In vivo safety study using radiation at wavelengths and dosages relevant to intravascular imaging.

SIGNIFICANCE: Intravascular photoacoustic (IVPA) imaging can identify native lipid in atherosclerotic plaques in vivo. However, the large number of laser pulses required to produce 3D images is a safety concern that has not been fully addressed. AIM: We aim to evaluate if irradiation at wavelengths and dosages relevant to IVPA imaging causes target vessel damage. APPROACH: We irradiate the carotid artery of swine at one of several energy dosages using radiation at 1064 or 1720 nm and use histological evaluation by a pathologist to identify dose-dependent damage. RESULTS: Media necrosis was the only dose-dependent form of injury. Damage was present at a cumulative fluence of 50 J / cm2 when using 1720 nm light. Damage was more equivocally identified at 700 J / cm2 using 1064 nm. CONCLUSIONS: In prior work, IVPA imaging of native lipid in swine has been successfully conducted below the damage thresholds identified. This indicates that it will be possible to use IVPA imaging in a clinical setting without damaging vessel tissue. Future work should determine if irradiation causes an increase in blood thrombogenicity and confirm whether damaged tissue will heal over longer time points.

Dual-Illumination Ultrasound/ Photoacoustic System for Cervical Cancer imaging.

Early stage cancer detection technologies can provide functional information and potentially decrease the mortality rate caused by cervical cancer. In our previous work, a miniaturized ultrasound and photoacoustic endoscopic system has been developed to image the cervical tissue through the cervical canal to fulfills the need for a safe, low-cost, and high-resolution functional diagnostic system. However, the miniaturized size of endoscope and American National Standards Institute safety limits cause constraints of using high-intensity illumination during imaging. In addition, the strong light scattering of tissues limits the light penetration depth. Fortunately, the cervix anatomy allows for the delivery of additional light from the ectocervix by using an external illumination system. Here we propose a dual, co-planar illumination system, which can provide adequate illumination to the cervical tissue via combined internal and external light delivery strategies. Therefore, an increase in the area of light-tissue interaction allows us to raise the laser light energy while keeping fluence under safety limits. Thus, a reliable PA imaging can be obtained for the whole cervical tissue thickness. The system performance was tested using a Monte Carlo simulation, and laser-light fluence was calculated and compared at different depths within a simulated cervical-tissue model. The results indicated a higher and more uniform fluence in the Monte Carlo simulations. In addition, the photoacoustic imaging of the proposed system was evaluated by two cervical tissue-mimicking phantoms with human blood and graphite rods as inclusions inside it. In accordance with the simulations, the phantom study revealed a more reliable photoacoustic signal for the entire depth of the phantoms with an improved contrast to noise ratio and signal to noise ratio, and a higher coverage ratio of the imaging field of view. In summary, the dual-mode illumination system can provide more realistic information of inclusions within the tissue while considering safety limits, which can lead to more accuracy in biomarker detection for cervical cancer diagnostics.

Laser threshold and cell damage mechanism for intravascular photoacoustic imaging.

OBJECTIVES: Intravascular photoacoustic (IVPA) imaging is being developed to image atherosclerotic plaques, a leading cause of morbidity and mortality in the United States. However, the safety of this imaging modality, which requires repeated irradiation with short laser pulses, has not yet been investigated. This study has two objectives. First, determine in vitro the limit of cumulative fluence that can be applied to cells before death at IVPA relevant wavelengths. Second, evaluate if high single pulse fluences are a potential cause of cell death during IVPA imaging. MATERIALS AND METHODS: Experiments were conducted using endothelial cells, macrophages, and smooth muscle cells. The cumulative fluence experiments were conducted at 1064 and 1197 nm, using a high pulse repetition frequency laser. Cells were irradiated with a wide range of cumulative fluences and evaluated for cell death. The thresholds for death were compared to the maximum expected clinical cumulative fluence. To evaluate the effect of single pulse fluences, cells were irradiated at 1064, 1210, and 1720 nm. Light was delivered at a range of pulse energies to emulate the fluences that cells would be exposed to during clinical IVPA imaging. RESULTS: At 1064 nm, all three cell types remained viable at cumulative fluences above the maximum expected clinical cumulative fluence, which is calculated based on common IVPA imaging protocols. At 1197 nm, cells were viable near or just below the maximum expected clinical cumulative fluence, with some cell type to cell type variation. All three cell types remained viable after irradiation with high single pulse fluences at all three wavelengths. CONCLUSION: The cumulative fluence experiments indicate that safety considerations are likely to put constraints on the amount of irradiation that can be used in IVPA imaging protocols. However, this study also indicates that it will be possible to use IVPA imaging safely, since cumulative fluences could be reduced by as much as two orders of magnitude below the maximum expected clinical cumulative fluence by varying the imaging protocol, albeit at the expense of image quality. The single pulse fluence experiments indicate that cell death from single pulse fluence is not likely during IVPA imaging. Thus, future studies should focus on heat accumulation as the likely mechanism of tissue damage. Lasers Surg. Med. 51:466-474, 2019. © 2018 Wiley Periodicals, Inc.

Integrated optical coherence tomography and multielement ultrasound transducer probe for shear wave elasticity imaging of moving tissues.

Accurate measurements of microelastic properties of soft tissues in-vivo using optical coherence elastography can be affected by motion artifacts caused by cardiac and respiratory cycles. This problem can be overcome using a multielement ultrasound transducer probe where each ultrasound transducer is capable of generating acoustic radiation force (ARF) and, therefore, creating shear waves in tissue. These shear waves, produced during the phase of cardiac and respiratory cycles when tissues are effectively stationary, are detected at the same observation point using phase-sensitive optical coherence tomography (psOCT). Given the known distance between the ultrasound transducers, the speed of shear wave propagation can be calculated by measuring the difference between arrival times of shear waves. The combined multitransducer ARF/psOCT probe has been designed and tested in phantoms and ex-vivo studies using fresh rabbit heart. The measured values of shear moduli are in good agreement with those reported in literature. Our results suggest that the developed multitransducer ARF/psOCT probe can be useful for many in-vivo applications, including quantifying the microelasticity of cardiac muscle.

Laser-activated perfluorocarbon nanodroplets: a new tool for blood brain barrier opening.

A major obstacle in the monitoring and treatment of neurological diseases is the blood brain barrier (BBB), a semipermeable barrier that prevents the delivery of many therapeutics and imaging contrast agents to the brain. In this work, we explored the possibility of laser-activated perfluorocarbon nanodroplets (PFCnDs) to open the BBB and deliver agents to the brain tissue. Specifically, near infrared (NIR) dye-loaded PFCnDs comprised of a perfluorocarbon (PFC) core with a boiling point above physiological temperature were repeatedly vaporized and recondensed from liquid droplet to gas bubble under pulsed laser excitation. As a result, this pulse-to-pulse repeated behavior enabled the recurring interaction of PFCnDs with the endothelial lining of the BBB, allowing for a BBB opening and extravasation of dye into the brain tissue. The blood brain barrier opening and delivery of agents to tissue was confirmed on the macro and the molecular level by evaluating Evans Blue staining, ultrasound-guided photoacoustic (USPA) imaging, and histological tissue analysis. The demonstrated PFCnD-assisted pulsed laser method for BBB opening, therefore, represents a tool that has the potential to enable non-invasive, cost-effective, and efficient image-guided delivery of contrast and therapeutic agents to the brain.

Optimization of dual-wavelength intravascular photoacoustic imaging of atherosclerotic plaques using Monte Carlo optical modeling.

Coronary heart disease (the presence of coronary atherosclerotic plaques) is a significant health problem in the industrialized world. A clinical method to accurately visualize and characterize atherosclerotic plaques is needed. Intravascular photoacoustic (IVPA) imaging is being developed to fill this role, but questions remain regarding optimal imaging wavelengths. We utilized a Monte Carlo optical model to simulate IVPA excitation in coronary tissues, identifying optimal wavelengths for plaque characterization. Near-infrared wavelengths (≤1800 nm) were simulated, and single- and dual-wavelength data were analyzed for accuracy of plaque characterization. Results indicate light penetration is best in the range of 1050 to 1370 nm, where 5% residual fluence can be achieved at clinically relevant depths of ≥2 mm in arteries. Across the arterial wall, fluence may vary by over 10-fold, confounding plaque characterization. For single-wavelength results, plaque segmentation accuracy peaked at 1210 and 1720 nm, though correlation was poor (<0.13). Dual-wavelength analysis proved promising, with 1210 nm as the most successful primary wavelength (≈1.0). Results suggest that, without flushing the luminal blood, a primary and secondary wavelength near 1210 and 1350 nm, respectively, may offer the best implementation of dual-wavelength IVPA imaging. These findings could guide the development of a cost-effective clinical system by highlighting optimal wavelengths and improving plaque characterization.

Real-Time Intravascular Ultrasound and Photoacoustic Imaging.

Combined intravascular ultrasound and intravascular photoacoustic (IVUS/IVPA) imaging is an emerging hybrid modality being explored as a means of improving the characterization of atherosclerotic plaque anatomical and compositional features. While initial demonstrations of the technique have been encouraging, they have been limited by catheter rotation and data acquisition, displaying, and processing rates on the order of several seconds per frame as well as the use of off-line image processing. Herein, we present a complete IVUS/IVPA imaging system and method capable of real-time IVUS/IVPA imaging, with online data acquisition, image processing, and display of both IVUS and IVPA images. The integrated IVUS/IVPA catheter is fully contained within a 1-mm outer diameter torque cable coupled on the proximal end to a custom-designed spindle enabling optical and electrical coupling to system hardware, including a nanosecond-pulsed laser with a controllable pulse repetition frequency capable of greater than 10 kHz, motor and servo drive, a US pulser/receiver, and a 200-MHz digitizer. The system performance is characterized and demonstrated on a vessel-mimicking phantom with an embedded coronary stent intended to provide IVPA contrast within content of an IVUS image.

The dynamic deformation of a layered viscoelastic medium under surface excitation.

In this study the dynamic behavior of a layered viscoelastic medium in response to the harmonic and impulsive acoustic radiation force applied to its surface was investigated both theoretically and experimentally. An analytical solution for a layered viscoelastic compressible medium in frequency and time domains was obtained using the Hankel transform. A special incompressible case was considered to model soft biological tissues. To verify our theoretical model, experiments were performed using tissue-like gel-based phantoms with varying mechanical properties. A 3.5 MHz single-element focused ultrasound transducer was used to apply the radiation force at the surface of the phantoms. A phase-sensitive optical coherence tomography system was used to track the displacements of the phantom surface. Theoretically predicted displacements were compared with experimental measurements. The role of the depth dependence of the elastic properties of a medium in its response to an acoustic pulse at the surface was studied. It was shown that the low-frequency vibrations at the surface are more sensitive to the deep layers than high-frequency ones. Therefore, the proposed model in combination with spectral analysis can be used to evaluate depth-dependent distribution of the mechanical properties based on the measurements of the surface deformation.

Visualization of molecular composition and functionality of cancer cells using nanoparticle-augmented ultrasound-guided photoacoustics.

Assessment of molecular signatures of tumors in addition to their anatomy and morphology is desired for effective diagnostic and therapeutic procedures. Development of in vivo imaging techniques that can identify and monitor molecular composition of tumors remains an important challenge in pre-clinical research and medical practice. Here we present a molecular photoacoustic imaging technique that can visualize the presence and activity of an important cancer biomarker - epidermal growth factor receptor (EGFR), utilizing the effect of plasmon resonance coupling between molecular targeted gold nanoparticles. Specifically, spectral analysis of photoacoustic images revealed profound changes in the optical absorption of systemically delivered EGFR-targeted gold nanospheres due to their molecular interactions with tumor cells overexpressing EGFR. In contrast, no changes in optical properties and, therefore, photoacoustic signal, were observed after systemic delivery of non-targeted gold nanoparticles to the tumors. The results indicate that multi-wavelength photoacoustic imaging augmented with molecularly targeted gold nanoparticles has the ability to monitor molecular specific interactions between nanoparticles and cell-surface receptors, allowing visualization of the presence and functional activity of tumor cells. Furthermore, the approach can be used for other cancer cell-surface receptors such as human epidermal growth factor receptor 2 (HER2). Therefore, ultrasound-guided molecular photoacoustic imaging can potentially aid in tumor diagnosis, selection of customized patient-specific treatment, and monitor the therapeutic progression and outcome in vivo.

Correspondence: Spatial variations of viscoelastic properties of porcine vitreous humors.

Using a microbubble-based acoustic radiation force approach, spatial variations of Young's modulus and shear viscosity of the porcine vitreous humors in two groups--young pigs (6 months old) and mature pigs (2 to 3 years old)--were measured in situ. The measurements in these groups (4 specimens in each group) were performed in several positions along an anterior-to-posterior direction. At each position, microbubbles were generated by focusing a nanosecond pulsed laser beam and the displacement of each microbubble in response to an impulsive acoustic radiation force was measured every 10 µs using a custom-made high-pulse-repetition-frequency ultrasound system. Based on measured dynamics of the microbubble, Young's modulus and shear viscosity at various locations of the vitreous were reconstructed. Young's moduli of the young and mature porcine vitreous at anterior region were the highest, whereas the central region had the lowest values, indicating the clear spatial variations in the vitreous humor elasticity in both groups.

The mechanical properties of ex vivo bovine and porcine crystalline lenses: age-related changes and location-dependent variations.

The mechanical properties of ex vivo animal lenses from three groups were evaluated: old bovine (25-30 mo old, n = 4), young bovine (6 mo old, n = 4) and young porcine (6 mo old, n = 4) eye globes. We measured the dynamics of laser-induced microbubbles created at different locations within the crystalline lenses. An impulsive acoustic radiation force was applied to the microbubble, and the microbubble displacements were measured using a custom-built high pulse repetition frequency ultrasound system. Based on the measured dynamics of the microbubbles, Young's moduli of bovine and porcine lens tissue in the vicinity of the microbubbles were reconstructed. Age-related changes and location-dependent variations in the Young's modulus of the lenses were observed. Near the center, the old bovine lenses had a Young's modulus approximately fivefold higher than that of young bovine and porcine lenses. The gradient of Young's modulus with respect to radial distance was observed in the lenses from three groups.

Assessing the mechanical properties of tissue-mimicking phantoms at different depths as an approach to measure biomechanical gradient of crystalline lens.

We demonstrate the feasibility of using the dominant frequency of the sample surface response to a mechanical stimulation as an effective indicator for sensing the depthwise distribution of elastic properties in transparent layered phantom samples simulating the cortex and nucleus of the crystalline lens. Focused ultrasound waves are used to noninvasively interrogate the sample surface. A phase-sensitive optical coherence tomography system is utilized to capture the surface dynamics over time with nanometer scale sensitivity. Spectral analysis is performed on the sample surface response to ultrasound stimulation and the dominant frequency is calculated under particular loading parameters. Pilot experiments were conducted on homogeneous and layered tissue-mimicking phantoms. Results indicate that the mechanical layers located at different depths introduce different frequencies to the sample surface response, which are correlated with the depth-dependent elasticity of the sample. The duration and the frequency of the ultrasound excitation are also investigated for their influences on this spectrum-based detection. This noninvasive method may be potentially applied for localized and rapid assessment of the depth dependence of the mechanical properties of the crystalline lens.

Intravascular photoacoustic imaging of exogenously labeled atherosclerotic plaque through luminal blood.

Combined intravascular ultrasound and intravascular photoacoustic (IVUS/IVPA) imaging has been previously established as a viable means for assessing atherosclerotic plaque morphological and compositional characteristics using both endogenous and exogenous contrast. In this study, IVUS/IVPA imaging of atherosclerotic rabbit aortas following systemic injection of gold nanorods (AUNRs) with peak absorbance within the tissue optical window is performed. Ex vivo imaging results reveal a high photoacoustic signal from localized AUNRs in regions with atherosclerotic plaques. Corresponding histological staining further confirms the preferential extravasation of AUNRs in atherosclerotic regions with compromised luminal endothelium and acute inflammation. The ability to detect AUNRs using combined IVUS and photoacoustic imaging in the presence of luminal saline and luminal blood is evaluated using both spectroscopic and single wavelength IVPA imaging techniques. Results demonstrate that AUNR detection within the arterial wall can be achieved using both methods, even in the case of imaging through luminal blood.

In vivo intravascular ultrasound-guided photoacoustic imaging of lipid in plaques using an animal model of atherosclerosis.

We present a preliminary study demonstrating the capability of ultrasound-guided intravascular photoacoustic (IVPA) imaging to visualize the depth-resolved distribution of lipid deposits in atherosclerotic plaques in vivo. Based on the characteristic optical absorption of lipid in the near infrared wavelength range, IVPA imaging at a single, 1720 nm, wavelength was used to provide a spatially-resolved, direct measurement of lipid content in atherosclerotic arteries. By overlaying an IVPA image with a spatially co-registered intravascular ultrasound (IVUS) image, the combined IVPA/IVUS image was used to visualize lipid distribution within the vessel wall. Ultrasound-guided IVPA imaging was performed in vivo in the abdominal aorta of a Watanabe heritable hyperlipidemic (WHHL) rabbit. Subsequently, the excised rabbit aorta filled with a solution of red blood cells (RBC) was then imaged ex vivo, and histology was obtained in the section adjacent to the imaged cross-section. To demonstrate the potential for future clinical application of IVPA/IVUS imaging, a sample of diseased human right coronary artery (RCA) was also imaged. Both in vivo and ex vivo IVPA images clearly showed the distribution of lipid in the atherosclerotic vessels. In vivo IVPA imaging was able to identify diffuse, lipid-rich plaques in the WHHL rabbit model of atherosclerosis. Furthermore, IVPA imaging at a single wavelength was able to identify the lipid core within the human RCA ex vivo. Our results demonstrate that ultrasound-guided IVPA imaging can identify lipid in atherosclerotic plaques in vivo. Importantly, the IVPA/IVUS images were obtained in presence of luminal blood and no saline flush or balloon occlusion was required. Overall, our studies suggest that ultrasound-guided IVPA imaging can potentially be used for depth-resolved visualization of lipid deposits within the anatomical context of the vessel wall and lumen. Therefore, IVUS/IVPA imaging may become an important tool for the detection of rupture-prone plaques.

Feasibility of in vivo intravascular photoacoustic imaging using integrated ultrasound and photoacoustic imaging catheter.

Pilot studies of in vivo combined intravascular ultrasound (IVUS) and intravascular photoacoustic (IVPA) imaging are reported. A recently introduced prototype of an integrated IVUS/IVPA imaging catheter consisting of a single-element ultrasound transducer and a light delivery system based on a single optical fiber was adapted and used for in vivo imaging of a coronary stent deployed in a rabbit's thoracic aorta in the presence of luminal blood. The results suggest that in vivo IVUS/IVPA imaging is feasible using the integrated IVUS/IVPA imaging catheter. The challenges of in vivo combined IVUS/IVPA imaging are discussed, and further improvements on the design of the catheter and the clinical imaging system are proposed.

A high pulse repetition frequency ultrasound system for the ex vivo measurement of mechanical properties of crystalline lenses with laser-induced microbubbles interrogated by acoustic radiation force.

A high pulse repetition frequency ultrasound system for an ex vivo measurement of mechanical properties of an animal crystalline lens was developed and validated. We measured the bulk displacement of laser-induced microbubbles created at different positions within the lens using nanosecond laser pulses. An impulsive acoustic radiation force was applied to the microbubble, and spatio-temporal measurements of the microbubble displacement were assessed using a custom-made high pulse repetition frequency ultrasound system consisting of two 25 MHz focused ultrasound transducers. One of these transducers was used to emit a train of ultrasound pulses and another transducer was used to receive the ultrasound echoes reflected from the microbubble. The developed system was operating at 1 MHz pulse repetition frequency. Based on the measured motion of the microbubble, Young's moduli of surrounding tissue were reconstructed and the values were compared with those measured using the indentation test. Measured values of Young's moduli of four bovine lenses ranged from 2.6 ± 0.1 to 26 ± 1.4 kPa, and there was good agreement between the two methods. Therefore, our studies, utilizing the high pulse repetition frequency ultrasound system, suggest that the developed approach can be used to assess the mechanical properties of ex vivo crystalline lenses. Furthermore, the potential of the presented approach for in vivo measurements is discussed.

Intravascular photoacoustic imaging of lipid in atherosclerotic plaques in the presence of luminal blood.

Intravascular photoacoustic (IVPA) imaging can characterize atherosclerotic plaque composition on the basis of the optical absorption contrast between different tissue types. Given the high optical absorption of lipid at 1720 nm wavelength, an atherosclerotic rabbit aorta was imaged at this wavelength ex vivo using an integrated intravascular ultrasound (IVUS) and IVPA imaging catheter in the presence of luminal blood. Strong optical absorption of lipid combined with low background signal from other tissues provides a high-contrast, depth-resolved IVPA image of lipid. The ability to image lipid at a single wavelength without removing luminal blood suggests that in vivo detection of lipid in atherosclerotic plaques using combined IVUS/IVPA imaging is possible.

Estimation of mechanical properties of a viscoelastic medium using a laser-induced microbubble interrogated by an acoustic radiation force.

An approach to assess the mechanical properties of a viscoelastic medium using laser-induced microbubbles is presented. To measure mechanical properties of the medium, dynamics of a laser-induced cavitation microbubble in viscoelastic medium under acoustic radiation force was investigated. An objective lens with a 1.13 numerical aperture and an 8.0 mm working distance was designed to focus a 532 nm wavelength nanosecond pulsed laser beam and to create a microbubble at the desired location. A 3.5 MHz ultrasound transducer was used to generate acoustic radiation force to excite a laser-induced microbubble. Motion of the microbubble was tracked using a 25 MHz imaging transducer. Agreement between a theoretical model of bubble motion in a viscoelastic medium and experimental measurements was demonstrated. Young's modulii reconstructed using the laser-induced microbubble approach were compared with those measured using a direct uniaxial method over the range from 0.8 to 13 kPa. The results indicate good agreement between methods. Thus, the proposed approach can be used to assess the mechanical properties of a viscoelastic medium.

Photoacoustic imaging of prostate brachytherapy seeds.

Brachytherapy seed therapy is an increasingly common way to treat prostate cancer through localized radiation. The current standard of care relies on transrectal ultrasound (TRUS) for imaging guidance during the seed placement procedure. As visualization of individual metallic seeds tends to be difficult or inaccurate under TRUS guidance, guide needles are generally tracked to infer seed placement. In an effort to improve seed visualization and placement accuracy, the use of photoacoustic (PA) imaging, which is highly sensitive to metallic objects in soft tissue, was investigated for this clinical application. The PA imaging properties of bare (i.e., embedded in pure gelatin) and tissue-embedded (at depths of up to 13 mm) seeds were investigated with a multi-wavelength (750 to 1090 nm) PA imaging technique. Results indicate that, much like ultrasonic (US) imaging, an angular dependence (i.e., seed orientation relative to imaging transducer) of the PA signal exists. Despite this shortcoming, however, PA imaging offers improved contrast, over US imaging, of a seed in prostate tissue if sufficient local fluence is achieved. Additionally, although the PA signal of a bare seed is greatest for lower laser wavelengths (e.g., 750 nm), the scattering that results from tissue tends to favor the use of higher wavelengths (e.g., 1064 nm, which is the primary wavelength of Nd:YAG lasers) when the seed is located in tissue. A combined PA and US imaging approach (i.e., PAUS imaging) shows strong potential to visualize both the seed and the surrounding anatomical environment of the prostate during brachytherapy seed placement procedures.

Photoacoustic imaging of clinical metal needles in tissue.

The ability to visualize and track temporarily or permanently implanted metal devices is important in many applications ranging from diagnosis to therapy. Specifically, reliable imaging of metal needles is required in today's clinical settings. Currently, ultrasound is utilized to image a needle inserted into tissue in real time. However, the diagnostic value and tracking ability of these images depends highly on the orientation of the needle, and also its proximity to regions of interest in the tissue. We examine the use of photoacoustic imaging combined with current ultrasound imaging methods to obtain high-contrast images of commonly used needles in the body. Experiments were performed using 21 G and 30 G needles inserted into ex vivo porcine tissue and tissue-mimicking phantoms. The needles and surrounding tissue were imaged using an ultrasound imaging system interfaced with the pulsed laser source necessary for photoacoustic imaging. The results suggest that photoacoustic imaging, combined with ultrasound imaging, is capable of real-time, high-contrast, and high-spatial-resolution visualization of metal implants within anatomical landmarks of the background tissue.