurement, while the other group of specimens was untreated. The HIFU system consisted of an HIFU transducer (2 MHz center frequency, $F=0.98$, Blatek Inc., State College, Pennsylvania), a delay/pulse generator (Model 565, Berkeley Nucleonics Corp., San Rafael, California), a function generator (33220A, Agilent, Santa Clara, California), and a power amplifier (325LA, ENI, Rochester, New York), as shown in Fig. 1(a). A tissue specimen ($22\mathrm{mm}\times 22\mathrm{mm}\times 13\mathrm{mm}$) was placed in a plastic holder with the front and the back sides covered by acoustically transparent membranes (Tegaderm, 3M, St. Paul, Minnesota) to prevent possible changes in hydration and concentration of chromophores in the tissue specimen when submersed in water. Each ablation was conducted using a 20 s pulsed HIFU exposure ($1500\mathrm{W}/{\mathrm{cm}}^2$ focal intensity, 80% duty cycle, 10 Hz pulse repetition frequency), with the HIFU focus aimed near the tissue surface for visual confirmation of the ablation outcome. The HIFU transducer was moved using translation stages to generate a bigger volume of coagulated tissue.

Fig. 1

Schematic illustration of experimental setup for (a) HIFU ablation and (b) PA sensing in a temperature-controlled water bath. (c) and (d) Typical PA signal and photodiode signal. Vp-p denotes the peak-to-peak amplitude.

Figure 1(b) shows the schematic experimental setup to obtain PA signals from the HIFU lesions and nontreated tissues at various temperatures. The system consisted of a temperature-controlled water bath (Precision 280, Thermo Scientific, Waltham, Massachusetts), a circulating pump (Peri-Star Pro, World Precision Instruments Inc., Sarasota, Florida), a focused imaging transducer (10 MHz center frequency, V311, Panametrics, Waltham, Massachusetts), and a laser source. Tissue temperatures were controlled using the water bath and measured with a K-type thermocouple (0.08 mm diameter, Omega Engineering, Stamford, Connecticut), which was inserted in the specimen and sampled at 20 Hz by a data acquisition system (OMB-DAQ-3000, Omega Engineering). An Nd:YAG (Brilliant B, Quantel, Bozeman, Montana) pumped optical parametric oscillator (OPO) system (Vibrant 532 I, Opotek Inc., Carlsbad, California) provided laser pulses at 700 and 800 nm wavelengths with a pulse duration of 5.5 ns and a pulse repetition rate of 10 Hz. These two wavelengths were chosen for two main reasons: (1) the output energy from the laser system is strong and stable in this range and (2) the major chromophores at these two wavelengths include oxygenated hemoglobin (${\mathrm{HbO}}_2$), deoxygenated hemoglobin (Hb), and MetHb, and their optical absorptions are different between 700 and 800 nm,^38,39 which allows detection of changes in ratio $k$ when the concentrations of these chromophores change due to thermal coagulation. The energy of the laser beam was measured using a photodiode (Model 2031, New Focus, Santa Clara, California) for later normalization. The PA signals received from the imaging transducer, which worked on the transmission mode, were first amplified by 40 dB with a preamplifier (5052PR, Panametrics) and then recorded with a 200 MHz sampling rate on a digital oscilloscope (54380B, Agilent). In this experiment, the temperature was only increased from room temperature to 43°C to prevent any tissue alteration. For all measurements, the laser beam was centered at the lesion center and the beam size (3 mm in diameter) was smaller than the lesion cross-section. PA signals were acquired for 10 s at each wavelength at each temperature. Gross photos of the tissue specimens were taken after the experiment.

2.3.

Thermal Treatments Using Water Bath Heating

To investigate the changes in signal ratio $k$ during thermal treatments, PA sensing was performed on tissue specimens undergoing water bath heating using the same setup in Fig. 1(b). The temperature of the water tank was gradually increased from 35 to 60°C to ensure that tissue coagulation occurred during treatment. The heating rate was $0.20\pm 0.03°\mathrm{C}/\min$ from 43 to 54°C and $0.06\pm 0.02°\mathrm{C}/\min$ from 54 to 60°C. The circulating temperature-controlled system required a longer time to raise water temperature at higher temperatures, resulting in the slower heating rate above 54°C. At each temperature/time measurement point, PA signals were acquired for 10 s at each wavelength, first at 700 nm and then at 800 nm. The time interval between the start of PA signal acquisition using 700 and 800 nm illuminations was $14.11\pm 1.05\mathrm{s}$, with the absolute difference of the average tissue temperature being $0.07\pm 0.05°\mathrm{C}$. The small time and temperature differences and the slow heating rate allowed our assumption that changes in the tissue status or composition during signal acquisition were negligible.

2.4.

Data Processing

The normalized PA amplitude was obtained by dividing the peak-to-peak PA signal amplitude [Fig. 1(c)] by the peak amplitude of the photodiode signal [Fig. 1(d)] to minimize the effect due to the fluctuation of the laser energy over time. The measured PA signals were averaged over 10 s to improve signal-to-noise ratio. We denote the averaged normalized PA amplitude as $P$. The ratio $k$ (which may differ from the $k$ in Eq. (3) by some constant) was computed using

To investigate how PA signals depend on temperature and to compare different wavelengths, the relative signal change (%) of $P$ over temperature was computed based on Eq. (4) using

3.1.

Ratio $k$ is Temperature Independent

As an example, Fig. 2(a) shows the gross photo of an HIFU lesion on the surface of a tissue specimen. As shown in Fig. 2(b) and 2(c), the $\text{mean}\pm \text{standard}$ deviation (S.D.) of the relative signal change (%) of the averaged normalized PA amplitude $P$ in the native (no coagulated lesion) tissues ($N=9$) and in the HIFU lesions ($N=8$) computed using Eq. (6) exhibits the same trend for both wavelengths used in the experiments. Since the temperature range was kept below 43°C, and measurements were made within 2 h for each sample, no tissue alteration occurred as observed from the gross photo and calculated using the thermal damage model.^40–42 Thus these results confirmed that the relative percentage change in the PA signal intensity is independent of the wavelength at any temperature when the tissue properties remain unchanged. Therefore, the ratio $k$ between these two wavelengths is expected to be constant at different temperatures, as demonstrated in Fig. 3, where $k=P_{700\mathrm{nm}}/P_{800\mathrm{nm}}$ is constant from 25 to 43°C. Combining all data obtained from individual specimens and at 10 different temperatures, $k=2.28\pm 0.20$ in native tissues ($N=90$) and $2.70\pm 0.18$ in HIFU lesions ($N=80$). The difference in the $k$ value between these two groups is statistically significant ($p$ value $<0.001$). The threshold for differentiating the two groups is $k=2.52$, as determined using the receiver-operating characteristic curve,⁴³ providing an 85.3% accuracy with an area under curve of 0.93.

Fig. 2

(a) Gross photo of a tissue specimen with an HIFU lesion generated before PA experiment. (b) and (c) The relative signal change (%) of the averaged normalized PA amplitude $P$ as a function of temperature in native tissues and in HIFU lesions. $T_0=25.3\pm 0.3°\mathrm{C}$.

Fig. 3

Comparison of the dual-wavelength ratio $k$ ($700\text{n}\mathrm{m}/800\text{n}\mathrm{m}$) in HIFU lesions and native tissues as a function of temperature.

3.2.

Ratio $k$ Increases with Tissue Changes During Thermal Treatment

The averaged normalized PA signal $P$ at 700 and 800 nm from one tissue specimen subjected to water bath heating are shown in Fig. 4(a) and 4(b) to illustrate the change of PA signals with increasing temperature. Both of the PA signal amplitudes obtained with 700 and 800 nm laser excitations increased with increasing tissue temperature, with a relatively slower increase below 50°C and a much faster increase when temperature increased further. However, from either of these single wavelength results it is difficult to determine exactly when coagulation or changes in tissue status/content occurred, as both increased temperature and tissue alteration caused the PA signals to increase in amplitude.

Fig. 4

An example of coagulation during water bath heating. (a) and (b) The averaged normalized PA amplitude $P$ as a function of temperature at 700 and 800 nm wavelengths. (c) Corresponding dual-wavelength ratio $k$ ($700\text{n}\mathrm{m}/800\text{n}\mathrm{m}$).

By taking the ratio of the PA signals from the two wavelengths to eliminate the effect of temperature in the PA signals, the change in tissue status can be better identified as shown in Fig. 4(c). Unlike $P$, which is temperature dependent, $k$ is nearly constant with increasing temperature until a sharp increase appeared at 56°C, which allows clear identification of the temperature at which changes in tissue occurred.

Figure 5(a) shows the $\text{mean}\pm \mathrm{S.D.}$ of ratio $k$ from five independent measurements. Again, it can be seen that $k$ is nearly constant before it increases sharply at 55°C, suggesting that changes in the optical properties of the tissues during the coagulation process occurred at this temperature. Coagulation of the tissue samples was confirmed at the end of each experiment. Coagulated tissue appeared pinkish and white in contrast to its original dark red color. Individual curves of $k$ for all five specimens are shown in Fig. 5(b). $k$ in each experiment follows a similar trend, exhibiting a constant value before it increases sharply at a temperature between 55 and 58°C.

Fig. 5

Dual-wavelength ratio $k$ ($700\text{n}\mathrm{m}/800\text{n}\mathrm{m}$) during the coagulation process: (a) $\text{Mean}\pm \mathrm{S.D.}$ ($N=5$) and (b) individual plots as a function of temperature (°C).

In two sets of measurements, the coagulated tissues were allowed to cool down using the temperature-controlled water bath and $k$ values were obtained in the coagulated tissues at a lower temperature range of 37 to 44°C. Our experiments confirmed that $k$ remained constant in this temperature range, similar to the native tissue samples, except that $k=5.00\pm 0.27$ in the coagulated tissues ($N=19$), which is statistically significantly different from their native state when $k=2.55\pm 0.13$ ($N=14$) with a $p$ value $<0.001$, indicating that the tissue status alone affects the values of $k$.