Photoacoustic temperature measurements for ...

Photoacoustic temperature measurements for monitoring of thermal therapy Shiou-Han Wang*ab, Chen-Wei Wei c, Shiou-Hwa Jee b, Pai-Chi Li ac a Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan; b Department of Dermatology, National Taiwan University Hospital, Taipei, Taiwan; c Department of Electrical Engineering, National Taiwan University, 1 Sec.4 Roosevelt Rd, Taipei 106, Taiwan ABSTRACT Plasmonic photothermal therapy is a new cancer thermotherapy method based on surface plasmon resonance of nanoparticles. It is important to measure the temperature during thermotherapy for safety and efficacy. In this study, we apply a photoacoustic (PA) method for real-time, non-invasive temperature measurements. In particular, this method can be effectively combined with a photothermal therapy system that we developed in parallel. The method is based on the fact that the PA pressure amplitude is linearly related to temperature. To explore its potential, a home-made, 20 MHz PA transducer was used, in which an optical fiber was inserted in its center for emitting laser pulses while the PA signal was simultaneously detected. Continuous wave (CW) laser was used to heat the subject, including both phantoms and mice. The temperature of the region of interest was also measured by a fine-needle thermal couple. Results show that the temperature was linearly proportional to the PA signal with good correlation with the CW laser irradiation. The in vivo study also demonstrated potential of this technique. . Keywords: Laser, photoacoustics, plasmonic photothermal therapy, surface plasmon resonance, temperature measurement

1. INTRODUCTION Thermotherapy can be used to treat cancer by heating the tissue to 41-47 oC for tens of minutes. Plasmonic photothermal therapy is based surface plasmon resonance (SPR) of nanoparticles, and heat is generated when irradiated with laser. Gold nanoparticles absorb light at specific wavelengths effectively. The SPR can make the temperature of hot electrons of the nanostructures reach thousands of kelvins on the timescale of about 100 ps. The lattice temperature is then able to reach tens of degrees Celsius for hyperthermia [1]. During thermotherapy, it is essential to monitor the tissue temperature for safety and efficacy. Ideally, the spatial resolution of thermal mapping should be at the order of submillimeter, and temperature resolution should be less than 1oC [2]. The most accurate temperature monitoring method is direct measurement with a thermocouple. However, it is invasive and generally is not preferred and often it is not feasible. Several non-invasive temperature monitoring methods have been developed. Infrared thermography is a real-time method with 0.1 oC accuracy, but it is limited to superficial temperature only. On the other hand, ultrasound (US) can also be applied for real-time measurements at depth based on speckle tracking, but the accuracy is poor and the temperature range is limited to 5 oC. Magnetic resonance has the advantage of high resolution and accuracy, but it takes a long acquisition time and thus not real-time [2, 3, 4, 5]. Photoacoustic (PA) effect is based on generation of acoustic waves induced by optical irradiation. Two conditions should be met. The first one is thermal confinement, meaning that the heat diffusion is minimal during the excitation pulse, and thus the time for the subject to absorb the pulse energy τp should be smaller than the thermal dissipation duration τth. The second one is stress confinement, referring the time for absorbing the stress τp should be smaller than the time for the stress to transit. When both the conditions are satisfied, the pulsed laser energy can produce thermal expansion, which then induces a pressure rise P(z) and subsequently a photoacoustic (PA) signal. The PA technique has been widely applied on in biomedical fields, such as breast tumor imaging, brain functional and structural imaging, real-time blood oxygen monitoring, measurements of hemoglobin, and tumor angiogenesis. It was

Photons Plus Ultrasound: Imaging and Sensing 2009, edited by Alexander A. Oraevsky, Lihong V. Wang, Proc. of SPIE Vol. 7177, 71771S · © 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.809973

Proc. of SPIE Vol. 7177 71771S-1

also used to monitor the tissue temperature during cryotherapy and thermotherapy [6, 7]. This method can satisfy the requirements of spatial and temperature resolution. It can also be made real-time in the clinical scenario, such as ophthalmologic laser surgery [8, 9], and photothermal cancer therapy [4].

2. THEORETICAL BACKGROUND AND EXPERIMENTAL SETUP 2.1 Background The PA signal can be used for non-invasive imaging. It is produced after the subject is irradiated with pulsed laser which in turns causes a pressure rise. The pressure rise P(z) is proportional to dimensionless the Grüneisen parameter Γ:

P( z ) = (

β Cs 2 Cp

) μ aF ( z ) = Γμ aF 0e( − μ az ) ,

(1)

where β [1 oC-1] is the thermal volume expansion coefficient, Cs [ms-1] is the speed of sound, Cp [Jg-1 oC-1] is the heat capacity at constant pressure, μa [cm-1] is the absorption coefficient, and F [Jcm-2] is the laser fluence. The thermal expansion coefficient of volume is a linear function of temperature for water-based tissue in the range from 10 to 55 oC [4], and the Grüneisen parameter of water is linearly proportional to the temperature [2]:

Γ = a + bT ,

(2)

where a and b are constants, and T is the tissue temperature. Equation (1) can be rewritten as:

P ( z ) = (a + bT ) μ aF 0e( − μ az ) ,

(3)

We can rearrange the temperature-pressure relationship as:

T ( z) = (

a + bT 0( z ) P ( z ) a + bT 0( z ) ) ), + (T 0( z ) − b P 0( z ) b

(4)

Simplifying equation (4), one can obtain

T ( z ) = cP( z ) + d , where c =

(5)

a + bT 0( z ) a , d = − , and T0 and Po stand for the initial temperature and the pressure, respectively. bP 0( z ) b

Thus, we can use the PA pressure signal to measure the temperature during thermotherapy. 2.2 Experimental Setup We built a home-made PA transducer with a focal depth of 9.5 mm and a central frequency of 20 MHz (fractional bandwidth 50%). The center of the transducer was made available for an optical fiber with a diameter of 600 μm (Thorlabs, Newton, NJ) for confocal laser irradiation (LOTIS TII Ltd., Minsk, Belarus) and acoustic detection (Fig. 1). The subjects were also irradiated with a continuous wave (CW) mode laser for thermotherapy. Different concentrations of graphite in agarose, black hair and gold nanorods with absorption peaks at 800 nm (AuNR800) were used in the phantom study, and a thermal bath tank was used to stabilize the temperature of the phantoms. The gold nanoparticles were synthesized with an electrochemical method, and suspended in an aqueous solution [10]. The energy of our laser system was monitored with a laser power meter (Ophir Laser Measurement Group, North Logan, UT, USA). The NOD-scid male mice subcutaneously inoculated with oral cancer cells OECM1 (oral squamous cell carcinoma) were used for in vivo study. Under general anesthesia with isoflurane (2% with Matrx VIP-3000 veterinary anesthetic vaporizer) and oxygen supply (700 ml/min flow rate), the nanoparticles were injected subcutaneously into the tumor, and subsequently irradiated with the CW diode laser (808 nm, 660 mW, ONSET Electro-optics, Taipei, Taiwan) for plasmonic photothermal therapy. The pulsed wavelength-tunable Ti-sapphire laser (800 nm, 2.5 mJ/ pulse, pulse duration 16 ns, CF-125, SOLAR TII, Minsk, Belarus) simultaneously produced PA signals. The PA transducer was positioned by a precision translation stage (HR8, Nanomotion, Yokneam, Israel) with a step size of 0.2 mm and


controlled by a motion controller (DMC-1842, Galil Motion Control, Rocklin, CA) for one-dimensional cross-sectional scanning to reconstruct the PA imaging. The temperature of region of interest (ROI) was also measured with a fine-needle thermal couple with 0.1 oC accuracy (EDL NCF-06GS4TL3M, Danville, VA, USA) and a digital multimeter (Fluke Model 189 True RMS Multimeter, Everett, WA). The PA signal was sent to a pre-amplifier (5077PR, Panametrics, Waltham, MA) and sampled by a 12-bit data acquisition card (CompuScope 12100, Gage, Lachine, QC, Canada) with a sampling rate of 200MHz which was triggered by a photodetector (ET-2020, Electro-Optics Technology, Inc., Traverse City, MI). The system was coordinated with a program compiled with LabVIEW®, and the data were analyzed with MATLAB®. The experimental setting is shown in Fig. 2.

SMC EIectrica Connector

S'naI Wfr -

eras F1usnCanducttve Epoxy Backing

Fig. 1. The home-made photoacoustic transducer. A coaxial optical fiber is passed through the center of the transducer to confocally transmit the laser and receive the backward photoacoustic signal.

Pulsed laser

Prdct*r

Fine needle

thermocouple & multimctcr

CW4Iode laser

Fig. 2. Schematic experimental setup. For the phantom study, thermal bath tank was used to control the ambient temperature condition.


3. RESULTS AND DISCUSSION 3.1 Thermal effects and PA signal amplitude Measurements were done with a graphite phantom, black hair and gold nanoparticles in a fine tube. The temperature affects both the time shift (propagation velocity) and amplitude of the PA signal (Fig. 3) [4]. The peaks of the radiofrequency (RF) PA signal in Fig. 3(a) were located at different positions due to time shifts of the acoustic wave. They can be aligned [Fig. 3(b)] by calibrating with speed of sound C as the Marczak’s fifth order polynomial C = 1.402385*103 + 5.038813T - 5.799136*10-2T2 + 3.287156*10-4T3 - 1.398845*10-6T4 + 2.787860*10-9T5 (valid between 0-95 oC) [11]. Similarly, positions of the RF PA signals of the hair phantom can be calibrated according to this formula, but the peaks were unable to be aligned [Fig. 3(c)]. This was caused by thermal expansion of the phantom and the water tank. It was not seen in the optical fiber tip [Fig. 3(b)] because of insignificant thermal expansion effect. Nonetheless, the PA amplitude is also related to the temperature change, and thus PA amplitude is used for temperature estimation in this study. PA signal of the tip of optical fiber

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Fig.3. PA signal of the optical fiber tip and hair. (a) PA signal of the optical fiber tip before calibration with the speed of sound in different temperatures. (b) Calibrated PA signal of the optical fiber tip. The peaks of the RF signal were adjusted to the same position after calibration. (c) PA signal of the hair phantom. The positions of the RF signal varied even after calibration. It was due to the thermal expansion effect of the phantom and the thermal water tank.

Good linear relationship between the temperature and PA amplitude was found, but the deviation gets larger when the temperature was higher than 65 oC (Fig. 4). This may be due to the thermal volume expansion coefficient. The coefficient is one of the major factors of the Grüneisen parameter, and it is a linear function of temperature if the temperature goes beyond the range of 10 and 55 oC [4], and thus the Grüneisen parameter is no longer linearly proportional to the temperature in this condition [2]. However, the end point of temperature for thermotherapy are likely to be lower than 60 oC, and hyperthermia is generally heating the tissue to the temperature range 41-47 oC for tens of minutes [1]. This non-linear deviation of the PA pressure profile would not affect the applicability of this study.

PA signal (0.40% graphite in agarose) 30

25 -J

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E15 0

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20 40 60 Temperature (degree Celsius)


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PA signal (gold nanoparticles) 20 18 16 14

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40 60 80 Temperature (degree Celsius)

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Fig. 4. The temperature is linearly proportional to the PA amplitude in both the 0.40% graphite in agarose (a) and gold nanoparticles (b). The linear relationship showed deviation when the temperature was higher than about 65 oC.

When the gold nanoparticles were irradiated with the CW laser, the PA amplitude can be used for both the short-term irradiation (less than 600 s) [Fig. 5(a)] and long-term therapy (more than 30 min) [Fig. 5(b)]. In other words, the PA signal was sufficiently stable for the long duration thermotherapy. For the safety of plasmonic photothermal therapy, it is important to determine the temperature in real-time, and thus the ability of the PA signal to be rapidly responsive to the change of heat flux is critical. However, for the efficacy of thermal therapy, the target temperature has to maintain for tens of minutes [1]. This result demonstrated that the propose method meet the requirements of safety and efficacy of photothermal therapy.


PA signal of the gold nanorods 7.5

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irn off on

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Fig.5. Gold nanoparticles irradiated with CW laser. (a) Short duration (b) Long-term therapy.

.


40

3.2 in vivo Study After subcutaneous injection of gold nanoparticles into the tumor, we obtained both the US and PA images and display the fused image. The US images were displayed on gray scale, and the PA images were on pseudo-color [Fig. 6(a)]. With this combined, multi-modality imaging method, the clinician can locate the tumor for better treatment planning before starting the photothermal therapy. In addition, the therapeutic CW laser can be turned on only when the nanoparticles are targeted to the tumor. This practice can further ensure the safety.

Fig.6. Injecting gold nanorods into the tumor subcutaneously. (a) Without CW mode laser irradiation. The US and PA images were superposed. The tumor infiltrated with nanoparticles can express high PA signal before irradiated with CW mode laser. (b) With CW mode laser irradiation, the surface temperature profile was recorded with an infrared camera.

The room temperature was controlled at 20.0 oC. Before performing the in vivo PA experiments, four groups of surface temperature profiles of the tumor on the same mouse were recorded with an infrared (IR) camera (frame rate of 10 pictures/ s). Before injection of gold nanorods, the surface temperature of the tumor was kept on 24.86 + 0.15 oC (group 1), and increased minimally from 25.32 to 29.04 oC (ΔT = 3.72 oC) when irradiated with CW mode laser (group 2). After injection of nanoparticles (40 μl, 3 nM), the temperature was kept on 25.45 + 0.12 oC (group 3), but increased from 26.89 to 68.89 oC (ΔT = 42.00 oC) after irradiation for 2 minutes (group 4) [Fig. 6(b)]. By analyzing the temperature data from these four conditions, we can elucidate the thermal effect of CW laser and gold nanoparticles. The results manifested that it is insufficient to make the hyperthermic condition by mere irradiation of CW laser without nanoparticles. Nevertheless, if nanoparticles were injected into the tumor and then irradiated with CW laser at the same power, the elevated degree of temperature is more than 10-fold, and the condition of hyperthermia can be easily reached. This difference was induced by the SPR oscillation of the nanoparticles, one of the intrinsic properties of nanostructures [1]. The gold nanoparticles can be excited by a broad white light spectrum, but only wavelengths of the light corresponding to the SPR are strongly scattered [12]. During thermotherapy, the position of the mouse was fixed under general anesthesia with supply of mixture gas of isoflurane/ oxygen. The pulsed laser and CW mode laser irradiated the tumor simultaneously, and the temperature near the ROI was recorded with a fine needle thermocouple [Fig. 7(a)]. The PA image of the cross-section of the tumor was obtained, and the center of the highest intensity of signal was chosen for further analysis [Fig. 7(b)]. The less the size of the region of interest, the better accuracy was obtained to represent the hottest spot during thermotherapy. In this study, we may identify the different signals with the image resolution (200-300 μm), and the temperature resolution may reach as small as 0.1 oC. It was superior to the suggested requirements of noninvasive temperature measuring tools for thermal therapy which should reach submillimeter spatial resolution, and temperature resolution less than 1oC [2]. The PA image during thermotherapy was displayed with pseudo-color [Fig. 7(b)]. Most PA signals were distributed on the region of tumor. This is mainly caused by our local subcutaneous injection. However, the tumor may also express increased affinity to gold nanoparticles. Increased tumor metabolism may induce tumor angiogenesis, and thus the injected nanoparticles may be trapped in the tumor tissue. This biological behavior may provide more specific ability to destroy the tumor. Besides, it has been shown that gold nanoparticles may be conjugated with tumor-specific antibodies, such as anti-EGFR, anti-Her2 and anti-CXCR4 conjugated gold nanoparticles [1, 10] According to this rationale, multiple mixed targeting nanoprobes to carcinoma may be useful for simultaneous PA imaging and treatment with photothermal therapy [10].


10

I

2

LMirI mli)

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IS) Fig.7. In vivo study of PA temperature measurements to monitor laser-irradiated photothermal therapy assisted with gold nanoparticles. (a) Experiment setup. Under general anesthesia with isoflurane/ oxygen mixture, the mouse was fixed. The tumor was irradiated with pulsed laser (up) and CW mode laser (left) simultaneously, and the temperature near the ROI was directly evaluated with a fine needle thermocouple (right). (b) superposed US (displayed with gray scale) with PA (displayed with yellow pseudo-color) images during thermotherapy.

PA pressure profiles for the four events mentioned above were analyzed: (1) without injection of nanorods/ no CW mode laser irradiation (mean μ = 0.0174, standard deviation σ = 0.0054) (2) without injection of nanorods/ irradiated with CW mode laser (μ = 0.0139, σ = 0.0034) (3) with injection of nanorods/ no CW mode laser irradiation (μ = 0.1726, σ = 0.0785, T = 26.4 oC) (4) with injection of nanorods/ irradiated with CW mode laser (μ = 0.5286, σ = 0.1237, T = 40.0 oC) (Fig. 8). No significant difference between groups 1 and 2. It implies that no hyperthermic effect can be achieved if irradiated the tumor with CW mode laser without assistance of nanoparticles. A ten-fold increase in the PA amplitude was noted between the group 3 and group 1. This result indicated that for the absorption of pulsed laser energy to produce PA signal, the gold nanoparticles are superior to the intrinsic tissue chromophores, and thus gold nanoparticles can work as the “contrast medium“ for PA imaging. Significant difference existed between group 3 and 4, which was due to CW mode laser irradiation to provoke the SPR effect and resulted in tissue hyperthermia. The results were consistent with the findings shown with IR camera [Fig. 6(b)], and it also supported the reliability and stability of the temperature monitoring ability of PA system.


Maximal PA signal of the tumor 0.7 0.6 CWlaser on (with nanorods)

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Eve nit Fig.8. in vivo study of PA temperature measurements of plasmonic photothermal therapy. The four events represented CW laser off/ no nanorods injected, CW laser on/ no nanorods injected, CW laser off/ nanorods injected, and CW laser on/ nanorods injected to the tumor. Without the assistance of nanorods, no significant difference of the PA amplitude was noted even heated with CW laser (event 1 & 2). After injection of nanorods, the intensity of PA signal increased even without CW laser irradiation. It was due to the ability of nanoparticles to effectively absorb to the pulsed laser energy (event 1 & 3). With the assistance of nanorods, the PA amplitude increased significantly if the tumor was irradiated with CW laser (event 2, 3 & 4).

4. CONCLUSIONS A non-invasive real-time photoacoustic technique for multi-modality US/PA imaging and temperature monitoring during hyperthermia is presented. This combined modality can be used to study the anatomy of tumor before and during treatment, and to measure the temperature during photothermal therapy. It can be beneficial for the efficacy and the safety of thermotherapy. In our phantom and in vivo study, the PA amplitudes were correlated with the nanoparticles and CW laser irradiation. Comparing the findings measured by the thermocouple and IR camera, the PA system showed its potential. The results of this study suggest that the PA signal can be used not only for imaging purpose but also to monitor the temperature during photothermal therapy in real time.

5. ACKNOWLEDGEMENTS The authors would like to thank Dr. Churng-Ren Chris Wang, Ms. Carolina Poe, and Mr. Chi-Meng Chen for providing the gold nanoparticles, CW laser and IR camera. The authors are also grateful for support from NTU Center for Genomic Medicine, the NTU Nano Center for Science and Technology, the National Health Research Institutes, and the National Science Council through grant NSC 97-3011-P-002-009.


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