No cancer left behind: a testbed and demonstration of concept for photoacoustic tumor bed inspection

Approximately 19% of breast cancer patients undergoing breast conserving surgery (BCS) must return for a secondary surgery due to incomplete tumour removal. We propose a single sensor, low-frequency hand-held photoacoustic imaging (PAI) probe for detection of residual cancer tissue during BCS within the surgical cavity and on the excised specimen based on lipid content differences. The probe incorporated a single polyvinylidene fluoride acoustic sensor, a 1-to-4 optical fibre bundle and a polycarbonate axicon lens for light delivery. A phantom consisting of nylon strings was imaged to find an optimal scanning geometry and resolution of the probe. The effect of limited angular coverage was evaluated by comparing the PAI results of a phantom mimicking an ex-vivo breast cancer specimen obtained with the hand-held probe and near-full view PAI system. Translation of the probe with 4 mm steps and rotation over 6° steps resulted in lateral and axial resolution of 1.8 mm and 1 mm, respectively. Experiments with the prototype hand-held PAI probe at 930 nm resulted in excellent image contrast exclusively from lipids. Lipid-free gaps mimicking positive margins were clearly visible in the images. Compared to images from the near full-view PAI system, the hand-held PAI probe had a higher signal-to-noise ratio but suffered from more negativity image artefacts. Taken together, the results show that PAI with the hand-held probe has the potential for detection of residual breast cancer tissue during BCS.

Hybrid optoacoustic and pulse-echo ultrasound imaging is an attractive multi-modal combination owing to the highly complementary contrast of the two techniques. Efficient hybridization is often hampered by significant dissimilarities between their optimal data acquisition and image formation strategies. Herein, we introduce an approach for combined optoacoustic and ultrasound imaging based on a plano-concave detector array design with a non-uniform pitch distribution. The hybrid design optimized for both modalities allows for maintaining an extended field of view for efficient ultrasound navigation while simultaneously providing broad tomographic coverage for optimal optoacoustic imaging performance. Imaging sessions performed in tissue-mimicking phantoms and healthy volunteers demonstrate that the suggested approach renders an enhanced imaging performance as compared with the previously reported hybrid optoacoustic and ultrasound imaging approaches. Thus, it can greatly facilitate clinical translation of the optoacoustic imaging technology by means of its efficient combination with ultrasonography, a well-established clinical imaging modality.

Photoacoustic imaging is a novel biomedical imaging modality that has emerged over the recent decades. Due to the conversion of optical energy into the acoustic wave, photoacoustic imaging offers high-resolution imaging in depth beyond the optical diffusion limit. Photoacoustic imaging is frequently used in conjunction with ultrasound as a hybrid modality. The combination enables the acquisition of both optical and acoustic contrasts of tissue, providing functional, structural, molecular, and vascular information within the same field of view. In this review, we first described the principles of various photoacoustic and ultrasound imaging techniques and then classified the dual-modal imaging systems based on their preclinical and clinical imaging applications. The advantages of dual-modal imaging were thoroughly analyzed. Finally, the review ends with a critical discussion of existing developments and a look toward the future.

We have developed laser-activated perfluorocarbon nanodroplets containing copper sulfide nanoparticles (CuS NPs) for contrast-enhanced ultrasound and photoacoustic imaging. As potential clinical contrast agents, CuS NPs have favorable properties including biocompatibility, biodegradability, and enhance contrast in photoacoustic images at clinically relevant depths. However, CuS NPs are not efficient optical absorbers when compared to plasmonic nanoparticles and therefore, contrast enhancement with CuS NPs is limited, requiring high concentrations to generate images with sufficient signal-to-noise ratio. We have combined CuS NPs with laser-activated perfluorocarbon nanodroplets (PFCnDs) to achieve enhanced photoacoustic contrast and, more importantly, ultrasound contrast while retaining the favorable clinical characteristics of CuS NPs. The imaging characteristics of synthesized CuS-PFCnD constructs were first tested in tissue-mimicking phantoms and then in in vivo murine models. The results demonstrate that CuS-PFCnDs enhance contrast in photoacoustic (PA) and ultrasound (US) imaging. Upon systemic administration in vivo, CuS-PFCnDs remain stable and their unique vaporization provides sufficient PA/US contrast that can be further exploited for contrast-enhanced background-free imaging. The conducted studies provide a solid foundation for further development of CuS-PFCnDs as PA/US diagnostic and eventually therapeutic agents for clinical applications.

<div>Abstract<p>Breast-conserving surgery (BCS) is commonly used for the treatment of early-stage breast cancer. Following BCS, approximately 20% to 30% of patients require reexcision because postoperative histopathology identifies cancer in the surgical margins of the excised specimen. Quantitative micro-elastography (QME) is an imaging technique that maps microscale tissue stiffness and has demonstrated a high diagnostic accuracy (96%) in detecting cancer in specimens excised during surgery. However, current QME methods, in common with most proposed intraoperative solutions, cannot image cancer directly in the patient, making their translation to clinical use challenging. In this proof-of-concept study, we aimed to determine whether a handheld QME probe, designed to interrogate the surgical cavity, can detect residual cancer directly in the breast cavity <i>in vivo</i> during BCS. In a first-in-human study, 21 BCS patients were scanned <i>in vivo</i> with the QME probe by five surgeons. For validation, protocols were developed to coregister <i>in vivo</i> QME with postoperative histopathology of the resected tissue to assess the capability of QME to identify residual cancer. In four cavity aspects presenting cancer and 21 cavity aspects presenting benign tissue, QME detected elevated stiffness in all four cancer cases, in contrast to low stiffness observed in 19 of the 21 benign cases. The results indicate that <i>in vivo</i> QME can identify residual cancer by directly imaging the surgical cavity, potentially providing a reliable intraoperative solution that can enable more complete cancer excision during BCS.</p>Significance:<p>Optical imaging of microscale tissue stiffness enables the detection of residual breast cancer directly in the surgical cavity during breast-conserving surgery, which could potentially contribute to more complete cancer excision.</p></div>

<div>Abstract<p>Breast-conserving surgery (BCS) is commonly used for the treatment of early-stage breast cancer. Following BCS, approximately 20% to 30% of patients require reexcision because postoperative histopathology identifies cancer in the surgical margins of the excised specimen. Quantitative micro-elastography (QME) is an imaging technique that maps microscale tissue stiffness and has demonstrated a high diagnostic accuracy (96%) in detecting cancer in specimens excised during surgery. However, current QME methods, in common with most proposed intraoperative solutions, cannot image cancer directly in the patient, making their translation to clinical use challenging. In this proof-of-concept study, we aimed to determine whether a handheld QME probe, designed to interrogate the surgical cavity, can detect residual cancer directly in the breast cavity <i>in vivo</i> during BCS. In a first-in-human study, 21 BCS patients were scanned <i>in vivo</i> with the QME probe by five surgeons. For validation, protocols were developed to coregister <i>in vivo</i> QME with postoperative histopathology of the resected tissue to assess the capability of QME to identify residual cancer. In four cavity aspects presenting cancer and 21 cavity aspects presenting benign tissue, QME detected elevated stiffness in all four cancer cases, in contrast to low stiffness observed in 19 of the 21 benign cases. The results indicate that <i>in vivo</i> QME can identify residual cancer by directly imaging the surgical cavity, potentially providing a reliable intraoperative solution that can enable more complete cancer excision during BCS.</p>Significance:<p>Optical imaging of microscale tissue stiffness enables the detection of residual breast cancer directly in the surgical cavity during breast-conserving surgery, which could potentially contribute to more complete cancer excision.</p></div>

Photoacoustic (PA) imaging offers depth-resolved images of optical absorbers with the spatial resolution of ultrasound imaging. To enhance tumour contrast, tumour-specific probes are used as contrast agents. We synthesised a colourless PA probe that is activated in the presence of γ-glutamyltranspeptidase, a cancer-associated enzyme, to show its original colour and fluorescence. We have acquired high specificity fluorescence images of small tumours, using a fluorescent probe based on similar enzymatic reactions. Here, we developed a PA imaging technique to detect the PA probe. In PA imaging, depending on the concentration and excitation wavelength of the probe, the intensities of the probe signals may be lower than those of the background signals produced by intrinsic optical absorbers such as haemoglobin. For probe imaging in the presence of strong background signals, multispectral photoacoustic (MS-PA) imaging was evaluated. In MS-PA imaging, the spectral fitting method, which distinguishes the probe signals from background signals using reference spectra, has been widely used. To compensate for the decrease of fluence due to optical attenuation in biological tissue, we used a simplified compensation method that calculates fluence inside biological tissues by the Monte-Carlo model using published data on optical properties of biological tissues. The validity of the method was confirmed using tissue-mimicking phantoms. Finally, MS-PA imaging of a mouse subcutaneous tumour injected with the activatable probe was demonstrated. In conclusion, our MS-PA imaging technique afforded successful detection of the activated probe in the tumour, and time-increase of PA signals were successfully observed.

Photoacoustic (PA) imaging can revolutionize medical ultrasound by augmenting it with molecular information. However, clinical translation of PA imaging remains a challenge due to the limited viewing angles and imaging depth. Described here is a new robust algorithm called Superiorized Photo-Acoustic Non-NEgative Reconstruction (SPANNER), designed to reconstruct PA images in real-time and to address the artifacts associated with limited viewing angles and imaging depth. The method utilizes precise forward modeling of the PA propagation and reception of signals while accounting for the effects of acoustic absorption, element size, shape, and sensitivity, as well as the transducer's impulse response and directivity pattern. A fast superiorized conjugate gradient algorithm is used for inversion. SPANNER is compared to three reconstruction algorithms: delay-and-sum (DAS), universal back-projection (UBP), and model-based reconstruction (MBR). All four algorithms are applied to both simulations and experimental data acquired from tissue-mimicking phantoms, ex vivo tissue samples, and in vivo imaging of the prostates in patients. Simulations and phantom experiments highlight the ability of SPANNER to improve contrast to background ratio by up to 20 dB compared to all other algorithms, as well as a 3-fold increase in axial resolution compared to DAS and UBP. Applying SPANNER on contrast-enhanced PA images acquired from prostate cancer patients yielded a statistically significant difference before and after contrast agent administration, while the other three image reconstruction methods did not, thus highlighting SPANNER's performance in differentiating intrinsic from extrinsic PA signals and its ability to quantify PA signals from the contrast agent more accurately.

Three-dimensional (3D) handheld photoacoustic (PA) and ultrasound (US) imaging performed using mechanical scanning are more useful than conventional 2D PA/US imaging for obtaining local volumetric information and reducing operator dependence. In particular, 3D multispectral PA imaging can capture vital functional information, such as hemoglobin concentrations and hemoglobin oxygen saturation (sO2), of epidermal, hemorrhagic, ischemic, and cancerous diseases. However, the accuracy of PA morphology and physiological parameters is hampered by motion artifacts during image acquisition. The aim of this paper is to apply appropriate correction to remove the effect of such motion artifacts. We propose a new motion compensation method that corrects PA images in both axial and lateral directions based on structural US information. 3D PA/US imaging experiments are performed on a tissue-mimicking phantom and a human wrist to verify the effects of the proposed motion compensation mechanism and the consequent spectral unmixing results. The structural motions and sO2 values are confirmed to be successfully corrected by comparing the motion-compensated images with the original images. The proposed method is expected to be useful in various clinical PA imaging applications (e.g., breast cancer, thyroid cancer, and carotid artery disease) that are susceptible to motion contamination during multispectral PA image analysis.

The next generation of intelligent robotic systems has been envisioned as micro-scale mobile and externally controllable robots. Visualization of such small size microrobots to track their motion in nontransparent medium such as human tissue remains a major challenge, limiting translation into clinical applications. Herein, we present a novel, non-invasive, real-time imaging method by integrating ultrasound (US) and photoacoustic (PA) imaging modalities for tracking and detecting the motion of a single microrobot in deep biological tissue. We developed and evaluated a prototyped PA-guided magnetic microrobot tracking system. The microrobots are fabricated using photoresist mixed with nickel (Ni) particles. The microrobot motion was controlled using an externally applied magnetic field. Our experimental results evaluated the capabilities of PA imaging in visualizing and tracking microrobots in opaque tissue and tissue-mimicking phantoms. The results also demonstrate the ability of PA imaging in detecting a microrobot with the sizes less than the minimum detectable size by US imaging (down to 50 µm). The spectroscopic PA imaging studies determined an optimal wavelength (700 nm) for imaging microrobots with embedded Ni particles in oxygenated (fresh) human blood. In addition, we examined the ability of PA imaging to detect the microrobots through a nontransparent tissue mimic and at a depth of 25 mm, where conventional optical methods are unable to be used in tracking the objects. These initial results demonstrate the feasibility of an integrated US and PA imaging method to push the boundaries of microrobot applications into translational applications.

Photoacoustic (PA) tomography (PAT) is a promising technology for noninvasive temperature sensing. However, traditional PA thermometry can measure only the temperature changes relative to a baseline. Here we report a new thermal-energy-memory-based PA thermometry (TEMPT) to quantify the Gruneisen parameter and recover the absolute temperature distribution in deep tissues. We have validated the feasibility of TEMPT on tissue-mimicking phantoms and achieved a measurement accuracy of ~0.5 °C at 1.5 cm depth. As proof-of-concept, we applied TEMPT for temperature mapping during focused ultrasound treatment in mice in vivo. TEMPT is expected to find applications in thermotherapy on small animal models.

Gold nanocage decorated pH-sensitive micelle for highly effective photothermo-chemotherapy and photoacoustic imaging.

This chapter (the contents of this chapter build upon the paper: K.M. Meiburger, S.Y. Nam, E. Chung, L.J. Suggs, S.Y. Emelianov, and F. Molinari, “Skeletonization algorithm-based blood vessel quantification using in vivo 3D photoacoustic imaging”, In: Physics in Medicine and Biology, 61(22), 2016) closes the second section of this work, which is based on emphasizing quantitative imaging techniques for the assessment of architectural parameters of vasculature that can be extracted from 3D volumes. A skeletonization technique for the quantitative assessment of vascular architecture in burn wounds was developed and validated using completely non-invasive photoacoustic imaging, thus not requiring any contrast agent administration. It was shown how this technique can provide quantitative information about the vascular network from photoacoustic 3D images that can distinguish healthy from diseased tissue. Blood vessels are the only system to provide nutrients and oxygen to every part of the body. Many diseases have significant effects on blood vessel formation, so the vascular network can be a cue to assess malicious tumor and ischemic tissues. Various imaging techniques can visualize blood vessel structure, but their applications are often constrained by expensive costs, contrast agents, ionizing radiations, or a combination of the above. Photoacoustic imaging combines the high-contrast and spectroscopic-based specificity of optical imaging with the high spatial resolution of ultrasound imaging, and image contrast depends on optical absorption. This enables the detection of light absorbing chromophores such as hemoglobin with a greater penetration depth compared to purely optical techniques. A skeletonization algorithm for vessel architectural analysis using non-invasive photoacoustic 3D images acquired without the administration of any exogenous contrast agents is presented in this chapter. 3D photoacoustic images were acquired on rats (n \(=4\)) in two different time points: before and after a burn surgery. A skeletonization technique based on the application of a vesselness filter and medial axis extraction is proposed to extract the vessel structure from the image data and six vascular parameters (number of vascular trees (NT), vascular density (VD), number of branches (NB), 2D distance metric (DM), inflection count metric (ICM), and sum of angles metric (SOAM)) were calculated from the skeleton. The parameters were compared (1) in locations with and without the burn wound on the same day and (2) in the same anatomic location before (control) and after the burn surgery. Four out of the six descriptors were statistically different (VD, NB, DM, ICM, \(p<0.05\)) when comparing two anatomic locations on the same day and when considering the same anatomic location at two separate times (i.e., before and after burn surgery). The study demonstrates how it is possible to obtain quantitative characterization of the vascular network from 3D photoacoustic images without any exogenous contrast agent which can assess microenvironmental changes related to disease progression.

Recent clinical studies have demonstrated that photoacoustic (PA) imaging, in conjunction with pulse echo (PE) ultrasound is a promising modality for diagnosing breast cancer. However, existing devices are unwieldy and are hard to integrate into the clinical environment. In addition, it is difficult to illuminate thick samples because light must be directed around the transducer. Conventional PA imaging designs involve off-axis illumination or transillumination through the object. Whereas transillumination works best with thin objects, off-axis illumination may not uniformly illuminate the region of interest. To overcome these problems we have developed an attachment to an existing clinical linear array that can efficiently deliver light in line with the image plane. This photoacoustic enabling device (PED) exploits an optically transparent acoustic reflector to co-align the illumination with the acoustic waves, enabling realtime PA and PE imaging. Based on this concept, we describe results from three types of PEDs in phantoms and rat tissue. The most recent version is fabricated by rapid prototyping, and attached to a 10 MHz linear array. Real-time PA and PE images of a 127-μm diameter wire were consistent with our expectations based on the properties of the ultrasound transducer. Comparisons with and without the PED of another test phantom printed on transparency demonstrated that the PED does not appreciably degrade or distort image quality. The PED offers a simple and inexpensive solution towards a real-time dual-modality imaging system for breast cancer detection. It could also be adapted for virtually any kind of ultrasound transducer array and integrated into routine ultrasound exams for detection of cancerous lesions within 1-2 cm from the probe surface.

To noninvasively assess vascular hemodynamics with photoacoustic imaging (PAI) and blood oxygenation level-dependent (BOLD) magnetic resonance (MR) imaging in phantoms and in an animal model. In vivo studies were performed with institutional animal care and use committee approval. In vitro experiments were performed by using a tissue-mimicking phantom in multiple oxygenation conditions (n = 6) to compare PAI measurements and BOLD MR imaging measurements. PAI and T2-weighted spin-echo-based BOLD MR imaging were performed to assess tumor response to carbogen (95% O2, 5% CO2) in mice with head and neck tumors before (n = 11) and after (n = 9) treatment with a vascular disrupting agent (VDA). Two-tailed Pearson correlation analysis was performed to determine the correlation between the parameters measured with PAI and BOLD MR imaging in vitro. Two-tailed paired t tests were used to compare change in tumor hemoglobin oxygen saturation (sO2) levels and BOLD signal in response to carbogen. Changes in PAI and BOLD signal intensity before and after VDA treatment were analyzed for significance by using analysis of variance with repeated measures. Phantom measurements yielded good correlation between photoacoustically derived sO2 levels and BOLD signal intensity (r = 0.937, P = .005) and partial pressure of oxygen (r = 0.981, P = .005). In vivo hemodynamic response to carbogen was characterized by a significant increase in tumor sO2 levels (P = .003) and BOLD signal (P = .001). When compared with pretreatment estimates, treatment with VDA resulted in a significant reduction in the tumor hemodynamic response to carbogen at PAI (P = .030). Carbogen-based functional imaging with PAI and BOLD MR imaging enables monitoring of early changes in tumor hemodynamics after vascular targeted therapy.