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. Author manuscript; available in PMC: 2020 Aug 15.
Published in final edited form as: Opt Lett. 2019 Aug 15;44(16):4063–4066. doi: 10.1364/OL.44.004063

Real-time Photoacoustic Sensing for Photo-mediated Ultrasound Therapy

Wei Zhang 1,&, Yu Qin 1,2,&, Xinyi Xie 3, Zizhong Hu 3, Yannis M Paulus 1,4, Xinmai Yang 5,*, Xueding Wang 1,*
PMCID: PMC6907727  NIHMSID: NIHMS1061621  PMID: 31415547

Abstract

Photo-mediate ultrasound therapy (PUT) is a novel, non-invasive anti-microvascular approach that can treat neovascularization with high precision. We developed a photoacoustic (PA) sensing (PAS) system for PUT, and achieved real-time PAS-guided PUT. Experiments performed on chicken yolk sac membrane model demonstrated that PAS could monitor the treatment effect in a microvessel during PUT. Vessel shrinkage induced a decrease in PA signal amplitude, while vessel rupture induced an abrupt increase in PA signal amplitude. The integrated PUT and PAS system can significantly improve the safety and effectiveness of PUT, and may assist with clinical translation of this novel anti-microvascular technique.

1. INTRODUCTION

Angiogenesis and vasculogenesis, which are characterized by the formation of new micro-size blood vessels, are hallmarks during embryogenesis and contribute to a broad range of pathological conditions such as cancer, arthritis, and ocular diseases [1-5]. Currently available anti-vascular treatments, such as anti-VEGF therapy [6] and photodynamic therapy (PDT) [7, 8], have drawbacks such as drug toxicity and resistance, financial burden, and collateral damage to surrounding tissues [9-12]. New vessel-targeted therapeutic approaches with reduced side effects are in great demand.

Recently, a novel anti-vascular technique named photo-mediated ultrasound therapy (PUT) has been developed [13,14]. PUT employs synchronized nanosecond-laser pulses and ultrasound bursts to achieve highly selective and precise treatment of blood vessels. The underlying mechanism of PUT is photoacoustic (PA) cavitation, or more generally, the photospallation effect [15, 16]. After a nanosecond laser pulse is absorbed by blood, strong transient thermal-elastic stress wave, or PA wave is produced. For a spherically or cylindrically shaped object (e.g. a blood vessel), the laser-induced photoacoustic wave can converge into the center and produce a rarefaction wave with significantly high amplitude. When the rarefaction wave overlaps with an ultrasound burst applied at the same time, cavitation in the vessels can occur [17]. The resulted bubbles, when further driven by the ultrasound burst, can induce different anti-vascular effects, including vessel shrinkage and rupture, depending on the laser and ultrasound parameters applied.

The high selectivity of PUT is facilitated by the large optical absorption contrast between hemoglobin and other tissues [18, 19]. When working in the visible to near-infrared spectrum, hemoglobin absorbs significantly more optical energy than other tissues. Hence, PUT treatment effect targets at the vessels containing high concentration of hemoglobin; while other tissues with much less optical absorption will be left intact. The high precision of PUT is due to the fact that the cavitation produced is only located in the vessels. Unlike thermal-based therapy, cavitation only damages a thin layer of tissue that is in direct contact, and can potentially achieve treatment accuracy better than 100 μm [20, 21]. PUT is a synergistic effect of the light pulses and the ultrasound bursts. Neither the light pulses nor the ultrasound bursts alone can produce any treatment effect. In addition, both the laser energy level and the ultrasound pressure used in PUT are much lower than those in traditional laser thermal therapies and high intensity focused ultrasound (HIFU) therapy [13].

During PUT treatment of a blood vessel, the vascular response is not only determined by the laser and the ultrasound parameters, but also a function of the vessel size and type (e.g. vein or artery). Therefore, to further improve the effectiveness, minimize the side-effect, and achieve personalized treatment by using PUT, a sensing system that can provide real-time feedback reflecting the response of treated vessels is desired. Some commercially available imaging approaches including color photography, optical coherence tomography angiography (OCTA), confocal microscopy, and Doppler ultrasound imaging have all been used to visualize microvasculature and responses to treatment [22-25]. However, each of these imaging modalities requires additional equipment to be added upon the PUT setup, and will make the monitoring and treatment process cumbersome and more costly.

Biomedical PA imaging and sensing (PAI/PAS) technology, based on the detection of laser-generated ultrasound signals, can provide optical absorption information in deep biological samples with excellent ultrasonic resolution [26]. As an emerging imaging tool, the capability of PAI for visualizing microvasculature and evaluating angiogenesis had been explored [27]. The feasibility of integrating PAI with a HIFU system has also been demonstrated previously [28-30], where the combined treatment and PAI system was used for pretreatment planning and post-treatment evaluation. In this work, for the first time, PAS technology has been integrated with PUT to achieve real-time PAS-guided treatment of microvessels without interrupting the treatment process. This integration is natural, as both PUT and PAS are fundamentally based on the PA effect. The nanosecond laser employed as the light source for PUT also induces detectable PA signals for evaluating the treatment. At the same time, the HIFU transducer used to generate the ultrasound bursts for PUT can be used as the receiver for PAS. Hence, in a PAS-guided PUT system, both the laser and the ultrasound transducer are shared by the sensing setup and the treatment setup, largely reducing the size and the cost of the system. During the PUT treatment of vessels, the response of targeted vessels can be monitored in real-time by PAS to facilitate personalized treatment with efficiency and safety improved. To validate the feasibility and effectiveness of such a system, the experiments were conducted on a chicken yolk sac membrane model in vivo.

2. METHOD

A. System setup

An integrated system combining PA sensing and PUT treatment, as shown in Figure 1a, was developed to investigate the feasibility of using the same laser and the same ultrasound transducer for PAS-guided PUT in real time. An Nd:YAG laser (Continuum Powerlite DLS 8010, Santa Clara, CA) working at 532-nm wavelength was used as the light source, providing laser pulses with 5-ns pulse duration and 10-Hz pulse repetition rate. The jitter of the laser pulsing is less than 1 ns. The applied light energy on the sample was continuously and precisely controlled by a pair of polarizers. Part of the treatment beam energy, split by a dichroic mirror, was directed to a photodiode for real-time monitoring of the laser energy applied to the sample. The treatment laser beam was coaxially aligned with a guiding laser beam. The aiming beam was from a continuous wave (CW) helium neon laser working with a low power of 7.3 mW. The aiming beam facilitated accurate positioning of the treatment area. Another illumination light beam, after being expanded by a convex lens and then reflected by a dichroic mirror (DM), was also merged with the treatment beam and directed to the sample. The reflection of the illumination light from the sample was detected by a charge coupled device (CCD) camera for pretreatment positioning and after treatment validation [13]. The strong reflection of the 532-nm treatment light from the sample was blocked by a filter placed in front of the CCD camera.

Fig. 1.

Fig. 1.

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Schematic diagram of real-time PA-guided PUT system and timing sequence. (a) Schematic diagram of the system; (b) Timing sequence of the system. DM: Dichroic Mirror; PH: Pin Hole; DG: Delay Generator; FG: Function Generator; PR: Pulser/Receiver.

A 0.5-MHz therapeutic ultrasound transducer (H107, Sonic Concepts, Bothell, WA) with a geometric focal distance of 63.2 mm, a focal depth of 21.42 mm, and a focal width of 3.02 mm, was used to deliver ultrasound bursts to the treatment area, and also detect the PA signals induced by the laser. The ultrasound transducer was immersed in the water for acoustic coupling, facing toward the sample which was placed on the water surface and fixed by a translation stage. The temperature of water was maintained at 37.5°C degree. The focal area of the transducer was coaxially aligned with the laser treatment spot on the sample. The transducer worked on the transmission mode (for PUT) and the receiving mode (for PAS) alternatively, controlled by single pole double throw (SPDT) electromechanical relay failsafe switch (PE71S6052, Pasternack, Irvine, CA). The transducer and the switch were connected through an impedance coupling circuit to match the impedance. In the transmission mode, a power amplifier (240L, Electronics & Innovation, Rochester, NY), which was controlled by a function generator (DS345, Stanford Research System, Sunnyvale, CA ), was used to drive the transducer to transmit ultrasound bursts. In the receiving mode, the PA signals were detected by the transducer and then amplified by a pulser/receiver (PR 5072, Olympus, Japan). Then, following the sampling clock from the delay generator, the PA signals and the output of the photodiode used to monitor the laser energy were acquired by a 14-bit digitizer card (Razor 14, GaGe), and finally transferred to a PC.

The timing sequence of the system, which was controlled by a low jitter delay generator (DG 535, Stanford Research System, Sunnyvale, CA) with <100ps rms jitter, is shown in Figure 1b. For both PUT and PAS, the treatment laser beam delivered to the sample surface had a 1.5-mm beam diameter controlled by an adjustable pinhole. The Q-switch sync output of laser system was used as the external trigger of delay generator. Every two laser pulses, which contained one for PAS and the other for PUT treatment, were treated as a sequence. In each sequence, the first laser pulse was used for PA sensing, while its corresponding trigger signal was used to trigger the ultrasound burst with a given delay. The delay was designed to ensure the ultrasound burst can arrive at the target vessel right before the second laser pulse for PUT treatment. The distance between the HIFU transducer and blood vessel can be measured accurately by detecting the PA signal from the vessel. After finish one PA sensing sequence, HIFU transducer was switched to the transmission mode and maintain for 100 ms, then switched back to the receiving mode under the control of the second channel of the delay generator. During the transmission mode, the HIFU transducer delivered an ultrasound tone burst at 0.5 MHz, with 10% duty cycle and 5-Hz pulse repetition rate. The sampling clock, synchronized with the laser pulses on the receiving mode, was used to trigger the DAQ card to acquire the produced PA signals and the photodiode output in real time with 200 MHz sampling rate. A LabView program was used to control the digitizer and display the PA signals. The PA signal amplitude at each laser pulse was normalized by the output of photodiode before it was displayed on the monitor. To study the relative change in PA signal amplitude for monitoring the PUT treatment effect, the initial PA signal amplitude before the PUT treatment of treated area was recorded as a baseline for later normalization.

B. Chicken yolk sac membrane model

Fertilized chicken eggs were purchased from a local farm (Townline Poultry Farm, Inc., Zeeland, MI) and cultured in a 38.0°C humidified incubator as reported [31, 32]. In brief, on the embryo development day 3-5 (EDD3-5), eggs were cleaned with 70% ethanol, gently cracked, and the chick yolk sac membranes were transferred to a sterilized petri dish. The petri dish was cut to form a 6 cm-diameter hole at the bottom and was covered by a piece of plastic wrap (Glad Cling Wrap) to allow the ultrasound bursts or the PA signals to propagate through. For all experiments, the posterior vitelline vein (p.v. vein) was selected as the target vessel because it barely has any branches and its size is relatively consistent on the same EDD.

3. RESULT

A. PA detection of different vascular responses

By controlling the optical and ultrasound settings during PUT, different treatment outcomes can be achieved. When using 10 mJ/cm2 fluence light pulse and 0.15 MPa negative peak pressure of ultrasound burse, no vascular change (i.e. no effect) happened in chicken yolk sac membrane. When using 10 mJ/cm2 fluence light pulse and 0.25 MPa negative peak pressure of ultrasound burse, vessel shrinkage was noticed. When using 20 mJ/cm2 fluence light pulse and 0.25 MPa negative peak pressure of ultrasound burse, vessel rupture happened. These different vascular responses to the PUT treatment under different optical and ultrasound settings could be subsequently detected by PAS. During our experiment, PA signals were acquired before and right after the treatments leading to different treatment outcomes (no effect, vessel shrinkage, and vessel rupture) to analyze the relation between PA signal amplitude and treatment effect. Figure 2 shows the results correspondent to these three different treatment outcomes, including the photographs of the treated chicken yolk sac membranes, and the normalized PA signal amplitudes before and after the treatment. In the situation of vessel shrinkage as a result of the treatment, a decrease in PA signal amplitude was observed; in the situation of the vessel rupture as a result of the treatment, an abrupt increase in PA signal amplitude was observed; while in the situation of no vascular change (i.e. no effect) in response to the treatment, no obvious change in PA signal amplitude was noticed. To evaluate the statistical significance, this experiment was competed with forty-five samples in total (shrinkage, no effect, and rupture were measured 15, 10, and 20 times, respectively). For each group, a paired t-test was conducted to compare the PA measurements before and after the treatment. Statistical significance (p<0.001) was achieved for the shrinkage and the rupture groups, demonstrating that PA measurement successfully detected the vascular response, including vessel shrinkage and vessel rupture, to the PUT treatment. No statistically significant difference was observed in the no-effect group.

Fig. 2.

Fig. 2.

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Correlation between the PA signals and vessel sizes. (a)-(c) show the situation of vessel shrinkage. (d)-(f) show the situation of no effect. (g)-(i) show the situation of vessel rupture. (j) shows the statistical analysis of the changes in PA signal amplitude in respond to PUT leading to three different treatment outcomes (i.e. shrinkage, no effect, and rupture). The black circles indicate the treated area of PUT.

B. Real-time monitoring the treatment effect

To further investigate the changes in blood vessels during PUT, the PA signals were acquired in real time during the treatment. The measurement results were divided into three groups based on the three different treatment outcomes (i.e. vessel shrinkage, no effect, and vessel rupture). The typical patterns presenting the changes in PA signal amplitude over time are shown in Figure 3(a)-(c) for the three outcomes. The vessel shrinkage led to a rapid decrease in PA signal amplitude until reaching a steady state indicating that the vessel did no shrink any more. For the vessel that did not show any observable treatment effect, the PA signal as a function of time was stable in amplitude. In the result from a vessel ruptured as a result of the treatment, the PA signal amplitude was initially stable. Once the rupture happened, the PA signal amplitude kept increasing over time until the local hemorrhage became stable. The normalized changes in PA signal amplitude at the end of the treatment were averaged for each of the treatment outcomes, as shown in Figure 3(d). With the results from a total of 15 measurements for each group, t-test was performed to distinguish the three different groups. A statistically significant difference (p<0.001) was achieved for any two of the three groups, indicating that PA measurement is capable of differentiating the different outcomes from PUT.

Fig. 3.

Fig. 3.

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Real-time PA monitoring of the vascular response to PUT treatment. (a) PA signal amplitude over time during a PUT treatment leading to vessel shrinkage. (b) PA signal amplitude over time during a PUT treatment without leading to any noticeable treatment effect. (c) PA signal amplitude over time during a PUT treatment leading to vessel rupture. (d) Statistic analysis of the PA signal amplitude change at the end of the PUT treatment leading to three different treatment outcomes. Red dashed lines indicate the start time of the ultrasound bursts, i.e. start of PUT treatment.

C. Threshold Analysis

As shown in Figure 4, in the result from the no-effect group, the instability of the laser output energy and the detection system led to less than 10% variation of the PA signal amplitude. Both the decrease of PA signal amplitude due to the vessel shrinkage and the increase of PA signal amplitude due to the vessel rupture led to variations of the PA signal amplitude above 10%. When setting a threshold at +10%, as shown by the red line in Figure 4, the entire rupture group could be separated from the other two groups, demonstrating that we could separate the rupture from the other treatment outcomes to minimize unwanted vessel ruptures.

Fig. 4.

Fig. 4.

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The ranges of PA signal amplitudes during the PUT treatment leading to three different treatment outcomes (i.e. shrinkage, no effect, and rupture). A threshold at +10%, as marked by the red line, can separate the rupture group from the other two groups.

4. Discussion

The technical development described in this work focuses on addressing several barriers faced by PUT aiming at safely and efficiently treating blood vessels in biological samples. First, an integrated PAS and PUT system was developed so that the therapeutic effect can be monitored in real time. By sharing the same HIFU transducer and the same laser source, the PAS area and the PUT treatment area are naturally co-registered, avoiding the need for alignment which is necessary when using an independent imaging system for treatment monitoring. When working on biological samples with complex structure and inhomogeneous optical and ultrasound parameters, this alignment can be challenging. Second, the system design can also verify the good ultrasound coupling between the transducer and the sample surface. This is due to the fact that a good ultrasound coupling will ensure good PA signal detection, while a poor ultrasound coupling will result in weak or none PA signal. As noticed in our previous study on rabbit eye model [13], a poor ultrasound coupling during PUT can lead to deposition of the ultrasound energy on the interface between the transducer and the biological sample, leading to unwanted damage of the tissue (e.g. the surface of the eye). When the ultrasound coupling can be verified by PA detection, the safety of PUT is further improved. Third, real-time monitoring the treatment outcome will further contribute to the safety and effectiveness of PUT. An advantage of PUT is that the treatment effect can be controlled by adjusting the optical and ultrasound parameters applied during the treatment. However, the optimal parameters may vary from patient to patient, making it difficult to achieve optimal outcomes for all patients using a set of universal parameters. PAS can monitor the vascular response to PUT in real time, providing an immediate online feedback during the treatment. As shown in the threshold analysis, a threshold can be set to avoid the unwanted outcome of vessel rupture. For some clinical applications in ophthalmology, local hemorrhage may cause serious complications. By setting the specified threshold for the PAS-guided PUT, vessel rupture can be distinguished early before serious damages can occur, and the treatment can be stopped immediately to prevent further damage. In these regards, the safety of PUT can be further improved.

In conclusion, an integrated PAS and PUT treatment system was developed to realize PAS-guided PUT in real time. Experiments performed on the chicken yolk sac membrane model demonstrated the feasibility and effectiveness of this technique in monitoring the treatment effect without interrupting the treatment process, which means that this technique development can be directly transferred to in vivo application. With the capability to address several technical barriers of PUT, the integrated real-time PAS could further enhance the safety and efficacy of the newly invented PUT technology, which is crucial for quick translation to clinic. To further demonstrate its clinical feasibility and values, studies on animal models of diseases need to be conducted. The effectiveness of this technique in minimizing the side effects, such as preventing the rupture of neoangiogenic vessels during the treatment, needs to be further investigated.

Acknowledgment

This study is supported in part by NIH 1R01EY029489 (XY), the National Eye Institute 1K08EY027458 (YMP), the Alliance for Vision Research (YMP), and unrestricted departmental support from Research to Prevent Blindness (YMP). This work utilized the Vision Research Core Center funded by P30EY007003 from the National Eye Institute.

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