This PDF file contains the front matter associated with SPIE Proceedings Volume 10878, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

Delay and Sum (DAS) is one of the most common beamforming algorithms for photoacoustic imaging reconstruction that can function well in real-time imaging for its simplicity and quickness. However, high sidelobes and intense artifacts usually appear in the reconstructed image using DAS algorithm. To solve this problem, a novel beamforming algorithm called Multiple Delay and Sum with Enveloping (multi-DASE) is introduced in this paper, which can suppress sidelobes and artifacts efficiently. Compared to DAS, multi-DASE beamforming algorithm calculates not only the initial beamformed signal but also the N-shaped photoacoustic signal for each pixel. Firstly, Delay and Sum is performed multiply based on time series to recover the N-shaped photoacoustic signal for each pixel in the reconstructed image. And then, the recovered signal is enveloped to transform the N-shaped wave into a pulse wave to remove the negative part of the signal. Finally, signal suppression is performed on the enveloped signal which can lead to the suppression of sidelobes and artifacts in the reconstructed image. The multi-DASE beamforming algorithm was tested on the simulated data acquired with MATLAB k-Wave Toolbox. Experiment was also conducted to evaluate the efficiency of the multiDASE algorithm for clinical application. Both in computer simulation and experiment, our multi-DASE beamforming algorithm showed great performance in removing artifacts and improving image quality. In our multi-DASE beamforming algorithm, only fundamental operations and Discrete Fourier Transform (DFT) are performed, which means it can be a promising method for real-time clinical application.

Photoacoustic imaging instruments based on the Fabry Perot ultrasound sensing concept have been used extensively for the preclinical assessment of mouse models and shown to provide high fidelity images to sub-cm depths. In a new development, a 3D high resolution scanner based on the same technology has now been specifically engineered into a format comprising a mobile platform and a convenient hand-held imaging probe for clinical use. A number of key engineering developments designed to advance the clinical translation of the technology have been implemented. The system now employs a novel 32-channel optical scanning architecture and a 1kHz PRF excitation laser providing an order-of-magnitude faster acquisition than previous pre-clinical embodiments. 3D images can now be acquired within 1 second, and video rate 2D synthetic aperture imaging is achievable. Image acquisition speed can be further accelerated by employing sub-sampling techniques based on total variation and deep learning image reconstruction, e.g. 3D images can be obtained at the rate of 4Hz with a typical 25% sub-sampling factor. To further aid clinical utility, the scanner allows rapid switching between the two imaging modes. This enables the ROI to be searched for and located in real-time using the 2D video rate mode prior to 3D image acquisition. Additional recent technical developments include bias wavelength tracking for temperature compensation, synthetic 1.5D array based receive beam forming for out-of-plane signal rejection, fast image reconstruction and visualisation and the implementation of an intuitive user-friendly interface. To confirm clinical applicability, proof-of-concept studies both in healthy volunteers and patients have been conducted using the system. Following ethical and local regulatory approval, consenting patients were recruited from a single tertiary care hospital. Participants had previously been diagnosed with peripheral vascular disease (PVD), head and neck malignant tumours (including nodal deposits), inflammatory arthritis, or were under active clinical investigation for these conditions. We obtained mutliwavelength 3D images of the superficial vasculature in critically-ischaemic and normally perfused regions in patients with PVD. In both cases, the photoacoustic images were compared to clinical B-mode and Doppler ultrasound scans. The results show that the scanner is able to visualise the spatial-temporal changes in human microvasculature and thus may be able to identify regions of ischaemia otherwise undetectable using existing modalities. Images of small joint arthopathies, and malignant lymph nodes were also obtained, and compared with contemporaneous high resolution ultrasound. Patients found the use of the scanner highly acceptable, both in degree of comfort and the duration of the scan procedure. This exploratory phase clinical study represents an initial step towards establishing the clinical utility of photoacoustic imaging in a range of clinical conditions.

Non-invasive imaging plays an important role in diagnosing and monitoring peripheral artery disease (PAD). Doppler ultrasound imaging can be used for measuring blood flow in this context. However, this technique frequently provides low contrast for flow in small vessels. Photoacoustic imaging can allow for the visualization of blood in small vessels, with direct contrast from optical absorption of hemoglobin. In this work, we investigate the potential applications of a compact LED-based photoacoustic (850 nm) and ultrasound imaging system for visualizing human peripheral blood vessels during cuff occlusion. Each measurement comprised three stages. First, a baseline measurement of a digital artery of a human finger from a volunteer without a diagnosis of PAD was performed for several seconds. Second, arterial blood flow was stopped using an occlusion cuff, with a rapid increase of pressure up to 220 mm Hg. Third, the occlusion cuff was released rapidly. Raw photoacoustic and ultrasound image data (frame rate: 70 Hz) were recorded for the entire duration of the measurement (20 s). The average photoacoustic image amplitude over an image region that enclosed the digital artery was calculated. With this value, pulsations of image amplitudes from the arteries was clearly visualized. The average photoacoustic image amplitude decreased during the increase in cuff pressure and it was followed by a rapid recovery during cuff release. With real-time non-invasive measurements of peripheral blood vessel dynamics in vivo, the compact LED-based system could be valuable for point-of-care imaging to guide treatment of PAD.

Vasculatures enable nutrient transportation, waste disposal, and immune surveillance. Due to these diverse functions, abnormal changes in vascular morphology are commonly associated with the development of various diseases, including tumor growth and metastasis, inflammatory disorders, and pulmonary hypertension. Many models linking vascular morphogenesis to the development of a particular disease have been developed for prognosis, diagnosis, or disease management. To apply these models in clinical assessment, however, a tissue biopsy of the lesion is needed. This procedure is invasive and occasionally clinically infeasible. Photoacoustic endoscopy offers in vivo label-free examination of visceral vascular morphology, but its volumetric imaging process is vulnerable to breathing movement and peristalsis, because its typical B-scan rate is ~ 10 Hz, restricted by the speed of the scanning unit and the laser pulse repetition rate. Here, we present a transvaginal fast-scanning optical-resolution photoacoustic endoscope with a 250 Hz B-scan rate over a 3 mm scanning range. After demonstrating its imaging capability and safety, we not only illustrate the morphological differences in the vasculatures of the human ectocervix, uterine body, and sublingual mucosa, but also show the longitudinal and cross-sectional differences in the cervical vasculatures of pregnant women. This technology is promising for screening the visceral pathological changes associated with angiogenesis.

Port-wine stain (PWS) is a discoloration of human skin caused by a vascular anomaly (i.e., capillary malformation in the skin). In the past years, several techniques have been developed for characterization and treatment evaluation of PWS. However, each of them has some limitations. Optical methods working in the ballistic regime, such as dermoscopy and VISIA, do not have sufficient penetration to cover the entire scale of PWS. High frequency ultrasound, although with better imaging depth, does not offer sufficient contrast to differentiate PWS and normal skin tissue. Therefore, current endpoint clinical assessment for PWS still relies on physicians’ subjective judgement. In this study, photoacoustic (PA) imaging utilizing light emitting diodes (LED) as the light source was adapted to the evaluation of PWS and response to photodynamic therapy (PDT). PA images as well as US images of the targeted skin area before and at different time points after the treatment were acquired. The imaging results from adults and children were also compared. The imaging findings demonstrate that the PWS levels of adult patients are significantly higher than children (p<0.01), which fits well with the knowledge that the vessel malefaction degree develops with patients’ age. The 2-month follow-up study on four children shows that the average PWS level reduced for 33.60%onstrat (p<0.01) as a result of 3-4 times of PDT treatment. This initial clinical trial on patients suggests PA imaging holds potential for quantitative assessment of PWS in clinical settings.

Our previous research has demonstrated that photoacoustic (PA) imaging is capable of evaluating the pathological condition in human peripheral joints affected by inflammatory arthritis. In this work, we tested the performance of a PA imaging system based on the LED light source and its performance for arthritis imaging. The LED-based PA imaging system not only has less cost but also has smaller footprint and, hence, is more portable and convenient for use in rheumatology clinic. 2D B-scan PA and US images of each metacarpophalangeal (MCP) joint were acquired along the sagittal sections. Along the same sections, US Doppler images were also acquired. Images from 12 joints with clinically active arthritis (i.e., positive on Doppler US), 5 joints with subclinically active arthritis (i.e., negative on Doppler US), and 12 joints of normal volunteers were compared. The blood volume in each joint reflecting hyperemia was quantified by counting the density of the color pixels in each pseudo-color PA image. T-tests were conducted to evaluate whether PA imaging can differentiate the three groups. The results from this study suggest that LED-based PA imaging is capable of detecting hyperemia as an important biomarker of joint inflammation. In addition, PA imaging could differentiate the subclinically active arthritis group and the normal group while Doppler US could not, suggesting that PA imaging has higher sensitivity to mildly hyperemia when compared to Doppler US. The imaging technique presented may contribute to rheumatology clinic by providing a new tool for early diagnosis and treatment evaluation of joint inflammation.

Transrectal ultrasound (TRUS) guided biopsy is the standard procedure for evaluating the presence of prostate cancer. TRUS, however, has limited sensitivity to prostate tumors, nor can it differentiate aggressive cancer from non-aggressive ones. The emerging photoacoustic (PA) imaging combined with TRUS offers a great promise to solve this overarching issue, especially when powered by tumor-targeting contrast agent. In this work, we studied the feasibility of PA imaging to cover the entire prostate by using light illumination via the urethral track. Experiment was conducted on whole human prostates ex vivo. The light source was an array of light emitting diodes (LED) which has many advantages compared to solid state laser. The LED array was placed in the urethra, delivering light with fluence within the ANSI safety limit. A PA and ultrasound (US) dual modality system acquired the images in the same way as in TRUS. The imaging target was a 1-mm tube filled with ICG solution, mimicking the situation of a prostate tumor labeled with ICG contrast agent. The imaging results demonstrated that PA imaging can detect the ICG-filled tube at any place in the prostate, with an imaging depth over 20 mm. This study validated that PA imaging, when performed in a transrectal manner and combined with transurethral light illumination, is capable of molecular level imaging of the entire prostate noninvasively. The high sensitivity offered by PA imaging in detecting aggressive prostate cancer may contribute to prostate cancer management, e.g., enabling more accurate guidance for needle biopsy.

Multispectral photoacoustic (PA) imaging is a prime modality to monitor hemodynamics and changes in blood oxygenation (sO₂). Although sO₂ changes can be an indicator of brain activity both in normal and in pathological conditions, PA imaging of the brain has mainly focused on small animal models with lissencephalic brains. Therefore, the purpose of this work was to investigate the usefulness of multispectral PA imaging in assessing sO₂ in a gyrencephalic brain. To this end, we continuously imaged a porcine brain as part of an open neurosurgical intervention with a handheld PA and ultrasonic (US) imaging system in vivo. Throughout the experiment, we varied respiratory oxygen and continuously measured arterial blood gases. The arterial blood oxygenation (SaO₂) values derived by the blood gas analyzer were used as a reference to compare the performance of linear spectral unmixing algorithms in this scenario. According to our experiment, PA imaging can be used to monitor sO₂ in the porcine cerebral cortex. While linear spectral unmixing algorithms are well-suited for detecting changes in oxygenation, there are limits with respect to the accurate quantification of sO₂, especially in depth. Overall, we conclude that multispectral PA imaging can potentially be a valuable tool for change detection of sO₂ in the cerebral cortex of a gyrencephalic brain. The spectral unmixing algorithms investigated in this work will be made publicly available as part of the open-source software platform Medical Imaging Interaction Toolkit (MITK).

High re-excision rates in breast conserving surgery call for a new intraoperative approach to the lumpectomy specimen evaluation problem. The unique intraoperative photoacoustic screening (iPAS) system, presented here, demonstrated the capability of photoacoustic tomography (PAT) to deliver optical sensitivity and specificity, along with over 2 cm imaging depth, in a true clinical setting. Highly specialized acoustic transducers enabled the evaluation of tumor extent, shape, morphology and position within lumpectomy specimens measuring up to 11 cm in diameter. Comparison with conventional ultrasound (US) and x-ray imaging validated the performance of iPAS. This approach shines a light towards more effective soft tissue abnormality assessment.

Ocular neovascularization occurs in various eye diseases such as diabetic retinopathy, neovascular macular degeneration, and retinopathy of prematurity. Current treatment methods including conventional laser ablation therapy and anti-vascular endothelial growth factor (VEGF) injection each has drawbacks including collateral tissue damages, frequent administration, high cost, and drug toxicity. We recently developed a novel noninvasive image-guided photo-mediated ultrasound therapy (PUT) which concurrently applies nanosecond laser pulses and millisecond ultrasound bursts to precisely and safely remove pathologic microvessels in the eye. Relying on the mechanism of photoacoustic cavitation, PUT takes advantages of high optical contrast among biological tissues, and can selectively remove microvessels without causing collateral tissue damage. To achieve personalized treatment with optimal treatment outcome, a multi-modality eye imaging system involving advanced photoacoustic microscopy (PAM) and optical coherence tomography (OCT) has been integrated with the PUT system to provide real-time feedback and online evaluation of the treatment outcome. To assess the performance of this image-guided PUT system, experiments have been conducted on rabbit eye models. During the treatment, cavitation signals were observed and monitored by OCT with good sensitivity, suggesting that OCT can be used to evaluate treatment effect in real time. The PAM was capable of mapping the 3D distributed microvessels with excellent image quality, demonstrating that PAM can help to quantitatively evaluate the treatment outcome. As indicated by the initial results from this study, imaging guidance involving both PAM and OCT could further improve the efficacy and safety of the newly invented PUT, accelerating its translation to ophthalmology clinic.

All-optical ultrasound imaging uses optical generation and detection of ultrasound to acquire pulse-echo images. Recent advances have resulted in efficient optical ultrasound sources, emitting pressures and bandwidths rivalling those generated by conventional electronic transducers. Two-dimensional imaging of biological tissues was achieved using a fibre-optic Fabry-Pérot cavity and a nanocomposite generator membrane in which ultrasound was generated photoacoustically. Using scanning mirrors, excitation light was steered to consecutive locations, thus synthesising an acoustic source aperture with a geometry that could be arbitrarily and dynamically reconfigured. This unique capability of implementing different geometries on the same hardware allows for a direct comparison of the image quality obtained with different aperture geometries, which is difficult to achieve using conventional electronic transducers. Here we explore how the source aperture geometry affects the image quality through a set of numerical simulations and experiments. First, we determined that the image artefacts and corresponding contrast level depend strongly on the total number of A-scans (increasing from 200 to 1800 A-scans improved the contrast from 30 to 50 dB), irrespective of the number and locations of the detectors. Second, we demonstrated how parametric optimisation of the spatial optical ultrasound source distribution allowed for local (within a user-defined region of interest) or global image optimisation achieving an additional reduction in artefact level of up to 8 dB. Finally, we demonstrated video-rate, real-time 2D image acquisition using optimised source aperture geometries.

Generation of ultrasound using the optoacoustic effect has received increasing attention in the field of imaging and translational medicine. However, none of the current optoacoustic converters has been used for neural modulation. Here, we developed a miniaturized Fiber-Optoacoustic Converter (FOC), which has a diameter of 600 μm, and can convert nano-second laser pulses into acoustic waves through the optoacoustic effect. The ball shaped FOC is composed of one ZnO /epoxy based diffusion layer and two graphite/epoxy based absorption layer. The radiofrequency spectrum of the generated US frequency ranges from 0.1-5 MHz, with multiple frequencies peaks at 0.5, 1 and 3MHz. Compared to traditional ultrasound transducers, the FOC system has the advantages of miniaturized size, superior spatial resolution, and produces omnidirectional propagating acoustic wave. Using this FOC system, we show that ultrasound can directly activate individual cortical neuron in vitro with a radius of 500 μm around the FOC tip, and generate intracellular Ca2+ transient without neural damage. Neural activation is the consequence of mechanical disturbance of neuronal membrane, rather than direct laser or photothermal stimulation. Finally, we combine FOC neural modulation with electrophysiology, and achieve direct and spatially confined neural stimulation in vivo. The FOC system opens new possibilities to use optoacoustic effect as a new method for precise neural modulation.

Silicon Photonics-based sensors can provide low cost, high sensitivity optical detection solutions in ultrasound and photoacoustic (PA) imaging. We demonstrate experimentally the measurement of ultrasound (2.5-8.5 MHz) in water using photonic crystal slab (PCS) nanostructure devices. Each PCS is composed of a periodic array of nanoholes, etched into silicon nitride (t=160 nm), on top of a silicon dioxide layer, and silicon substrate. The PCS devices have guided resonances that peak at ~ 1550 nm, with linewidths that vary from 0.7 to 5.5 nm. One type of PCS device includes a PCS nanostructure located above a thin micro-fabricated silicon membrane (~ 10 micron thick). Membrane deformation by incoming ultrasound waves induce resonance changes in the PCS spectral peak location (i.e., drum effect). We observe these drum-effect PCS devices to have acoustic sensitivities that are very narrowband (with bandwidths ~ 1 MHz), with a 300-micron diameter drum device found to have a peak sensitivity at 5 MHz and a noise equivalent pressure (NEP) of 2.0 kPa (72 Pa/rt Hz). In another mechanism, the sensitivity of the PCS nanostructures to changes in the ambient index of refraction is used. A pressure wave in water that impinges the PCS is accompanied by changes in the water's index of refraction, which causes the resonance peak of the PCS to shift. The acoustic sensitivities of these PCS devices is found to be broadband (> 6 MHz), in contrast to the drum-effect devices, with an NEP of less than 0.5 kPa (6.7 Pa/rt Hz). These devices can potentially allow for optics-based monolithic ultrasound sensor arrays, optimized for PA imaging.

Fabry-Pérot (FP) sensors have enabled high resolution 3D photoacoustic (PA) imaging in backward mode. However, raster-scanning of the interrogation laser beam across the sensor can result in slow 3D image acquisition. To overcome this limitation, parallelized PA signal acquisition can be used for which FP sensors with uniform optical thickness are required. In this work, the optical thickness is tuned a) irreversibly through the use of a photopolymer host matrix and b) actively using embedded electro-optic (EO) chromophores. Polymer spacers (5 μm) were deposited using spin coating and sandwiched between two dielectric mirrors and transparent ITO electrodes. The employed polymer guest-host system consists of an EO chromophore (2-methyl-4-nitroaniline) and poly(vinyl cinnamate). EO tuneability was induced using contact poling and a tuneability of 68 pm was demonstrated. The optical thickness was homogenised by raster scanning a UV beam whilst varying the exposure time across a 4 mm2 detection aperture.

In recent years, Polymer optical fibre (POF) has been receiving increasing attention for sensing applications. For applications such as endoscopic ultrasound and photo-acoustics, PMMA polymer fibres deliver ever improving performance and more types of polymer are being trialled for these applications. The fundamental properties of POF, when correctly leveraged, deliver at least an order of magnitude in improvements over silica fibres. POF delivers lower acoustic impedance, a reduced Young’s Modulus and a higher acoustic sensitivity within the megahertz region. In contrast, existing piezo-electric transducers have an inherent narrow acoustic bandwidth and a proportionality to size that causes difficulties for applications such as endoscopy within the biomedical domain. With the increasing take-up of POF, improvements have been made in fibre dopant distribution, the range of polymers available and connectorisation techniques. While newer polymer fibres are under preliminary study, PMMA has shown itself to be the most sensitive fibre to date despite historically higher levels of dopant inconsistency and an implementation around 830 nm that has a lower signal-to-noise ratio than possible. The prior approach of edge filtering a Bragg grating with a high speed photodiode has been shown to function and is therefore maintained. We present a step index PMMA Bragg grating ultrasonic probe in the L-band for the first time. Detection is achieved using a Bragg grating less than 1cm in length in a fibre under 10cm long. We examine the temporal and frequency response of the sensor over a 1-15 MHz range.

Acoustic imaging modalities such as multispectral optoacoustic tomography (MSOT) have recently matured to a point which allows retrieval of anatomical, molecular and dynamic information with resolutions of several tens of microns These advancements are going hand in hand with the continuous development of ultrasound detectors. Yet, the traditional piezoelectric transducers have a distinct disadvantage: the detector size is proportional to its sensitivity. This limits miniaturization and prevents the development of point-like detectors as well as the subsequent construction of ultra-dense detector arrays. Consequently, research has shifted towards all-optical ultrasound detectors, such as Fabry-Pérot resonators, where miniaturization does not affect detector sensitivity. In this context, we present a novel optical resonator on a silicon chip with a sensing area of 220 x 500 nm that - to the best of our knowledge – is the smallest ultrasound detector ever created. These dimensions are 77 and 34 times smaller than the acoustic wavelength at the central detection frequency, hence our detector is truly a point detector. Using the scalable silicon photonics platform we constructed an array of eight detectors. The archived density of 125 detectors/mm2 is larger by orders of magnitude compared to arrays of piezoelectric and capacitive micromachined ultrasound transducers. We describe the working principle of the detector and characterize its sensitivity, spatial response, and bandwidth. We demonstrate its applicability for optoacoustic tomography and perform the first SOI based tomography ever reported, by imaging micron-sized phantoms at light fluences well below the ANSI limit for human skin.

Polymer film Fabry-Perot (FP) sensors are commonly used to detect ultrasound for Photoacoustic (PA) imaging providing high resolution 3D images. Such high image quality is possible due to their low Noise Equivalent Pressure (NEP) because of their broadband response and small acoustic element size. The acoustic element size is small (<100 μm) as defined, to first approximation, by the spot size of the focused interrogation beam. However, it has been difficult until now to gain an accurate intuitive understanding of the working principle of FP sensors interrogated with a focused beam. To overcome this limitation a highly realistic rigorous model of the FP sensor’s optical response has used to establish a new intuitive understanding. The origin of fringe depth reduction and asymmetry associated with the FP sensors optical response is explained using the model developed.

An estimated ~250,000 new cases of both invasive and non-invasive breast cancer were diagnosed in US women almost every year. To reduce the local recurrence rate, the breast conserving surgery (BCS) is widely used as the initial therapy, which is to excise the tumor with a rim of normal surrounding tissue such that no cancer cells remain at the cut margin, Patients with positive margin commonly require a second surgical procedure to obtain clear margins. To this end, optical-resolution photoacoustic microscopy (OR-PAM) with ultraviolet (UV) laser illumination (OR-UV-PAM) has been developed for providing label-free, high-resolution, and histology like imaging of fixed, unprocessed breast tissue. To further improve the performance of OR-UV-PAM, here, we introduce dual-view UV-PAM (DV-UV-PAM) to significantly improve the axial resolution, achieving three-dimensional (3D) resolution isotropy. We first use 0.5 μm polystyrene beads and carbon fibers to validate the resolution isotropy improvement. Imaging of mouse brain slices further demonstrates the improved resolution isotropy, revealing the 3D structure of cell nuclei in detail, which facilitates quantitative cell nuclear analysis.

This lecture will discuss history of optoacoustic imaging from pioneering works that set the basic principles of the technology to the first in vivo images, to the most recent advances, and finally to the future medical imaging modalities and their applications in the main stream medicine and surgery. We also present the design features and technical parameters of the optoacoustic imaging systems required for clinically viable devices.

We proposed to use optoacoustics (photoacoustics) for biomedical applications and for more than 25 years have been working on it. In this overview we present our major biomedical optoacoustics achievements over these years. Optoacoustic diagnostic modality is based on thermoelastic generation of optoacoustic waves and combines high optical contrast and ultrasound spatial resolution. We proposed to use the optoacoustic technique for a number of applications including cancer detection in breast, prostate, and other organs; hematoma detection and characterization; monitoring of thermotherapy (hyperthermia, coagulation, freezing); monitoring of cerebral blood oxygenation in adults, neonatal patients, fetuses during late stage labor; monitoring of central venous oxygenation and total hemoglobin concentration. In early 90s we started from ideas, basic science, and first in vitro studies. In mid 90s we demonstrated optoacousitc wave: 1) detection from tissues at cm-depths (well beyond the light diffusion limit); 2) detection from microscopic tissue volumes; 3) diffraction and attenuation effects in tissues. Then we reconstructed first optoacoustic images of tissue phantoms and tissues. We developed optoacoustic methods and systems (including highly-compact laser diode systems) for monitoring, mapping, and imaging in many organs (including the human brain) and tested them in small and large animal studies and in clinical studies in healthy volunteers and patients with traumatic brain injury, circulatory shock, and anemia as well as in neonatal and fetal patients. Recently, we proposed to use optoacoustic therapy and theranostics and tested them in animal studies. At present, biomedical optoacoustics is an emerging diagnostic imaging modality with a great potential to become an invaluable tool for diagnostics, therapy, and theranostics.

Photoacoustic computed tomography (PACT), also known as optoacoustic tomography, is an emerging imaging technique that holds great promise for biomedical imaging. PACT is a hybrid imaging method that can exploit the strong endogeneous contrast of optical methods along with the high spatial resolution of ultrasound methods. Similar to X-ray CT or MRI, PACT is a computed imaging modality that utilizes a reconstruction method for image formation. In this talk, we provide an overview of image reconstruction methods for PACT that have been proposed over the past 25 years. This review will include an overview of classic backprojection-based methods as well as modern optimization-based approaches to image reconstruction.

Photoacoustic tomography has been developed for in vivo functional, metabolic, molecular, and histologic imaging by physically combining optical and ultrasonic waves. Broad applications include early-cancer detection and brain imaging. High-resolution optical imaging—such as confocal microscopy, two-photon microscopy, and optical coherence tomography—is limited to superficial imaging within the optical diffusion limit (~1 mm in the skin) of the surface of scattering tissue. By synergistically combining light and sound, photoacoustic tomography conquers the optical diffusion limit and provides deep penetration at high ultrasonic resolution and high optical contrast. Photoacoustic tomography has two major embodiments: photoacoustic computed tomography and photoacoustic microscopy. In photoacoustic computed tomography, a pulsed broad laser beam illuminates the biological tissue to generate a small but rapid temperature rise, which leads to emission of ultrasonic waves due to thermoelastic expansion. The unscattered pulsed ultrasonic waves are then detected by ultrasonic transducers. High-resolution tomographic images of optical contrast are then formed through image reconstruction. In photoacoustic microscopy, a pulsed laser beam is delivered into the biological tissue to generate ultrasonic waves, which are then detected with a focused ultrasonic transducer to form a depth resolved 1D image. Raster scanning yields 3D high-resolution tomographic images. Super-depths beyond the optical diffusion limit have been reached with high spatial resolution. The annual conference on PAT has grown exponentially since early 2000 and become the largest in SPIE’s 20,000-attendee Photonics West since 2010.

Premature cervical remodeling is an indicator of impending spontaneous preterm birth, however, current clinical measurements of cervical remodeling are mainly obtained by digital examinations, which are subjective and detect only late events, such as cervical effacement and dilation. The incompletely understood mechanisms of cervical remodeling lead to degradation of extracellular matrix proteins and inflammation, and these physiological changes are associated with increased tissue hydration. Near-infrared spectroscopy is routinely used in industrial applications to quantify the water content in various products, because this method does not require sample preparation and is nondestructive. Spectroscopic photoacoustic tomography is an embodiment of near-infrared spectroscopy and has been demonstrated in the quantification of various biochemical constituents. However, the dimensions of those tabletop systems in the previous demonstrations preclude in vivo use in the gastrointestinal tract and urogenital tract. Photoacoustic endoscopy (PAE) incorporates an acoustic detector, optical components, and electronic components in a millimeter-diameter-scale probe to image tissue that is inaccessible by the tabletop systems. Here, we present a near-infrared spectroscopic PAE system. We analyze the measured photoacoustic near-infrared (PANIR) spectra by linear regression. We demonstrate that this method successfully quantifies the water contents of tissue-mimicking phantoms made of gelatin hydrogel. Applying this method to the cervices of pregnant women, we observe their physiological water contents and a progressive increase throughout gestation. The application of this technique in maternal health care may advance our understanding of cervical remodeling and provide a sensitive method for predicting preterm birth.

White light endoscopy is widely used both as a diagnostic tool for the assessment of abdominal cancers and to help guide their surgical excision using minimally invasive procedures. However, the information it provides is limited to visual inspection of the tissue surface. Endoscopic ultrasonography provides depth-resolved morphological images but exhibits poor label-free microvascular contrast thus limiting its ability to identify and delineate deep seated tumours. These drawbacks can potentially be addressed by using a laparoscopic probe that provides co-registered photoacoustic (PA) and white light endoscopy images. With the help of PA contrast, the probe can visualise depth-resolved microvasculature and thus offers the prospect of more sensitive detection of tumours based on abnormal vascular anatomy and function. However, it is challenging to implement such a probe using conventional piezoelectric transducers. Their opaque nature makes it difficult to achieve forward-viewing capability in a small footprint as required for laparoscopic use as well as incorporate videoscopy. Furthermore, achieving sufficiently widebandwidth (tens of MHz) and λ/2 spatial sampling as required for high resolution endoscopic PA imaging present further challenges. To address these challenges, we present a rigid miniature forward-viewing endoscope that is based on a transparent optical ultrasound sensor which offers a wideband response up to 50 MHz with sub-100 µm spatial sampling. The probe is designed for laparoscopic use. It is 260 mm long and 9 mm in outer diameter to permit insertion via a standard 12 mm abdominal trochar and comprises a lens relay system with a high-finesse FP ultrasound sensor at its distal end. The sensor is designed to operate in the 1500 – 1600 nm spectral range with high transmission in the visible to near-infrared region (550 – 1200 nm). The latter not only enables delivery of near-infrared pulsed excitation light through the sensor to acquire PA images but also transmission of visible CW light for simultaneous acquisition of wide-field video images at the probe tip. A MEMS scanning mirror located at the proximal end of the probe scans the FP sensor via the optical relay with 8 focused beams from a CW tunable laser source (1550 nm centre wavelength) to map the generated photoacoustic waves. High-resolution 3D tomographic images are reconstructed using a time reversal algorithm and fused with the white light video images. The probe has 8 mm lateral field-of-view and the NEP is 200 Pa over 20 MHz bandwidth. The lateral spatial resolution is 52 µm at a depth of 1 mm decreasing to 110µm at a depth of 7 mm. The axial resolution is 29 µm over this depth range. To demonstrate potential clinical applicability, the probe was evaluated in an in vivo sheep study and shown to provide excellent high resolution 3D images of vascular structures in the liver, kidney and placentomes. This novel forward-viewing PAE probe could provide new opportunities for the photoacoustic assessment of tumours in the liver, cancer in the GI tract and guiding minimally invasive procedures in abdominal surgery and foetal medicine.

The first successful validation of a forward-looking Piezoelectric Micromachined Ultrasound Transducer (PMUT) ring array designed for photoacoustic endoscopic imaging applications is presented. PMUT ring arrays were fabricated with a 0.5 mm inner diameter, to allow insertion of an optical fiber for light delivery, and ~2.5 mm overall outer diameter. Each ring array consisted of 6 elements, with a total of 102 PMUT cells, or 17 cells per element. Each PMUT cell has a 100 μm diameter multi-layered diaphragm having a ~700 nm thick c-axis oriented aluminium nitride (AlN) thin film as the piezoelectric layer over a Si (handle layer) / SiO₂ (1 μm) / Si (10 μm) / SiO₂ (100 nm) / TiO₂ (40 nm) / Pt (150 nm) substrate to act as an ultrasound receive element. The resonant frequency was ~ 6 MHz in water. The output end of an optical fiber, coupled to a pulsed laser diode (PLD), was fitted with a 2.5 mm ferrule. The PMUT ring array was concentrically mounted on the ferrule to obtain a miniaturized endoscopic PAI device. The observed photoacoustic bandwidth was ~75%, and a strong photoacoustic signal of ~13 mV peak-to-peak output was observed from a light absorbing target kept 5 mm away from the PMUT array. 2D and 3D photoacoustic images of the targets were obtained via raster scanning of the phantom sample. In the future, the functionality of the PMUT ring array will be enhanced through multichannel acquisition to obtain variable acoustic focus and limited view photoacoustic images in real time.

Lipid composition of atherosclerotic plaques is considered to be one of the primary indicators of plaque vulnerability. Therefore, a specific diagnostic or imaging modality that can sensitively evaluate plaques’ necrotic core is highly desirable in atherosclerosis imaging. In this regard, intravascular photoacoustic (IVPA) imaging is an emerging plaque detection technique that provides lipid-specific chemical information from an arterial wall with great optical contrast and long acoustic penetration depth. Within the near-infrared window, a 1210-𝑛𝑚 optical source is usually chosen for IVPA applications as lipids exhibit a strong absorption peak at that wavelength due to the second overtone of the C-H bond vibration within the lipid molecules. However, other arterial tissues also show some degree of absorption near 1210 𝑛𝑚 and thus generate undesirably interfering PA signals. In this study, a theory of the novel Frequency-Domain Differential Photoacoustic Radar (DPAR) modality is introduced as an interference-free detection technique for accurate and reliable evaluation of vulnerable plaques. By assuming two low-power continuous-wave (CW) optical sources at ~ 1210 𝑛𝑚 and ~ 970 𝑛𝑚 in a differential manner, DPAR theory and the corresponding simulation study suggest a unique imaging modality that can efficiently suppress any undesirable absorptions and system noise, while dramatically improving PA sensitivity and specificity toward cholesterol contents of atherosclerotic plaques.

We present the Twente Photoacoustic Mammoscope 2 (PAM 2) based on a 3D tomographic geometry. A functional optical contrast map of breast vascularization can be obtained in a noninvasive, radiation-free and painless manner. A woman lies prone on a bed with one breast pendant in an imaging tank with water, where 12 curved ultrasound arrays are mounted. Each array extends from chest wall towards the nipple following the contour of the pendant breast, and carries 32 detector elements. The detectors’ center frequency is 1 MHz. The breast is illuminated from multiple directions: the ventral side of the breast from the bottom and the areas close to the chest wall from the sides. The excitation wavelengths are 755 nm and 1064 nm. By rotating the imaging tank in between measurements, multiple projections can be obtained, providing a 3D image of the breast after reconstruction by means of a filtered backprojection. So far, breasts of healthy volunteers were imaged. Three-dimensional images of the breast contour, the nipple and blood vessel networks within the breast could be observed with high contrast and unprecedented detail.

It has been shown that sub-diffraction structures can be resolved in acoustic resolution photoacoustic imaging thanks to norm-based iterative reconstruction algorithms exploiting prior knowledge of the point spread function (PSF) of the imaging system. Here, we demonstrate that super-resolution is still achievable when the receiving ultrasonic probe has much fewer elements than used conventionally (8 against 128). To this end, a proof-of-concept experiment was conducted. A microfluidic circuit containing five parallel microchannels (channel’s width 40μm, center-to center distance 180μm) filled with dye was exposed to 5ns laser pulses (=532nm, fluence=3.0mJ/cm2, PRF=100Hz). Photoacoustic signals generated by the sample were captured by a linear ultrasonic array (128 elements, pitch=0.1mm, fc=15MHz) connected to an acquisition device. The forward problem is modelled in a matrix form Y=AX, where Y are the measured photoacoustic signals and X is the object to reconstruct. The matrix A contained the PSFs at all points of the reconstruction grid, and was derived from a single PSF acquired experimentally for a 10-μm wide microchannel. For the reconstruction, we used a sparsity-based minimization algorithm. While the conventional image obtained by beamforming the signals measured with all the 128 elements of the probe cannot resolve the individual microchannels, our sparsity-based reconstruction leads to super-resolved images with only 8 elements of the probe (regularly spaced over the full probe aperture), with an image quality comparable to that obtained with all the 128 elements. These results pave the way towards super-resolution in 3D photoacoustic imaging with sparse transducers arrays.

Photoacoustic microscopy (PAM) is able to represent the light energy absorption of specific biological molecules (hemoglobin, melanin, DNA/RNA, lipid etc.) without the contrast agents. Especially, AR-PAM can overcome the optical diffusion limit and achieve much greater penetration depth up to a quasi-diffusive regime with low acoustic scattering. Therefore, AR-PAM has been significantly investigated in various applications for morphological, physiological, and molecular information. However, previously developed AR-PAM systems have limited field-of-view (FOV) and imaging speed. These barriers preclude AR-PAM systems from their application to exploratory preclinical and clinical trials. In this work, we introduce an ultra-wide-field AR-PAM system. We fabricated a water-proof microelectromechanical systems (MEMS) scanner for high-speed imaging integrated with two stepper-motors for wide-field scanning (30 × 80 mm²). Finally, we performed in-vivo experiments for preclinical and clinical applications to validate the developed AR-PAM system. For the preclinical experiment with mice, we visualized ventral, sagittal, and dorsal anatomical microstructures including vascular layers and inner organs non-invasively using one wavelength. An entire 3D volume data was acquired with the developed system and depth-encoded along the skin surface up to 2.3 mm. Subcutaneous multi-layers were differentiated on each layer according to distinct microstructures such as recognizable vasculatures and organs (mammary vessels, caudal vessels, popliteal vessels, intestine, heart, spline, etc.). Moreover, we have successfully obtained the distinct microstructures and microvascular networks of human fingers, palm and forearm in-vivo.

Optoacoustic imaging is a highly scalable and versatile method that can be used for optical resolution (OR) microscopy applications at superficial depth yet can be adapted for tomographic imaging with ultrasonic resolution at centimeter penetration scales. However, imaging speed of the commonly employed scanning-based microscopy methods is slow as far as concerned with acquisition of volumetric data. Herein, we propose a new approach using multifocal structured illumination in conjunction with a spherical matrix ultrasonic array detection to achieve fast volumetric optoacoustic imaging in both optical and acoustic resolution modes. In our approach, the laser beam is raster scanned by an acousto-optic deflector running at hundred hertz scanning rate with the beam then split into hundreds of mini-beams by a beamsplitting grating, which are subsequently focused by a condensing lens to generate multifocal structured illumination. Phantom experimental results show that 10 x 10 x 5 cm³ volumetric imaging can be accomplished with spatial resolution around 29 μm. We believe by further speeding up the data acquisition in the further, the system will be operated in full power, making it possible to study functional, kinetic and metabolic processes across multiple penetration scales.

We present an ultra-thin endoscope that combines a multimode optical fiber (MMF) attached to an optical hydrophone for simultaneous optical-resolution photoacoustic microscopy and fluorescence imaging. The MMF is used for light delivery and fluorescence collection and the hydrophone for acoustic detection; a digital micro-mirror device (DMD) modulates the amplitude of the optical wavefront of a pulsed laser coupled into the MMF, controlling the illumination at the distal tip. The DMD allows for fast calibration approaches to reach calibration and measurement times of a few seconds. We obtain optical-diffraction-limited images with full field illumination recording the intensity of a series of various calibrated speckle patterns produced by different configurations of the DMD at the input, with no wavefront shaping. The intensity fluctuations from speckle pattern to speckle pattern encodes for the position at which the signal is emitted. The fluorescence signal from the sample is collected with the MMF and detected with a PMT at the proximal side. For the acoustic detection, embedding the ultrasound detection within the device avoids the absorption of high-frequency ultrasound by the tissue and therefore removes any limitation on the insertion depth. The footprint of the probe is 250 um x 125 um making it thinner than common GRIN lenses used for endoscopy. To best of our knowledge, our approach provides the thinnest endoscope head capable of obtaining optical-resolution photoacoustic and fluorescence images simultaneously.

Photoacoustic (PA) computed tomography (PACT) is a non-invasive imaging technique offering optical contrast, high resolution, and deep penetration in biological tissues. PACT, highly sensitive to optical absorption by molecules, is inherently suited for molecular imaging using optically absorbing probes. Genetically encoded probes with photochromic behavior dramatically increase detection sensitivity and specificity of PACT through photoswitching and differential imaging. Starting with a DrBphP bacterial phytochrome, we have engineered a near-infrared photochromic probe, DrBphP-PCM, which is superior to the full-length RpBphP1 phytochrome previously used in differential PACT. DrBphP-PCM has a smaller size, better folding, and higher photoswitching contrast. We have also developed an advanced PACT technique, which combines the reversibly-switchable photochromic probes with single-impulse panoramic PACT, termed RS-SIP-PACT. Using RS-SIP-PACT, we have characterized DrBphP-PCM both in vitro and in vivo as an advanced near-infrared photochromic probe for PACT. We introduce two phytochromes into the same mammalian cells, resulting in a distinctive decay characteristic in comparison with the cells expressing DrBphP-PCM only. By discriminating the different decay characteristics, we successfully separate multiple cell types in deep tissues. The simple structural organization of DrBphP-PCM allows engineering a bimolecular PA complementation reporter, a split version of DrBphP-PCM, termed DrSplit. DrSplit enables PA detection of protein-protein interactions in deepseated mouse tumors and livers, achieving 125-μm spatial resolution and 530-cell sensitivity in vivo. The combination of RS-SIP-PACT with DrBphP-PCM and DrSplit holds great potential for non-invasive multi-contrast deep-tissue functional imaging.

We present laser-induced ultrasound (LIUS) imaging, using a conventional linear ultrasound probe as a receiver. The LIUS source consists of a 40 μm thick film of Carbon Black-doped PDMS. Illumination of this LIUS transmitter with a 10 ns pulsed Nd:YAG laser with a 10 Hz repetition rate leads to the generation of a short, unipolar ultrasound pulse as a consequence of the photoacoustic effect. Two synthetically focused imaging techniques will be presented: coherently compounded multi-angled plane wave imaging (PWI) and synthetic transmit aperture imaging (SAI) . In the PWI case a planar LIUS transmitter, matched in size to the conventional probe aperture, is used. In the SAI case, the same film is illuminated sequentially at different locations along the aperture by an array of multimode optical fibres. For both PWI and SAI a comparison between conventionally acquired and LIUS images is made, as well as a cross-comparison between PWI and SAI. Images of wire phantoms, speckle analysis and finally images of tissue-mimicking phantoms demonstrate the image quality and advantages offered by LIUS sources. Aside from generating shorter pulses for enhanced resolution, the continuous nature of the absorber and the illumination spot provides a cleaner, more homogeneous plane wave field. The outlook for these unconventional US sources and their relative advantages and disadvantages are discussed.

Photoacoustic microscopy (PAM) as an emerging microscopy system have shown the capability of anatomical, functional, metabolic and pathological imaging of animals and humans in vivo. However, conventional PAM systems have limited spatial and/or temporal resolutions. Microelectromechanical systems (MEMS) scanners and super-resolution techniques have been used, but the MEMS scanners were unstable and conventional ultra-high resolution imaging methods were slow and/or required exogenous contrast agents. Here, we present an agent-free high speed PAM using a stable commercial scanner (L-PAM) that improves both spatial and temporal resolution concurrently. A B-mode image rate is improved up to a B-mode image rate of 500 Hz with the scanner. By localizing endogenous contrasts, unresolved microvessels in general PAM system are resolved. These enhancements enable the observation of microvasculatures and functional hemodynamics of animals and humans in vivo. This L-PAM enables leading to new studies in a variety of fields including neurology, oncology, pathology, pharmacology, etc.

To date, most optical-resolution photoacoustic microscopy (OR-PAM) systems rely on mechanical scanning with confocally aligned optical excitation and ultrasonic detection. As a result, the imaging speed of these systems is limited by the scanning speed. Although several multifocal OR-PA computed tomography (MFOR-PACT) systems had been developed to address this limitation, they were hindered by the complex design in a constrained physical space. Here, we present a two-dimensional (2D) MFOR-PAM system based on a 2D microlens array and an acoustic ergodic relay. This system is able to detect PA signals generated from 400 optical foci in parallel with a single-element transducer, and then raster scan the optical foci patterns to form an image. This system has improved the imaging resolution of a conventional photoacoustic ergodic relay system from 220 μm to 13 μm. Moreover, this system has reduced the imaging time of a conventional OR-PAM system at the same resolution and laser repetition rate by 400 times. We demonstrated the ability of the system with both in vitro and in vivo experiments.

Classical non-learned algorithms for photoacoustic tomography (PAT) reconstructions are mathematically proven to converge, but they can be very slow and inadequate with respect to model and data assumptions. Recently, learned neural networks have shown to surpass the reconstruction quality of non-learned algorithms, but since analysis is challenging, convergence and stability are not guaranteed. To bridge this gap, we investigate the stability of algorithms in which we combine the structure of model-based algorithms with the efficiency of data-driven neural networks. In the last decade, primal-dual algorithms have become popular due to their ability to employ non-smooth regularisation, which is used to overcome the limited sampling problem in photoacoustic tomography. The algorithm performs updates in both the image domain (primal) and the data domain (dual). These are connected by the photoacoustic operator, which modelling is based on the laws of physics and system settings. In our approach, we replace the updates with shallow neural networks, while maintaining the primal-dual structure and the information from the photoacoustic operator. This greatly improves reconstruction quality, especially in cases of strong noise and limited sampling. This has the additional benefit that a hand-crafted regularisation does not have to be chosen, but is learned in a data-driven manner. We show its robustness in simulation and experiment against uncertainty and changes in PAT system settings. This includes the number, placement and calibration of detectors, but also changes in the tissue type that is imaged. The method is stable, computationally efficient and applicable to a generic photoacoustic system with universal applications.

k-Wave is an open-source MATLAB toolbox designed for the time-domain simulation of propagating acoustic waves in 1D, 2D, or 3D. The first release was in 2009, and focused on the simulation of photoacoustic initial value problems and the reconstruction of photoacoustic images from simulated or experimental data. In the ten years since, there have been eight major releases, extending both the functionality and the computational performance of the toolbox. There are now more than 10,000 registered users worldwide, and the toolbox has become the defacto standard for simulation studies in photoacoustic imaging. The development team responsible for k-Wave has also grown, with expertise now spanning physics, mathematics, inverse problems, numerical methods, software engineering, and high-performance computing. In this presentation, the major theoretical, algorithmic, and computational developments of k-Wave will be described, along with the underlying design inputs and decisions that led to these developments. A roadmap for the future development of k-Wave will also be presented. This includes new transducer classes, stair-case free sources, native support for multiple GPUs, adaptive grid refinement using moving mesh methods, gradient-based iterative photoacoustic image reconstruction, performance and accuracy improvements for the elastic wave models, and automatic job-submission to run k-Wave simulations remotely using HPC-as-a-service.

Limited-view and bandwidth-limited acquisition in acoustic–resolution photoacoustic imaging lead to known imaging artifacts. To eliminate these artifacts, it has first been proposed to use fluctuations induced by multiple speckle illuminations to recover otherwise invisible features. However, a very small size of the optical speckle grain at depth in tissue against the acoustic resolution makes this approach unrealistic in practice. Here, we demonstrate experimentally in vitro that fluctuations induced by blood flow at physiological concentration may be exploited to improve visibility in photoacoustic imaging. We first illustrate how our method reveals features otherwise invisible due to the source directivity: a bended capillary tube (inner diameter 100µm) filled with blood flowing at 1.7cm/s was illuminated by 5ns laser pulses (=532nm, fluence=3.0mJ/cm2, PRF=100Hz) and imaged with a linear ultrasound array (128 elements, pitch=0.1mm, fc=15MHz) connected to an acquisition device. Being partially invisible in the conventional image, the whole capillary is reconstructed by means of a second-order analysis of photoacoustic images. Second, we illustrate how our approach allows for visualization of the inside of large objects otherwise invisible due to highpass filtering: we performed a second-order analysis on photoacoustic data resulting from illumination (=800nm, fluence=9.0mJ/cm2, PRF=10Hz/100Hz) of a glass tube (inner diameter 1mm) with blood flowing at 1cm/s. Whether the tube is perpendicular to the imaging plane or is lying inside the imaging plane parallel to the probe, the whole blood volume is visible on the fluctuation-based image, whereas conventional imaging only reveals the blood stream boundaries.

There are occasions, perhaps due to hardware constraints, or to speed-up data acquisition, when it is helpful to be able to reconstruct a photoacoustic image from an under-sampled or incomplete data set. Here, we will show how Deep Learning can be used to improve image reconstruction in such cases. Deep Learning is a type of machine learning in which a multi-layered neural network is trained from a set of examples to perform a task. Convolutional Neural Networks (CNNs), a type of deep neural network in which one or more layers perform convolutions, have seen spectacular success in recent years in tasks as diverse as image classification, language processing and game playing. In this work, a series of CNNs were trained to perform the steps of an iterative, gradient-based, image reconstruction algorithm from under-sampled data. This has two advantages: first, the iterative reconstruction is accelerated by learning more efficient updates for each iterate; second, the CNNs effectively learn a prior from the training data set, meaning that it is not necessary to make potentially unrealistic regularising assumptions about the image sparsity or smoothness, for instance. In addition, we show an example in which the CNNs learn to remove artifacts that arise when a slow but accurate acoustic model is replaced by a fast but approximate model. Reconstructions from simulated as well as in vivo data will be shown.

In this work, speckle in acoustic-resolution photoacoustic (PA) imaging systems is discussed. Simulations and experiments were used to demonstrate that PA speckle carries structural information related to sub-resolution absorbers. Numerical simulations of phantoms containing spherical absorbers were performed using Green’s function solutions to the PA wave equation. A 21 MHz linear array was simulated (256 elements, 75×165 µm resolution, bandwidth 9-33 MHz) and used to record, bandlimit and beamform the generated PA signals. The effects of absorber size (10-270 µm) and concentration (10-1000/mm3) on PA speckle were examined using envelope statistics and radiofrequency spectroscopy techniques. To examine PA speckle experimentally, a VevoLAZR system was used to image gelatin phantoms containing 3 and 15µm polystyrene beads, a tissue mimicking radial artery phantom, and murine tumour vasculature in vivo. Fully developed speckle, as assessed by Rayleigh distribution fits to PA signal envelopes, was present in all images (simulated and experimental) containing at least 10 absorbers per resolution volume, irrespective of absorber size. Changes in absorber size could be detected using the spectral slope of the normalized power spectrum (4.5x decrease for an 80 µm increase in size). PA images of flowing blood in the radial artery phantom also revealed the presence of speckle with intensity that fluctuated periodically with beat rate (4 dB per cycle). Speckle was ubiquitous to all murine tumor vasculature images. During treatment-induced vascular hemorrhaging, the spectral slope decreases by 80% compared to untreated mice. These results demonstrate that photoacoustic speckle encodes information about the underlying absorber distribution.

Photoacoustic breast imaging has been under development for more than a decade now, and is progressively moving towards the clinics to investigate its performance in tumor detection and diagnosis. Several system types have been built, all with their own characteristics and corresponding imaging performances. Different studies have observed variations in tumor appearances. Some works have seen peripheral blood vessels in the tumor region, while others showed mass-like appearances at the tumor site. These varying tumor presentations may be caused by the deviations in system characteristics, but can also be caused by anatomical differences of the tumors. While we can still learn a lot from in-vivo studies, accurate simulation studies are needed to understand the tumor appearances in full detail. We have developed a simulation toolbox to perform 3D full breast photoacoustic simulations. To investigate the tumor appearance, several tumor models are embedded inside a MRI-segmented digital breast phantom [Y. Lou et al (2017)], which is pendant in a hemispherical imaging tank filled with water. Illumination fibers and US detectors are placed on the surface of the imaging bowl. The toolbox easily allows to adjust laser and detector characteristics. Illumination of the breast is simulated with GPU-accelerated Monte Carlo simulations [Q. Fang et al. (2009)]. The subsequent acoustic signal generation and propagation is modeled with k-wave (GPU-accelerated, [B.E. Treeby et al. (2010)]). Finally, images are reconstructed to evaluate the tumor appearance.

Quantitative photoacoustic (PA) tomography aims to recover absolute chromophore concentrations from multiwavelength PA images. Challenges include the accurate prediction of the fluence, the accuracy of the initial pressure distribution reconstructed from measured data, and the large scale of the inverse problem involving high resolution 3D images. In this study, a radiance Monte-Carlo (RMC) light model was used to predict the fluence inside tissue phantoms. Gradients of the scattering coefficient and the chromophore concentrations were calculated using the adjoint formalism. The gradient descent efficiency was significantly improved by using adaptive moment estimation. 3D maps of chromophore concentrations and the scattering coefficient were recovered from measured PA images. The inversion scheme was validated on measured images of a tissue phantom consisting of a scattering liquid and chromophore-filled polymer tubes immersed at different depths. The images were acquired at visible and near-infrared wavelengths using a Fabry-Perot scanner with a planar detection geometry. Amplitude mismatches in the reconstructed initial pressure images due to limited view detection were corrected using an ad hoc correction method. The inversion was stabilized by introducing a calibrated absorber in the imaged volume, or an absolute calibration of the setup. 3D maps of absolute chromophore concentrations, their ratios, and the global scattering coefficient were accurately recovered. The recovery of chromophore concentrations in the image background where SNR is low was identified as a significant new challenge for quantitative PA imaging.

A myriad of optoacoustic imaging systems based on scanning focused ultrasound transducers or on tomographic acquisition of pressure signals are available. In all cases, image formation is based on the assumption that ultrasound waves undergo no distortion and propagate with constant velocity across the sample and coupling medium (typically water). Thereby, ultrasound time-of-flight readings from multiple time-resolved signals are required to form an image. Acoustic scattering is known to cause distortion in the signals and is generally to be avoided. In this work, we exploit acoustic scattering to physically encode the position of optical absorbers in the acquired time-resolved signals and hence reduce the amount of data required to reconstruct an image. This new approach was experimentally tested with an array of cylindrically-focused transducers, where a cluster of acoustic scatterers was introduced in the ultrasound propagating path between the sample and the array elements. Ultrasound transmission was calibrated by raster scanning a lightabsorbing particle across the effective field of view. The acquired calibrating signals were then used for the development of a regularized model-based iterative algorithm that enabled reconstructing an image from a relatively low number of optoacoustic signals. A relatively short acquisition time window was needed to capture the entire optoacoustic field, which demonstrates the high signal compression efficiency. The feasibility to form an image with a relatively low number of signals is expected to play a major role in the development of a new generation of optoacoustic imaging systems.

Photoacoustic imaging shows great promise for clinical environments where real-time position feedback is critical, including the guiding of minimally invasive surgery, drug delivery, stem cell transplantation, and the placement of metal implants such as stents, needles, staples, and brachytherapy seeds. Photoacoustic imaging techniques generate high contrast, label-free images of human vasculature, leveraging the high optical absorption characteristics of hemoglobin to generate measurable longitudinal pressure waves. However, the depth-dependent decrease in optical fluence and lateral resolution affects the visibility of deeper vessels or other absorbing targets. This poses a problem when the precise locations of vessels are critical for the application at hand, such as navigational tasks during minimally invasive surgery. To address this issue, a novel deep neural network was designed, developed, and trained to predict the location of circular chromophore targets in tissue mimicking a strong scattering background, given measurements of photoacoustic signals from a linear array of ultrasound elements. The network was trained on 16,240 samples of simulated sensor data and tested on a separate set of 4,060 samples. Both our training and test sets consisted of optical fluence-dependent photoacoustic signal measurements from point sources at varying locations. Our network was able to predict the location of point sources with a mean axial error of 4.3 μm and a mean lateral error of 5.8 μm.

Photoacoutic imaging of biological tissues is characterized by depth dependent optical fluence loss and acoustic variations. Here, we aim to correct for these inaccuracies aided by extrinsic imaging priors obtained through concurrent high-frequency ultrasound (US) imaging of tissue samples. We segmented the skin line and characterized tissue components using deformable model-based segmentation from the ultrasound images. The prior information from co-registered US images and tissue temperature was used to accurately model light fluence and speed of sound respectively. Methods applied here show significant improvement in beamforming performance, enhanced visual image quality and a higher PSNR.

The International Photoacoustic Standardisation Consortium (IPASC) emerged from SPIE 2018, established to drive consensus on photoacoustic system testing. As photoacoustic imaging (PAI) matures from research laboratories into clinical trials, it is essential to establish best-practice guidelines for photoacoustic image acquisition, analysis and reporting, and a standardised approach for technical system validation. The primary goal of the IPASC is to create widely accepted phantoms for testing preclinical and clinical PAI systems. To achieve this, the IPASC has formed five working groups (WGs). The first and second WGs have defined optical and acoustic properties, suitable materials, and configurations of photoacoustic image quality phantoms. These phantoms consist of a bulk material embedded with targets to enable quantitative assessment of image quality characteristics including resolution and sensitivity across depth. The third WG has recorded details such as illumination and detection configurations of PAI instruments available within the consortium, leading to proposals for system-specific phantom geometries. This PAI system inventory was also used by WG4 in identifying approaches to data collection and sharing. Finally, WG5 investigated means for phantom fabrication, material characterisation and PAI of phantoms. Following a pilot multi-centre phantom imaging study within the consortium, the IPASC settled on an internationally agreed set of standardised recommendations and imaging procedures. This leads to advances in: (1) quantitative comparison of PAI data acquired with different data acquisition and analysis methods; (2) provision of a publicly available reference data set for testing new algorithms; and (3) technical validation of new and existing PAI devices across multiple centres.

Photoacoustic tomographic breast imaging systems progressively move into the clinics for in-vivo studies. Next to tumor detection, studies also focus on extracting information about the tumor by performing multi-wavelength photoacoustics for quantitative oxygen saturation estimations. Until now, it has been difficult to compare the results from different systems due to the wide variability in system characteristics and image reconstruction algorithms. In order to do inter-system comparisons in photoacoustic breast imaging, and to validate oxygen saturation estimations, a standardized but realistic measurement object is required. In this study, we present the first 3D semi-anthropomorphic photoacoustic breast phantom and demonstrate its features both in ultrasound imaging as in photoacoustic tomography.

Opto-acoustic imaging involves using light to produce sound waves for visualizing blood in biological tissue. By using two different optical wavelengths, diagnostic images of blood oxygen saturation can be generated using endogenous optical contrast, without injection of any external contrast agent and without using any ionizing radiation. The technology has been used in recent clinical studies for diagnosis of breast cancer to help distinguish benign from malignant lesions, potentially reducing the need for biopsy through improved diagnostic imaging accuracy. To enable this application, techniques that can accurately map and effectively display small differences in oxygen saturation are necessary. We analyze the ability of an opto-acoustic imaging system to display a colorized parametric map for oxygen saturation of blood using biologically-relevant opto-acoustic phantoms. The relationship between colorized image output and known oxygen saturation values is examined. To mimic breast tissue, a material with closely matching properties for optical absorption, optical scattering, acoustic attenuation and speed of sound is used. The phantoms include two vessels filled with whole blood at oxygen saturation levels determined using a gold-standard sensor-based approach. A flow system with gas-mixer and membrane oxygenator adjusts the oxygen saturation of each vessel independently. Data is collected with an investigational Imagio breast imaging system. An opto- acoustic relative color map is generated using a novel statistical mapping approach. In addition, we propose a technique to characterize the ability to distinguish small differences in oxygen saturation as the oxygenation level is varied. When applied to the phantom, with reference vessel at 99% saturation, hematocrit of 42% and depth of 1.5cm, a contrast distinction threshold was reached when an adjusted vessel achieved a difference of approximately 4.6% saturation compared to the reference.

As a growing number of research groups exploit photoacoustic imaging (PAI), there is an increasing need to establish common standards for photoacoustic data and images in order to facilitate open access, use, and exchange of data between different groups. As part of a working group within the International Photoacoustic Standardisation Consortium (IPASC), we established a minimal list of metadata parameters necessary to ensure inter-group interpretability of image datasets. To this end, we propose that photoacoustic images should at least contain metadata information regarding acquisition wavelengths, pulse-to-pulse laser energy, and information regarding transducer design and illumination geometry. We also suggest recommendations for a standardized data format for both raw time series data as well as processed photoacoustic image data. Specifically, we recommend to use HDF5 as the standard data format for raw time series data, because it is a widely used open and scalable format that enables fast access times. To support long-term clinical translation of photoacoustics we propose to extend DICOM, the prevailing standardized medical image format, to officially support PA images. An international data format standard for photoacoustics will be an important first step towards accelerated system development by facilitating inter-group data exchange and inter-device performance comparison. This effort will thus form a foundation to integrate basic research with clinical translation of PAI.

Acoustic-resolution photoacoustic microscopy (AR-PAM) has great advantage over deep imaging depth when compared to optical-resolution PAM (OR-PAM). This is because that the point spread function (PSF) of AR-PAM is determined by the acoustic focus, which is relatively less scattered in biological tissues. In general, to maintain a high signal-to-noise ratio (SNR) and lateral resolution at a deep depth, AR-PAM uses an acoustic lens with a high numerical aperture (NA). The high NA lens provides high resolution and SNR in focal region, but significantly degrades the SNR and resolution in out-of-focus region. To overcome this problem, many researchers have introduced the synthetic aperture focusing technique (SAFT), which sums up the corresponding signals in the solid angle of the acoustic NA. However, the image enhancement of the conventional SAFTs has been limited because those techniques accumulate the signals without considering the actual photoacoustic (PA) wavefronts. In this study, we propose a novel SAFT that can overcome the existing limitation by exploiting each enhanced frequency components of the 1D SAFT images performed in multiple directions. As a result, we confirmed that the output AR-PAM image of our novel SAFT is superior to the existing SAFT image quality.

Optical-resolution photoacoustic microscopy can measure oxygen saturation () in vivo, offering an important tool to assess tissue oxygenation and health condition. Limited by available wavelengths for fast OR-PAM, the accuracy of sO2 imaging may be degraded by absorption saturation due to high absorption in the blood. Here, we report a nonlinear model to solve the saturation problem and increase the accuracy of measurement. The absorption saturation is analyzed by comparing a nonlinear and linear photoacoustic model using numerical simulation, which shows the nonlinear model has an improved accuracy than the linear model when the absorption is high. Phantom experiments on bovine blood further validate the accuracy of the nonlinear sO2 measurement method. In vivo experiments are conducted in the mouse ear. The values in a pair of arteries and veins are calculated using both linear and nonlinear methods, showing that the nonlinear method measures the arterial value closer to normal physiological condition than the conventional linear model. The nonlinear model requires the use of three or more wavelengths (532nm, 545nm, and 558nm in this work). As a result, we demonstrate the saturation effect in OR-PAM can be compensated via a nonlinear model, which may advance the application of functional optical-resolution photoacoustic microscopy.

A Frozen section examination is the conventional intraoperative histology method widely used in cancer surgery for tumor margin assessment. However, this method is necessary to perform complicated process including sectioning and staining, which require approximately 15 minutes. Particularly with the ultraviolet (UV) laser (266 nm), photoacoustic microscopy (PAM) showed the capability to visualize cell nuclei without the time consuming procedures by utilizing superior optical contrast of DNA/RNA at this wavelength, which can be a potential alternative of the frozen section. However, previously developed UV-PAM is limited to be applied in intraoperative scenarios because it has suffered from slow imaging speed because of 2D mechanical scanning with linear stepper motors. To overcome this limitation, we developed a fast UV-PAM system based on a 2-axis waterproof microelectromechanical systems (MEMS) scanner with the specially fabricated optical components for UV light. This MEMS scanner enables to scan 3 × 3 mm2 range and acquire 400 × 400 pixels image within 20 seconds. The measured spatial and axial resolutions of the developed system are 2.2 and 39 μm, respectively. Finally, we acquired the histology-like PA image of the mouse kidney with characteristic tubular structures of kidney epithelial cells. In the mouse brain, distinct microstructures such as hippocampus and dentate gyrus were differentiated with the validation of frozen section sample.

Model-based reconstruction techniques have been successfully implemented in optoacoustic tomography and acoustic-resolution microscopy to retrieve improved image quality over delay-and-sum methods. In scanning optical resolution optoacoustic microscopy (OR-OAM), no reconstruction methods are employed while post- processing is usually limited to basic frequency filtering and envelope extraction with the Hilbert transform. This results in considerable deterioration of the acoustically-determined resolution in the axial (depth) direction. In addition, when OR-OAM is used for transcranial mouse brain imaging, the skull strongly attenuates high ultrasonic frequencies and induces reverberations, which need to be accounted for during the reconstruction process to avoid image distortions and further deterioration of the axial resolution. Here we show a basic implementation of a model-based reconstruction to increase the axial resolution in OR-OAM. The model matrix is calculated using Field II for free field conditions, taking into account the shape and bandwidth of the spherically focused transducer. Assuming the confinement of the optoacoustic sources within the limits of the optical focus, one may calculate the model matrix by assuming a line source of small absorbing spheres equal in size to the optical beam. In addition, a plate model used in the recently reported virtual-craniotomy deconvolution algorithm is incorporated into the model matrix to tackle the transcranial acoustic transmission problem. The free-field model-based results are compared against the plate model for transcranial brain data obtained in-vivo.

Despite advancements in imaging and surgical methodology surgeons continue to face challenges in differentiating suitable margins in tumor resection sites and assessing the extent of cancer proliferation. Presently, histology is the gold standard used for determining margins post-operatively. However, currently no intraoperative tools can analyze entire resection sites. This results in unnecessary repeated surgeries and is especially critical to patient outcome for certain cancers. To address this critical need, we have developed a next-generation microscopy system intended to allow accurate intraoperative virtual histopathology for margin assessment. The modality, named ultraviolet photoacoustic remote sensing microscopy (UV-PARS), takes advantage of the intrinsic optical absorption contrast of DNA at 266 nm and a non-contact PARS technique. This approach measures time-dependent refractive index modulations at their subsurface origin resulting from thermo-elastic excitation from a pulsed excitation source. This enables the possibility of real time non-contact label-free visualization of cell nuclei in vivo. Preliminary results are presented including studies with live cell cultures, excised tissue samples, phantoms, and characterization of system parameters. Lateral-resolution was found to be 0.7 µm with a signal-to-noise ratio of 50 dB achieved in phantoms and 30 dB for HeLa cells. Simultaneously collected confocal reflectance microscopy using 1310 nm light provided cell body morphology. HeLa and PC3 cell cultures were imaged with good agreement to conventional H&E stained images of the same samples.

To study the structure and functions of the Drosophila brain, confocal microscopy is commonly used. However, surgical removal of the head cuticle of Drosophila is required because the cuticle hinders both the optical excitation and detection. Such invasive surgery may affect brain functions and prohibits long term monitoring. Targeting to the unmet need of surgery free procedure, here we propose laser scanning optical resolution photoacoustic microscopy (LSOR-PAM) for in vivo three dimensional cuticle intact Drosophila brain imaging. Cuticle intact Drosophila brains with cells in optic lobes expressing fluorescent protein DsRed, which serves as an optical absorber and thus a photoacoustic signal source, were imaged. Acquired in vivo 3D LSOR-PAM cuticle-intact brain images were cross-validated using their confocal microscopic counterparts with the cuticles being surgically removed. Acoustic and optical attenuation of the cuticles and degradation in spatial resolution caused by the cuticles were also measured, which explains the reason why LSOR-PAM outperforms confocal microscopy for cuticle intact brains. In addition, the optical absorption bleaching of DsRed expressing optic lobes as a function of the number of the repeated experiments was measured, verifying the LSOR-PAM long-term monitoring capability. In summary, we have demonstrated 3D LSOR-PAM of the Drosophila brain without invasive surgery for the first time. The focus of the future work will be on ways to explore its functional imaging capability on the cuticle intact Drosophila brain.

Retinal neovascularization is a major cause of vision loss and blindness, and is a common complication of numerous retinal diseases, including proliferative diabetic retinopathy, retinopathy of prematurity, sickle cell retinopathy, and retinal vein occlusions. Early diagnosis can be highly beneficial to the treatment of angiogenesis-related eye diseases. Due the limitations of current ocular imaging methods, a hybrid imaging approach that can combine the advantages of current imaging technologies with additional functional and molecular information is highly desired in the field of ophthalmology. A multimodality imaging system with integrated optical coherence tomography (OCT), photoacoustic microscopy (PAM), and fluorescence microscopy (FM) has been developed to evaluate the angiogenesis in clinically relevant larger animal eyes. Real-time, high resolution in vivo imaging was performed in live rabbit eyes with vascular endothelial growth factor (VEGF)-induced retinal neovascularization. PAM images demonstrate a network of tortuous neovascularization on the retina peaking at 7 days post-injection. Blood vessels and irregular vascular structures can also be indicated by OCT B-mode imaging. Leakage of retinal neovascularization is demonstrated by fluorescein sodium with FM. Quantitative analysis of retinal neovascularization has been achieved by PAM. The experimental results demonstrate that this multimodality imaging system can noninvasively visualize retinal neovascularization in both albino and pigmented rabbits for characterization of retinal pathology. This work presents the first description of a multimodality PAM, OCT, and FM system for high resolution, real-time visualization of angiogenesis in rabbits, and could be an important step toward the clinical translation of the technology.

Acquiring more comprehensive information of biological samples requires imaging multiple optical contrasts, which is not typically offered by a single imaging modality. Different optical imaging modalities providing absorption, scattering and molecular information of biological tissues, have been developed and used in many biomedical investigations in the past decades. Large field-of-view (FOV) and high imaging speed are desired for all these imaging techniques. Uneven surface of a sample can lead to uneven depth focus, resulting in images with non-uniform resolution and signal intensity especially in large FOV imaging. Here, we report on our newly developed OCT-guided opto-mechanical scanning multimodal imaging system with the capability of dynamic focusing. By taking advantage of the depth resolving capability of OCT, we developed a novel OCT-guided surface contour scanning methodology for dynamic focusing during entire scanning of an uneven sample. To achieve this, we combined a fully motorized three-dimensional mechanical stage with an X-Y galvanometer optical scanner which made the imaging system suitable for fast scanning of large area. This imaging system integrates photoacoustic microscopy (PAM), optical coherence tomography (OCT) and fluorescence microscopy in one platform providing absorption, structural and molecular information of biological tissue, respectively. Phantom, ex vivo, and in vivo imaging studies demonstrated the performance of the OCT-guided surface contour scanning scheme as well as the capability of our multimodal imaging system to provide comprehensive microscopic information on biological tissues with large FOV and fast scanning. We believe this novel multimodal imaging system has promising potential for preclinical research and clinical practice in the near future. Keywords: Multimodal optical imaging, optical microscopy, photoacoustic microscopy, optical coherence tomography, fluorescence microscopy, dynamic focusing

Multi-parametric photoacoustic microscopy (PAM) is uniquely capable of quantifying the cerebral hemodynamics and oxygen metabolism at the microscopic level. However, the limited depth of focus of conventional PAM is insufficient to encompass the depth variation of the mouse brain when imaging a large area. For instance, the surface contour of the mouse cortex is dome-shaped and spans several hundred microns along the depth direction. When out of focus, the resolution and sensitivity of PAM quickly degrades. Moreover, quantitative measurements (e.g., blood oxygenation and flow) are no longer accurate with the compromised resolution and sensitivity. Here, we report automated contour-scan multi-parametric PAM, which enables simultaneous imaging of blood perfusion, oxygenation and flow with high resolution and sensitivity over the entire mouse cortex. Different from the traditional contour-scan method that requires three steps (pre-scan, off-line calculation of the contour map, and contour scan), our technique can perform high-resolution wide-field contour scan without the first two steps, thereby significantly reducing the acquisition time. We first tested the feasibility of this technique by imaging a plastic ball coated with black ink. Then, we quantitatively analyzed the influence of out-of-focus on the measurement of blood flow in a vessel-mimicking phantom. Finally, we demonstrated cortex-wide multi-parametric PAM in the live mouse brain with high resolution and sensitivity.

In this work, we theoretically and experimentally deal with photoacoustic resolution enhancement by means of saturated modulation quenching. It is shown that experimental systems for resolution enhancement with saturated modulation quenching in fluorescence microscopy are not necessarily suited for photoacoustic modulation quenching. Here, we show that modulation quenching is not limited to fluorescent dyes but can be also applied to metallic nanoparticles. For modulation quenching in photoacoustic microscopy it is sufficient that the signal saturates with increasing excitation intensity.

Label-free functional photoacoustic microscopy (fPAM) has become a popular technology in small-animal hemodynamic studies. Here we report a stimulated-Raman-scattering-based (SRS) dual-wavelength high-speed fPAM that has achieved volumetric imaging at a 1 MHz A-line rate with capillary-level resolution. Potassium gadolinium tungstate (KGd(WO4)2) crystal is used as a Raman shifter to convert the pump 532 nm picosecond-pulsed laser to the first order Stokes line at 558 nm through the SRS effect with ~40% efficiency and a much narrower line width compared with previous fiber-based SRS PAMs. We also developed a water-immersible micro-electro-mechanical system scanner for scanning a ~4-mm range at a 500 Hz B-scan rate, while maintaining the optic-acoustic confocal alignment. This scanner is assembled entirely from commercially available components, facilitating replication. The detection sensitivity of our fPAM is also improved by employing a high numerical aperture polyvinylidene fluoride ultrasonic transducer, whose acoustic impedance matches better with tissue coupling medium than traditional ceramic transducers. The high sensitivity combined with ~2.4 µm resolution enabled our fPAM to image single red blood cells with a signal-to-noise ratio of ~27 dB. Compared with our previous laser-pulse-width based fPAM, we achieved simultaneous imaging of hemoglobin concentration and oxygenation with a 5-fold increase in imaging speed. Moreover, our system works in a convenient free-space manner compared to previous SRS-based PAMs. We applied it to imaging vasculature and blood oxygen saturation on mouse brains in both resting and stimulated states.

Label-free mid-infrared (MIR) imaging provides rich chemical and structural information of biological tissues without staining. Conventionally, the long MIR wavelength severely limits the lateral resolution owing to optical diffraction; moreover, the strong MIR absorption of water ubiquitous in fresh biological samples results in high background and low contrast. Here, we present a novel approach, called ultraviolet-localized MIR photoacoustic microscopy (ULM-PAM), to achieve high-resolution and water-background–free MIR imaging of fresh biological samples. In our approach, a pulsed MIR laser thermally excites the sample at the focal spot, and a pulsed ultraviolet (UV) laser photoacoustically detects the resulting transient temperature rise owing to the Grüneisen relaxation effect, thereby reporting the intensity of the MIR absorption by the sample. The imaging resolution of our method is determined by the wavelength of the UV laser, which is one order of magnitude shorter than that of the mid-IR laser (2.5 μm to 12 μm). In addition, in the UV region from 200 nm to 230 nm, most important organic molecules in biological tissues, including proteins, lipids and nuclei acids, have strong absorption, while water is transparent. Therefore, our method can achieve high resolution and water-background free IR imaging of fresh biological samples. For cell cultures, our method achieved high-resolution and high-contrast infrared imaging of lipids, proteins. The capability of label-free histology of this method is also demonstrated in thick biological tissues, such as brain slices. Our approach provides convenient high-resolution and high-contrast MIR imaging, which can benefit diagnosis of fresh biological samples.

Compared to piezoelectric based photoacoustic (PA) scanners, the planar Fabry-Perot (FP) scanner has several advantages. It can provide small element size with high sensitivity, a smooth broadband frequency response, and is transparent to excitation light. This enables the FP scanner to provide excellent high-resolution in vivo PA images of soft tissue to depths up to approximately 10 mm. However, unlike piezoelectric scanners, the FP scanner in its current form cannot provide a pulse-echo ultrasound (US) as well as a PA image, which is useful because of the additional tissue contrast it provides. To address this, a dual mode FP scanner-based system that, for the first time, can acquire co-registered 3D PA and US images has been developed. In order to provide an optical US generation capability, the FP ultrasound sensor was coated with a novel Gold-Nanoparticle-PDMS composite which was excited with nanosecond laser pulses to generate plane wave US pulses. By modifying the FP sensor in this way, it now acts as an US transmitter as well as a receiver. The coating is highly absorbing at the US generation wavelength (>95%) but transparent at the PA excitation wavelength, the latter to allow the system to also operate in PA imaging mode as before. The generated US pulses exhibited peak pressures in the MPa range, which is comparable to the output of conventional piezoelectric based medical US scanners. The pulses had a broad bandwidth (>40 MHz) and the emitted wavefront was planar to within λ/10 at 10 MHz. PA and pulse-echo US signals were mapped in turn by the FP scanner over centimetre scale areas with a step size of 100 μm and an element size of 64 μm. The -3dB bandwidth of the FP sensor was 30 MHz. Reconstruction methods using a k-space formulation recovered co-registered 3D PA and US images. The system’s lateral spatial resolution was evaluated by imaging a line target at depths up to 10 mm and ranged between 50 and 120 μm for both modes. Arbitrarily shaped 3D objects were imaged to demonstrate the volumetric US imaging capability of the scanner. Tissue mimicking phantoms, with impedance mismatches representative of soft tissues, and ex vivo tissue samples were imaged with the system as well as a conventional clinical US scanner for comparison. Finally, the system obtained promising high-resolution 3D dual mode PA-US images for a variety of phantoms with contrast based on both optical absorption and acoustic impedance. This novel all-optical system has the potential to add complementary morphological contrast to photoacoustic vascular images which could aid the clinical assessment of superficial tumours, lymph node disease and other conditions.

Photoacoustic (PA) imaging has had limited clinical applicability for many reasons but one primary barrier to clinical translation is the bulky, expensive, and low repetition-rate laser typically used, resulting in low frame-rate images and a system with a large physical footprint. We have previously demonstrated a fast-scan approach delivering the frame rates required for real-time integrated PA/ultrasound (PAUS) imaging. In this paper, we present a new real-time PAUS system based on a swept-scanning source approach using a compact, recently-developed laser, providing pulse-to-pulse wavelength tuning at kHz rates and a scanning fiber-optic delivery system integrated with a high-frequency (15 MHz) US linear array. An array of fibers spanning the array are arranged on two lateral sides of the transducer and scanned sequentially based on optimized pulse sequences. By coherent compounding of multiple sub-images associated with each fiber light source, PA imaging with sufficient SNR at a frame rate of 50 Hz is achieved. Real-time in vivo multi-spectral imaging of nano-drug delivery to mice is demonstrated. With the same scanner footprint, our compact PAUS system can provide not only conventional high-quality scanned US imaging with all associated modes, but interleaved, multispectral PA imaging at video rates appropriate for real-time clinical applications.

Photoacoustic imaging (PAI) can exploit near infrared (NIR) small molecule dyes to enhance contrast for the early identification of cancer with high biocompatibility and minimal toxicity. Unfortunately, the low optical absorption and rapid clearance of small molecule dyes impose considerable limitations for in vivo imaging applications, due to their limited PAI signal generation capabilities in the tumor bed. Using DNA nanotechnology it is possible to precisely position individual dye molecules to tune their intrinsic PAI signal generation capabilities and to engineer nano-carriers that enable targeted delivery and also prolong the lifetime of the contrast agent in vivo. Here, we report the synthesis, characterization and in vivo imaging of DNA nanotechnology-derived nano-carriers that exhibit superior tumor accumulation and photoacoustic (PA) signal generation capabilities when compared to free dyes. The nano-carrier (NC6Q+) comprised of closely positioned IRDye-800CW molecules offers good serum stability and biocompatibility. Absorption and emission measurements show that NC6Q+ exhibits a blue shifted absorption peak at 705nm due to exciton coupling and offers more than 80% fluorescence quenching efficiency. As a result, PAI data from NC6Q+ in tissue mimicking phantoms show a 72% enhancement in PA signal when compared to free IRDye-800CW. In vivo bio-distribution (n=2) and kinetics studies of the nano-carriers indicate 87 % increase in blood retention time compared to free dye, while maintaining renal clearance. Multispectral photoacoustic imaging of tumor bearing mice (n=3) showed 88% enhancement in tumor PA signal for NC6Q+ when compared to free IRDye-800CW. These results suggest that DNA nano-carriers hold promise to create PAI contrast agents with enhanced tumor uptake and PA signal generation capabilities

The interaction of methylene blue (MB) and sodium dodecyl sulfate (SDS) leads to a reversible spectral shift during SDS micellization, but the underlying mechanism has remained unclear. Here, we measured photoacoustic (PA) intensity, micelle concentration, and spectral shift of MB-SDS complex to elucidate this interaction mechanism. We observed a switchable PA effect of MB, which is sensitive to critical micelle concentration (CMC) of SDS (i.e. 8 mM). The addition of 3.47 mM SDS increased the PA intensity of 0.05 mM MB 492-fold because of fluorescence quenching. Then, the PA intensity decreased by 54-fold when the SDS concentration was increased above the CMC at 8.67 mM due to decrease of MB aggregation. Meanwhile, we observed increased number of non-micellar MB-SDS clusters, ranging from 80 to 400 nm, as the SDS concentration approaching to CMC and then the number decreased once the SDS concentration was above CMC. The correlation between PA intensity and nanoparticle number indicated that the formation of MB-SDS cluster was responsible for the PA enhancement. Further controlled studies using MB/hexadecyltrimethylammonium bromide, MB/sodium octyl sulfate, and MB/sodium chloride showed that the binding between MB and SDS occurred at the sulfate moiety of SDS. They also found that MB-SDS clusters disassociated to micelles MB-SDS monomers at the SDS micellar concentrations. These findings further elucidate the binding mechanism of MB and SDS and presented the potential for developing an activatable MB PA contrast agent.

We recently reported a real-time method to measure heparin in blood based on photoacoustic (PA) signal from methylene blue (MB). The PA enhancement was surprisingly accompanied by a decrease in absorbance. Here, we describe a mechanistic study of the MB-heparin binding in water and phosphate buffered saline. The addition of 0.79 mg/mL heparin decreased the nuclear magnetic resonance (NMR) magnitude of 0.90 mg/mL MB by 63% with a 0.25 ppm downshift—this indicated formation of MB aggregates due to π-π staking of MB. We also observed nanoscale MB/heparin aggregates under transmission electron microscopy (TEM). Spectroscopic analysis of the isolated aggregates found that the percentage of MB inside the MB-heparin aggregate increased from 3.6% to 82.5% when heparin concentration was increased from 0.16 mg/mL to 0.79 mg/mL. Meanwhile, the photoacoustic intensity increased 25-fold. The signal increase was largely due to the aggregates rather than free MB in the solution. These trends suggested that the MB-heparin aggregation was responsible for the PA enhancement likely due to the decreased degrees of freedom for MB. Molecular dynamics simulations revealed MB dimer formation on heparin and indicated that electrostatic binding occurred between the central thiazine ring of MB and the sulfates and glucosamines in heparin via electrostatic interaction. These findings elucidate the binding process of MB and heparin and provide strategies for immobilizing MB-like molecules on implantable devices for intravascular heparin sensing.

Chronic hypoxia in pulmonary diseases is known to have a severe negative impact on heart function, including right heart hypertrophy, increased workload on the heart and arrhythmia. Yet, the direct effect of the chronic hypoxic environment on the cardiovascular system is still not fully understood. Usual pre-clinical analytic methods analysing this effect are limited to ex vivo histology or highly invasive approaches such as right heart catheterisation, which inevitably interfere with cardiac tissue. In this work, we propose volumetric optoacoustic tomography as a method for assessing heart function in response to chronic hypoxia non-invasively. Hypoxic and normoxic murine hearts were imaged in vivo at high temporal (100 Hz) and spatial resolution (200 μm). Analysis of the murine models on a beat-to-beat scale enabled identifying and characterizing arrhythmic events in hypoxic models. In addition, blood flow was tracked using indocyanide green (ICG) contrast agent, which revealed a clear difference in the pulmonary transit time (PTT) between the hypoxic and normoxic models. Validation for presence of hypoxia in the lungs was carried out by α-smooth muscle actin staining for muscularization of the pulmonary vasculature. We expect that the novel capabilities offered by volumetric optoacoustic tomography for analysing impaired heart function under hypoxic conditions in pre-clinical models will provide important insights into early diagnosis and treatment methods for pulmonary diseases.

We developed and designed a near-infrared (NIR) absorbing diboronate xanthene dye ((E)-1,3,3-trimethyl-2-(2-(6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indol-1-ium) for photoacoustic imaging that is sensitive to reactive oxygen and nitrogen species (RONS). We initially used the probe with an OPO-based laser but found that the photoacoustic intensity degrades by 2.6-fold within 3150 laser pulses. Therefore, we adopted the probe to a LED-based excitation source (The average and standard deviation of photoacoustic intensity in presence of 3150 LED pulses is 118.08 and 1.67 (1.4% variation) respectively) and found that hydrogen peroxide (H2O2), superoxide radical (O2˙ −), and peroxynitrite (ONOO−) produce absorption at 700 nm, which was used for photoacoustic excitation. We observed a photoacoustic intensity increase of 2.1-, 1.9-, and 1.75-fold with addition of ONOO−, O2˙ −, and H2O2, (50 ), respectively. The dye is not sensitive to OCl − and ˙OH. At vascular compartment, formation of ONOO− is based on the reaction of nitric oxide (˙NO) with superoxide radical (O2˙ −) and formed ONOO− xidize plasmatic components as well as reaction with intracellular. We then tested the photoacoustic response of various concentrations of ONOO− (25, 50, 125, 185, 250, 375, and 500) in whole human plasma and blood. Concentrations of 50 ONOO− were also easily detectable with this probe. Finally, we examined the capability of new molecular probe for detection of endogenous RONS via SKOV3 (ovarian cancer) cell media. The RONS from these cells activated the probe but media treated with N-acetylcystein (NAC) (RONS scavenger) did not. In pre-incubated cells with NAC, we observed 2.5-fold decrease in photoacoustic intensity versus untreated cells.

We use a novel acoustic-based flow cytometer to detect individual nanobubbles flowing in a microfluidic channel using high-frequency ultrasound and photoacoustic waves. Each individual nanobubble (or cluster of nanobubbles) flowing through the foci of high-frequency ultrasound (center frequency 375 MHz) and nanosecond laser (532 nm) pulses interacts with both pulses to generate ultrasound backscatter and photoacoustic waves. We use in-house generated nanobubbles, made of lipid shells and octafluoropropane gas core, to detect ultrasound backscatter signals using an acoustic flow cytometer. Nanobubble solutions sorted in size through differential centrifugation are diluted to 1:10,000 v/v in phosphate buffered saline solution to maximize the probability that the detected signals are from individual nanobubbles. Nanobubble populations were sized using resonant mass measurement. Results show that the amplitude of the detected ultrasound backscatter signal is dependent on the nanobubble size. The average amplitude of the ultrasound backscatter signals from at least 950 nanobubbles with an average diameter of 150 nm, 225 nm, and 350 nm was 5.1±2.5 mV, 5.3±2.3 mV, and 6.4±1.8 mV, respectively. Similarly, we detected interleaved ultrasound backscatter and photoacoustic signals from nanobubbles tagged with Sudan Black B dye. The average amplitude of the ultrasound backscatter and photoacoustic signals from these black nanobubbles with an average diameter of 238 nm is 10±11 mV and 54±75 mV, respectively. The presence of the dye on the shell suppressed unique features seen in the ultrasound backscatter from the nanobubbles without dye. At present, there is no robust commercial technique able to analyze the ultrasonic response of individual nanobubbles. The acoustic flow cytometer can potentially be used to analyze physical parameters, such as size and ultrasonic response, of individual nanobubbles.

Nanobubbles are a new class of ultrasound contrast agents. Unlike conventional microbubbles, their sub-micron (~200nm) diameter allows them to extravasate outside the vasculature and accumulate in the tumor interstitium. In this study, nanobubbles with shells loaded with Sudan Black (BNB) and DiD fluorescent dye were synthesized. These nanobubbles can be used to simultaneously enhance ultrasound and photoacoustic signals for in vivo breast tumor imaging. The nanobubbles consisted of lipid shells with a C3F8 gas core and were formed via self-assembly driven by mechanical agitation and size isolation via centrifugation. Herceptin antibody was conjugated to the BNB for targeting HER2-positive cells via standard EDC/NHS coupling chemistry. Human breast cancer cell lines (BT474 as HER2-positive and MDA-MB-231 as HER2-negative) were inoculated in the flanks of BALB/c-B17-Scid mice. Ultrasound and photoacoustic imaging (VevoLAZR, 21MHz, 720nm) were performed pre-injection and post-injection of the Herceptin conjugated BNB. The impact of Herceptin targeting was assessed by computing the PA frequency spectra and the non-linear contrast US images of the tumor regions. Photoacoustic images of the HER2-positive tumor showed an average of 6 dB increase in contrast signal 2 mins post-injection, while the HER2-negative MDA tumors showed a negligible change in image contrast, suggesting increased uptake of Herceptin labelled BNBs. The enhanced contrast is also confirmed by the non-linear contrast signals between positive and negative tumors. The photoacoustic technique can potentially be used to examine the kinetics of BNB extravasation. This work shows the potential of BNBs as multi-modal contrast agents capable of specialized tumor imaging in vivo.

Imaging intracellular calcium dynamics to evaluate the cellular activity is crucial for an understanding of the related cell behavior and signal transduction. Identifying spatial-temporal activity patterns in three-dimensional (3D) tumor cell culture can provide novel insights into calcium-associated tumorigenesis and lead to optimizing the tumor-killing strategies. However, the information cannot be obtained without adequate penetration in 3D cell culture. Herein, we develop optical-resolution photoacoustic microscopy (OR-PAM) by tuning the laser wavelength to 627 nm to investigate calcium waves in 3D tumor cell culture using a novel photoacoustic contrast agent, Chlorophosphonazo-III (CPZ). CPZ has a peak optical absorbance at 650 nm. Not like another Bisazo derivative that can also be used for PA calcium imaging, Arsenazo-III, non-toxic and the high survival rate is observed even when cells are treated with 150 μM of CPZ (<97%). Moreover, CPZ can differentiate calcium changes using single wavelength like some conventional single wavelength calcium indicators used in optical imaging. Phantom results show that the photoacoustic (PA) signal intensity is highly correlated with calcium concentration using 100 μM or 150 μM CPZ, where R2 values are 0.94 and 0.97, respectively. To investigate the feasibility of live-cell PA calcium imaging in 3D cell culture, we evoke the intracellular calcium puffs of tumorspheres using thapsigargin and high concentration of extracellular calcium. A 2-fold enhancement of PA calcium intensity is observed after stimulating tumorspheres with thapsigargin or extracellular calcium. It demonstrates that the functional calcium imaging in 3D tumor cell culture can be detected by our OR-PAM system and CPZ can serve as a functional PA contrast agent.

A cell’s nucleus-to-cytoplasm (N:C) ratio is a histological metric used to stage malignant disease. Current N:C assessment methods, such as optical microscopy, are time-consuming, subjective, and low-throughput. Here, we compare the N:C ratios of prostate cancer (PC-3) cells measured by a novel microfluidic PhotoAcoustic Flow Cytometer (PAFC) to those obtained using an Imaging Flow Cytometer (IFC). PC-3 cells were stained with DRAQ-5 nuclear dye and divided into populations measured using the PAFC and IFC. The PAFC consisted of a microfluidic device integrated with a singleelement ultrasound transducer (375 MHz central frequency) and a sub-nanosecond pulsed laser (532 nm). Individual cells were 3D flow-focused through the overlapping focal region of the ultrasound and laser pulses. PAFC estimation of the cell and nucleus diameters were determined through power spectra fitting of backscattered US waves and emitted PA waves to established theoretical models. An ImageStreamX® IFC was used to acquire brightfield and fluorescent images of individual cells, which were masked, gated, and used to assess the cell (brightfield) and nucleus (fluorescence) diameter to validate the PAFC measurements. The average cell and nucleus diameters determined using the PAFC (n = 388) were 18.8 ± 3.3 μm and 14.3 ± 2.9 μm, respectively. The corresponding values from the IFC (n = 4651) were 18.3 ± 2.2 μm and 12.2 ± 1.9 μm. The N:C ratio (calculated as the ratio of the nucleus diameter to cell diameter) was 0.77 ± 0.10 using the PAFC and 0.67 ± 0.07 using the IFC. Our novel PAFC device has the potential to be used for circulation tumor cell detection using the N:C ratios of cells.

Due to its superior molecular sensitivity, surgical management of breast cancer now often includes preoperative dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). Nevertheless, statistics indicate that, in practice, tumor size is frequently misestimated leading to incomplete resections, and consequently, repeat surgeries. Our group developed a surgical specimen assessment technique, called intraoperative photoacoustic screening (iPAS), based on photoacoustic tomography and with the capability to visualize whole breast tumors. The system was deployed at a breast surgical center and used to scan freshly excised breast tissue specimens belonging to 12 patients. This report compares breast cancer imaging performance by iPAS to that of DCE-MRI, and pathology.

In recent years, conventional ultrasound (US) imaging devices have been adapted with the photoacoustic (PA) imaging capabilities to simultaneously provide both anatomical and molecular optical contrasts of soft biological tissues. To help optimize the design parameters of such dual modality imaging devices, we present a numerical simulation approach for Bmode beamformed US and multispectral PA imaging using a linear ultrasound transducer array surrounded by a light source. We combined the finite element based simulation platforms for ultrasound and light propagation, K-wave and NIRFast respectively, to model the ultrasound and photoacoustic effects in deep tissue, and created an effective hybrid platform for simulating US and multispectral PA imaging of different configurations. We also developed and applied a spectral unmixing algorithm on multispectral photoacoustic images, obtained from multiple optical wavelengths, to map different molecules (e.g., Indocyanogreen (ICG), Deoxyhemoglobin (Hb), and Oxyhemoglobin (HbO₂)) present inside the tissue background. The multi-spectral plots and unmixed spectral images clearly delineated the molecular contrast arising from different regions inside the tissue. The presented simulation platform allows for optimization of key design parameters of both US and PA imaging devices, such as the size of ultrasonic transducer array, and size and the distribution of light sources. Our results demonstrate that the ability to mimic the imaging performance of such dual modality deep tissue-imaging device will help to achieve high molecular sensitivity for the targeted clinical application, thus functioning as a powerful tool for medical device design.

Tissue elasticity is an important biomarker for early prediction of diseases, and elastography methods have been developed for several modalities. In practice, photoacoustic imaging (PAI) provides information about the optical properties of deep-seated tissue based on an ultrasonic readout. In this study, we demonstrate the use of PAI to measure tissue elasticity through high frequency acquisitions. The method was tested using tissue mimicking agar phantoms and transducers of different frequencies. The results show that elastic contrast can be recovered from PA signals only using high-frequency transducers, given significant differences of elasticity between the imaging target and background.

During RF ablation for atrial fibrillation, undesired tissue conductivity is interrupted by inducing lesions through applying RF current. Feedback on lesions formed is currently indirect, leading to unpredictable outcomes for patients. Previously we have shown that two-wavelengths photoacoustic imaging can distinguish between scarred and healthy tissue (Iskander-Rizk et al. BOE 2018). This principle may be translated to clinical imaging by integrating an optical fiber in the ablation catheter to generate photoacoustic signals of lesions using commonly present intracardiac echo (ICE) probe as a receiver. We modified a commercially available ablation catheter to fit a 400µm, 0.39 NA multi-mode optical fiber through the flushing channel. In a porcine passive beating heart model (Lifetec, Eindhoven), we inserted the modified ablation catheter and a St Jude ViewFlex ICE probe through the jointly tied pulmonary vein into the left atrium. We ablated around the mitral valve, and we used a 100Hz laser source (Innolas Spitlight EVO-OPO) constantly toggling between 790 and 930 nm to generate photoacoustic signals at the ablation site. The signals were digitized and processed with a Verasonics vantage 256 system. One acquisition frame consisted of 5 tilted diverging wave ultrasound acquisitions and 1 photoacoustic acquisitions per wavelength. We monitored lesion progression and continuity in a beating heart addressing motion artefacts concerns. In addition to that, the dual wavelength photoacoustic images successfully eliminate undesirable signals from the catheter tip, blood and healthy tissue, leaving only signals from lesions, enabling real-time intracardiac ablation monitoring that is readily translatable to an in vivo setting.

Optical imaging and photothermal therapy have been applied in biomedical field for decades. However, the strong scattering of light in biological tissue hinders the focal light delivery and thus restricts their clinical applications because of the resultant limited penetration. We hypothesize that the photon scattering is reduced in the cylindrical heating zone of high intensity focused ultrasound (HIFU) and thus the efficiency of light delivery can be improved via transmission of light through the heating cylindrical tunnel, enabling photoacoustic signal enhancement at the targeted region. In this study, Monte Carlo simulation and intralipid-phantom experiments were used to verify our hypothesis. The thermal effect could increase the laser fluence at the targeted region by at least 10% no matter in the simulation or the experiment. Similar results were also presented in the measured photoacoustic signal. Note that special care had been taken to keep the Gruneisen coefficient at the targeted region constant so that the photoacoustic signal change solely depended on delivered laser fluence. In addition, the simulation results indicate that with the local cylindrical heating tunnel, the fluence at the targeted region is at least 10% higher than that with global heating, suggesting that HIFU heating tissue tunnel owns the potential in enhancing the light delivery efficiency, the light penetration and thus the photoacoustic signal at the targeted region as well. It is expected that our finding is not only applicable to photoacoustic imaging but also photothermal therapy which also requires more focal light delivery.

Efficient monitoring of radiofrequency ablation procedures is essential to optimize the lesions induced to treat cancer, cardiac arrhythmias and other conditions. Recently, optoacoustic imaging and sensing methods have been suggested as a promising approach to address this challenge, offering unique advantages such as high sensitivity to temperature changes and chemical transformations in coagulated tissues, real-time operation and use of non- ionizing radiation. However, assessing how the ablation lesion boundary progresses is still challenged by changes in optical properties induced during the interventions. Herein, we suggest a new approach for dimensional characterization of the induced lesion based on detecting sharp positive variations in the time derivative of optoacoustic signals. Experiments in porcine tissue samples indicate that such variations are uniquely associated to the onset of ablation and that the method can robustly visualize the evolution of the lesion in three dimensions.

Liver surgeries carry considerable risk of injury to major blood vessels, which can lead to hemorrhaging and possibly patient death. Photoacoustic imaging is one solution to enable intraoperative visualization of blood vessels, which has the potential to reduce the risk of accidental injury to these blood vessels during surgery. This paper presents our initial results of a feasibility study, performed during laparotomy procedures on two pigs, to determine in vivo vessel visibility for photoacoustic-guided liver surgery. Delay-and-sum beamforming and coherence-based beamforming were used to display photoacoustic images and differentiate the signal inside blood vessels from surrounding liver tissue. Color Doppler was used to confirm vessel locations. Results lend insight into the feasibility of photoacoustic-guided liver surgery when the ultrasound probe is fixed and the light source is used to interrogate the surgical workspace.

Diffuse correlation spectroscopy (DCS) is an emerging technology that allows for the quantitative estimation of blood flow in tissue. By monitoring the autocorrelation of the time course of light speckle intensity, information about the motion of scattering particles, mostly red blood cells in the microvasculature of biological tissues, can be determined. The speckle fluctuations are due to motion of scatters along the entire path length of the photon from the source to the detector, which makes the determination of the location of the motion a difficult task. Multi-distance and tomographic methods have been employed to measure decorrelation times at different source detector separations, which helps to separate superficial blood flow from blood flow deeper in the tissue. DCS in the time-domain (TD-DCS) is being evaluated as a method to increase depth sensitivity by considering only the late arriving photons. Depth resolved quantification of blood flow is especially important when blood flow measurements of the brain are desired, as the superficial blood flow of the scalp is a known contaminant to the cortical signal. Recent demonstrations by other groups have shown the utility of ultrasound tagging of light to be an effective method to discriminate flow at different depths.1 Here we utilize ultrasound pulses to modulate the motion of particles at specific depths, which is dependent upon the time-of-flight of the ultrasound pulse. By analyzing the autocorrelation of the speckle intensity at different delay periods after the pulse, quantitative, depth specific information about the flow can be determined. References: 1. Tsalach, A. et al. Depth selective acousto-optic flow measurement. Biomed. Opt. Express 6, 4871–86 (2015).

Publisher’s Note: This video, originally published on 4 March 2019, was withdrawn per author request.

Photoacoustic microscopy with large depth of focus is significant to the biomedical research. Here, we developed a multifocus photoacoustic microscopy by using a tunable acoustic gradient (TAG) lens and optical delay pathways. We split a single laser pulse into three sub-pulses and introduce them into three multimode fibers with a length of 1 m, 26 m and 51 m, respectively. The sub-pulses out of the fibers were combined by a single-mode fiber thereafter. We then obtained a pulse train with a time interval of 120 ns. The output of the single-mode fiber is collimated by a fiber port, and then guided into homemade TAG lens vertically. A function generator generates a sinusoidal signal to drive the TAG lens at an eigenmode. The focusing power of the TAG lens will exhibit a sinusoidal oscillation at the frequency of the driving signal. By controlling the fire time of the pulse train and the driving signal of the TAG lens, the laser pulses out of three multimode fibers synchronize with three vibration states of the TAG lens. And we finally achieved three focal spots in one A line data acquisition using a single input laser pulse. The depth of focus (DoF) of the system was measured to be 360 μm, which is three times of that of single-focus system without the sacrifice of time resolution. A mouse cerebral vasculature were imaged in-vivo to demonstrate the feasibility of the extended DoF of our system.

Hemorrhagic shock, as an important clinical issue, is regarding as a critical disease with a high mortality rate. Unfortunately, existing clinical technologies are inaccessible to assess the hemorrhagic shock via hemodynamics in microcirculation. Here, we proposed an ultracompact photoacoustic microscope to assess hemorrhagic shock by using a rat model. In animal study, hemodynamic features of the microvascular network including concentration of total hemoglobin (C_HbT) and small vascular density (SVD) were derived to assess the microvascular hemodynamics of different organs, assessing the hemorrhagic shock via microcirculation. On the other hand, in vivo oral imaging of healthy volunteers also indicates the translational possibility of this technique for clinical evaluation of hemorrhagic shock.

Conventional photoacoustic tomography (PAT) systems use bulky, expensive Q-switched Nd:YAG lasers which limits its usage in clinics. The low repetition rate of these lasers makes them not suitable for real-time imaging as well. Compact pulsed laser diodes (PLD) are being currently used for PAT systems due to their high repetition rates and compact in size. These lasers can be mounted inside the PAT circular scanning geometry making the PAT system more portable. Using acoustic reflector based single-element ultrasound transducer (SUTR), the scanning radius can be reduced thereby making the PAT system more compact. In this work, we present a portable, high frame rate, more compact second generation PLD-PAT system using eight SUTRs in circular scanning geometry. This system provides high frame rate of ~1 Hz. We demonstrate the performance of this system using phantom imaging studies.

Monitoring of tissue temperature is necessary for guiding energy-based medical treatments. The local temperature information is also important for the safe deposition of light/heat energy into the surrounding healthy tissue. Existing imaging modalities fail to monitor tissue temperature with high accuracy and high resolution. Photoacoustic sensing of temperature was demonstrated using Q-switched Nd:YAG laser. A temperature sensitivity of ~0.15°C was obtained at a temporal resolution of ~2 s. Photoacoustic imaging is a high-speed, high-resolution, deep tissue imaging modality for both preclinical and clinical applications. In this work, we demonstrate photoacoustic sensing of temperature at high temporal resolution order of microseconds using high repetition rate (7000 Hz) near-infrared (~803 nm) pulsed laser diodes. The system will find applications in radiation therapy, photothermal therapy, photodynamic therapy, etc.

We report our experimental study on enhancement of photoacoustic (PA) signal from contrast dye by pre-illumination of the dye with continuous wave (CW) light beam other than optical pulse (pulse width ~ nsec) that is employed to irradiate tissue sample for inducing thermoelastic expansion and subsequently, generation of photoacoustic waves. This unique technique is in contrast to the conventional approaches of shift of optical absorption peak - in the characteristic absorption spectrum of (PA signal) contrast dyes, say, nano-particles by control of the physical parameters including structure, shape and size. Experiments were conducted employing home-built photoacoustic (acoustic resolution) microscopy imaging system for measurement of PA signal strength induced - under various conditions: (1) without any pre-illumination of the dye with CW laser beam, i.e., only pulse laser beam is employed to irradiate the dye and (2) with pre-illumination of the organic dye at various time durations of pre-illumination. In this second case, the dye is exposed, firstly, to CW laser beam (of wavelength, 642nm) for a pre-specified time interval that is followed by irradiation of the dye with pulse laser ( 6nsec) for inducing PA waves. The experimental results demonstrate that pre-illumination of the organic dye improves significantly the strength of pulse-laser induced photo-acoustic signal strength. It is a promising technique for end application in biomedical and clinical applications (more specifically, for enhancement of PA signal strength).

Microbubbles stabilized by surfactant shells have been established as ultrasound contrast agents for the past several decades. The microbubbles often get destroyed as these are delivered to the region of interest using catheters with different needle sizes. Optimizing the concentration of the surfactants on its shell is crucial for minimizing microbubble destruction. In terms of shell material for this work; polyoxyethylene glycol 40 (PEG-40) stearate which is a non-ionic surfactant, polypropylene glycol and glycerol were used to stabilize the microbubbles with a nitrogen gas core. Presence of surfactants greatly influence the size and stability of the microbubbles and thus four different surfactant concentrations (2, 5, 10 and 15%) of PEG-40 and two different polypropylene glycol + glycerol (GPW) mixtures (10% and 15%) were examined. Nitrogen microbubbles were synthesized through high-shear rotor homogenizer and pushed through three different needle sizes (23, 27 and 30 gauge) using a syringe pump to examine their sensitivity to needle injection. A sample volume of 100 μl containing microbubbles were collected at a constant flow rate of 43.63 ul/min which is the maximum flow rate of the syringe pump used in our experiments. The microbubbles collected at the outlet of the needles were sandwiched between two glass slides for their stability characterization using optical microscopy. The results demonstrated that solution containing 10% PEG-40, 10% polypropylene glycol and 10% glycerol had the highest concentration of microbubbles post injection for all three needle sizes. Finally, phantom experiments were conducted to calculate the signal-to-noise (SNR) ratios of the microbubbles with the different surfactant concentrations using a clinical ultrasound system.

Formation of blood clots or thrombus in healthy blood vessels can lead to serious or even life-threatening complications. Sonothrombolysis is a promising tool for lysing the blood clots non-invasively using focused acoustic waves. Ultrasound (US) imaging is commonly used to detect the blood clots presents in veins. In this work, we explore the use of a combined ultrasound and photoacoustic (PA) imaging clinical system during sonothrombolysis. PA imaging is a hybrid and emerging imaging modality which has garnered the attention of the biomedical imaging community in recent years. While US imaging has been used to visualize the blood clot, PA imaging enables the study of composition of the blood clot due to its optical absorption. Blood clots may be red, white or mixed due to the higher count of red blood cells (RBCs), white blood cells (WBCs) or it being a combination of RBCs and WBCs, respectively. Each clot type has a different photoacoustic signal. In our work, blood clots rich in RBCs are taken in transparent polyurethane tubes for sonothrombolysis. Meanwhile, the ultrasound and photoacoustic signal-to-noise ratios (SNR) are measured at fixed time intervals to evaluate the size and optical properties of the clot. Two cases were taken: blood clot + DI water and blood clot + blood and their US and PA SNR values were compared after 30 mins of sonothrombolysis treatment. The PA signal of the blood clot obtained after performing sonothrombolysis can be used to determine its final composition which may, in turn, help in the administration of clot-dissolving drugs.

We develop a data-driven regularization method for the severely ill-posed problem of photoacoustic image reconstruction from limited view data. Our approach is based on the regularizing networks that have been recently introduced and analyzed in [J. Schwab, S. Antholzer, and M. Haltmeier. Big in Japan: Regularizing networks for solving inverse problems (2018), arXiv:1812.00965] and consists of two steps. In the first step, an intermediate reconstruction is performed by applying truncated singular value decomposition (SVD). In order to prevent noise amplification, only coefficients corresponding to sufficiently large singular values are used, whereas the remaining coefficients are set zero. In a second step, a trained deep neural network is applied to recover the truncated SVD coefficients. Numerical results are presented demonstrating that the proposed data driven estimation of the truncated singular values significantly improves the pure truncated SVD reconstruction. We point out that proposed reconstruction framework can straightforwardly be applied to other inverse problems, where the SVD is either known analytically or can be computed numerically.

Filtered backprojection (FBP) is an efficient and popular class of tomographic image reconstruction methods. In photoacoustic tomography, these algorithms are based on theoretically exact analytic inversion formulas which results in accurate reconstructions. However, photoacoustic measurement data are often incomplete (limited detection view and sparse sampling), which results in artefacts in the images reconstructed with FBP. In addition to that, properties such as directivity of the acoustic detectors are not accounted for in standard FBP, which affects the reconstruction quality, too. To account for these issues, in this papers we propose to improve FBP algorithms based on machine learning techniques. In the proposed method, we include additional weight factors in the FBP, that are optimized on a set of incomplete data and the corresponding ground truth photoacoustic source. Numerical tests show that the learned FBP improves the reconstruction quality compared to the standard FBP.

We discuss several methods for image reconstruction in compressed sensing photoacoustic tomography (CS-PAT). In particular, we apply the deep learning method of [H. Li, J. Schwab, S. Antholzer, and M. Haltmeier. NETT: Solving Inverse Problems with Deep Neural Networks (2018), arXiv:1803.00092], which is based on a learned regularizer, for the first time to the CS-PAT problem. We propose a network architecture and training strategy for the NETT that we expect to be useful for other inverse problems as well. All algorithms are compared and evaluated on simulated data, and validated using experimental data for two different types of phantoms. The results one the hand indicate great potential of deep learning methods, and on the other hand show that significant future work is required to improve their performance on real-word data.

We consider image reconstruction in full-field photoacoustic tomography, where 2D projections of the full 3D acoustic pressure distribution at a given time T < 0 are collected. We discuss existing results on the stability and uniqueness of the resulting image reconstruction problem and review existing reconstruction algorithms. Open challenges are also mentioned. Additionally, we introduce novel one-step reconstruction methods allowing for a variable speed of sound. We apply preconditioned iterative and variational regularization methods to the onestep formulation. Numerical results using the one-step formulation are presented, together with a comparison with the previous two-step approach for full-field photoacoustic tomography.

Skin aging is characterized by color and wrinkle caused by degeneration of collagen and elastin in the dermis. Recently, the volume, diameter and branching of the micro vessels in the skin are proved to affect these biomechanical changes. Thus, high resolution imaging for both micro structure and micro vessels of the skin is desired. In the present study, dual-wavelength photoacoustic microscope (PAM) combined with high frequency ultrasound (HFUS) is developed to visualize both the morphology and microcirculation of the skin. Two Nd:YAG laser light sources with the wavelength of 532/556 nm, pulse width of 1.2/3.6 ns, pulse energy of 16 μJ/pulse and repetition rate of 1 kHz were equipped in the HFUS-PAM system. The optical fiber for laser delivery was inserted through the center hole of the concave ultrasound transducer with the central frequency of 75 MHz. Both HFUS and PA signals were acquired at the sampling rate of 500 MHz and the resolution of 12 bits. The transducer was scanned by voice coil actuators to obtain 3D dataset of HFUS and PA signals. Oxygen saturation of the micro circulation was calculated by the PA signals alternately obtained at 532 nm and 556 nm. 3D image of the layered structure and the micro vessels representing oxygen saturation in the 6 mm x 6 mm x 3 mm volume of the skin was successfully obtained with the system. HFUS-PAM will provide important information of skin morphology and microcirculation for assessment of skin aging.

Ultrasound (US) imaging is widely used for guiding minimally invasive procedures. However, with this modality, there can be poor visibility of interventional medical devices such as catheters and needles due to back-reflections outside the imaging aperture and low echogenicity. Photoacoustic (PA) imaging has shown promise with visualising bare metallic needles. In this study, we demonstrate the feasibility of a light emitting diode (LED)-based PA and US dual-modality imaging system for imaging metallic needles and polymeric medical catheters in biological tissue. Four medical devices were imaged with the system: two 20-gauge spinal needles with and without a multi-walled carbon nanotube / polydimethylsiloxane (MWCNT/PDMS) composite coating, and two 18-gauge epidural catheters with and without the MWCNT/PDMS composite coating. These devices were sequentially inserted into layers of chicken breast tissue within the US imaging plane. Interleaved PA and US imaging was performed during insertions of the needle and catheter. With US imaging, the uncoated needle had very poor visibility at an insertion angle of 45°. With PA imaging, the uncoated needle was not visible, but its coated counterpart was clearly visualised up to depths of 35 mm. Likewise, both catheters were not visible with US imaging. The uncoated catheter was not visible on PA images, but its coated counterpart was clearly visualised up to depths of 35 mm. We conclude that the highly absorbing CNT/PDMS composite coating conferred excellent visibility for medical devices with the LED-based PA imaging system and that it is promising for translation in minimally invasive procedures.

Surgery for rectal cancer is associated with significant side effects including wound infections, incontinence, sexual and bladder dysfunction, and long-term ostomies. Though studies have shown that patients who completely respond to preoperative treatment can safely avoid surgery, nonoperative options remain limited by the poor performance of MRI and endorectal ultrasound after initial therapy. Therefore, new imaging modalities are needed to improve posttreatment tumor assessment and enable the widespread adoption of nonoperative management in rectal cancer. An acoustic resolution photoacoustic microscope (AR-PAM) was constructed with high frequency ultrasonic transducer and near infrared laser. We performed initial phantom, and then imaged ex vivo human colorectal specimens to evaluate different AR-PAM characteristics in each tissue type (normal, untreated tumor, and treated tumor). Our data suggest that photoacoustic imaging can differentiate the distorted vasculature of rectal tumors from normal vascular patterns. However, the vascular distribution of rectal tissue in pathological complete responders showed similar distribution as the normal colorectal tissue; mucosa, submucosa and muscle layer are clearly presented in ultrasound images, while photoacoustic images have revealed that most vasculatures distribute in submucosa. Encouraged by these initial results, we are developed a high-speed scanning (1 second for 20mm B-scan) AR-PAM with laser pulse repetition rate of 1kHz for large field 3D imaging. Lateral resolution of 65μm, axial resolution of 45μm, and 8mm tissue imaging depth can be achieved.

To prevent complications from diabetes mellitus, patients need to control their blood glucose levels by referencing the level measured with invasive needle pricks, which is stressful. As a principle for a non-invasive blood glucose monitoring method, we have proposed resonant photoacoustic spectroscopy (PAS), wherein two lights with different wavelengths are amplitude-modulated to linearize the PAS signal against glucose concentration. We have investigated the characteristics of resonant PAS using glucose aqueous solutions and have performed an in-vivo study of resonant PAS with healthy volunteers. For the in-vivo study, the resonant PAS interface was attached to the earlobe. Blood glucose levels were monitored by a commercially available sensor as a reference. To induce an increase in blood glucose levels, oral loading of glucose was applied to (healthy) volunteers based on the 2-h 75-g oral glucose tolerance test protocol. There was a correlation between the signal of resonant PAS and blood glucose levels in terms of the error grid, correlation coefficients between reference blood glucose levels, and mean absolute relative difference. The results show the potential of resonant PAS for non-invasive blood glucose monitoring.

A Microelectromechanical Systems (MEMS)-based rapid scanning photoacoustic microscopy (PAM) is available to help life science research in neuroscience, cell biology, and in vivo imaging. MicroPhotoAcoustics (MPA; Ronkonkoma, NY), the only manufacturer and vendor of Optical Resolution (OR)-PAM systems, has developed a commercial PAM system with switchable optical and acoustic resolution (OR- and AR-PAM). To achieve real-time imaging capability without sacrificing high signal-to-noise ratios (SNRs), a 2-axis water-proofing MEMS scanner made of flexible polydimethylsiloxane (PDMS) was demonstrated by collaboration with Pohang University of Science and Technology (South Korea) that promises to dramatically increase the system’s imaging speed. This flexible scanner results in a wide scanning range and a fast imaging speed (5 B-scan images per second). Equipped with different excitation sources, in vivo PA images of microvasculatures in a mouse ear was obtained. The lateral and axial resolutions of the OR-PAM system are 4.38 μm. It is expected that this MEMS-based fast OR-PAM system can be significantly useful in both preclinical and clinical applications. With the continuation of new technological advancements and discoveries, MPA plans to further advance PAM to achieve faster imaging speed, higher spatial resolution at deeper tissue layer, and address a broader range of biomedical applications.

In this paper, a novel photoacoustic (PA) sensing probe design consisting of single optical fiber is reported. The same optical fiber is used for light delivery, which also serves as an acoustic delay line to relay the PA signal. As the key feature of the design, the ultrasound transducer is made optically-transparent to allow excitation light to pass through. This probe design provides three major benefits, including miniaturization, co-registered optical excitation and acoustic detection, and clear separation of PA signal from interference signals. Testing results show that PA probe provides good sensitivity and high linearity.

In this study, a low-cost procedure using evaporated milk is followed to make a gelatin-based phantom with ultrasound and optical properties close to soft tissues. To find out the effect of concentrations of gelatin and evaporated milk on the ultrasound properties, we first made two sets of phantoms. The first set was made by mixing different amounts of gelatin with deionized water (no evaporated milk in this set), while in the second set, evaporated milk concentration was changed (constant gelatin concentration). We measured the ultrasound attenuation of these phantoms at low and high frequency ranges and show that when the gelatin concentration is kept at 5 %, the ultrasound attenuation can vary from 0.4 to 0.6 dB/MHz/cm as the evaporated milk concentration increases from 20 % to 50 %. After getting some idea about the proper concentrations of evaporated milk and gelatin on ultrasound properties, n-propanol alcohol, glass microspheres, and Germall plus preservative were added to our recipe. We then measured the optical properties of the resulted phantom. A diffuse optical tomography system (DOT) was employed for this purpose to measure the optical absorption and reduced scattering coefficients of our phantom at four different wavelengths.

Photoacoustic imaging (PAI) is an emerging medical imaging modality which provides the resolution of ultra- sound imaging and contrast of optical imaging. Usually, in conventional PAI systems, high energy Nd:YAG lasers are used for illumination, but they are bulky and expensive. To address these problems, pulse laser diodes and light emitting diodes (LEDs) have been used in PAI imaging systems. LEDs have a lower energy, compared to Nd:YAG lasers. As a result, a lower signal-to-noise (SNR) is achieved. To address this problem, photoacoustic signal averaging is used necessitating a high number of LEDs illuminations. This method is not suitable for applications which the imaging sample is not motionless. In this paper, we propose to use an advanced image reconstruction method, known as double-stage delay multiply and sum (DS-DMAS), to address the low SNR of the data collected from an LED-based scanner. DS-DMAS addresses the low SNR inherent to the LED-based excitation. The results show that this algorithm compensates the low SNR of LED-based systems and provides a lateral resolution of about 60 %, 25 %, higher contrast ratio of about 97 %, 34 %, and better full-width-half- maximum of about 60 %, 25 %, in comparison with the delay-and-sum and delay-multiply-and-sum, respectively. Additionally, only 2 % of all the frames are used in DS-DMAS, which indicates that DS-DMAS uses a smaller number of frames to provide a high image quality.

It is of vital importance to understanding the relation between image quality and excitation light pulse characteristics in LED-based photoacoustic imaging system. We have tried to change pulse waveform of LED light source to detect several kinds of photoacoustic signals related to the image quality. As a result, in order to understand the photoacoustic signal, it is necessary to consider the characteristics of the optical pulse waveform and the ultrasonic probe well.

Colorectal cancer is the second most common malignancy diagnosed globally. Critical need exists for imaging and diagnosis of rectal tumors for both staging and therapeutic response evaluations. We have conducted a pilot study to image and characterize colorectal masses using a real-time co-registered photoacoustic (PAT) and ultrasound (US) system. A total of 8 tissue samples including pre- and post-treatment colorectal cancer, polyps have studied. Four different wavelengths (730, 780, 800, 830 nm) were used to illuminate the sample and a scanning stage was used to scan a large area and obtain a sequence of B-scans. For the pre-treatment colorectal cancer, photoacoustic images have shown significantly higher vascular level than neighbor benign regions of the same sample. The pre-treatment colorectal cancer PAT signal level is also higher than polyps and post-treatment colorectal cancer. Additionally, the quantitative features extracted from PAT and US power spectrum such as spectral slope, mid-band fit and zero MHz intercept have shown statistical significance between pre-treatment colorectal cancer and other 3 categories using t-test. Our initial results have demonstrated that PAT/US has a great potential to reveal tumor angiogenesis development or residual tumors after treatment.

We demonstrated the ultrasound modulated droplet lasers, in which the laser intensity from whispering gallery mode (WGM) of oil droplets can be reversibly enhanced up to 20-fold when the ultrasound pressure is beyond a certain threshold. The lasing enhancement was investigated with various ultrasound frequencies and pressures. Furthermore, the ultrasound modulation of the laser output was achieved by controlling the ultrasound pressure, the duty cycle, and the frequency of ultrasound bursts. Its potential application was explored via the study on a human whole blood vessel phantom. A theoretical analysis was also conducted, showing that the laser emission enhancement results from the directional emission from a deformed cavity under ultrasound pressure. Our studies reveal the unique capabilities of ultrasound modulated droplet lasers, which could lead to the development of laser emission-based microscopy for deep tissue imaging with high spatial resolution and detection sensitivity that may overcome the long-standing drawback of traditional fluorescence imaging.

A cell’s nucleus-to-cytoplasm ratio (N:C) can be used as a metric in histology for staging malignancy. Current techniques used for N:C assessment are time-consuming, low-throughput, and lack 3-D structure assessment. In this study, we assess the N:C ratio of MCF-7 cells using an ultra-high frequency acoustic/photoacoustic (PA) microscope and compare measurements to an imaging flow cytometer (IFC). MCF-7 cells were stained with the DRAQ-5 nuclear dye to facilitate production of fluorescence and PA signals. A PA microscope (PAM) consisting of a 375 MHz transducer and a pulsed 532 nm laser focused through a 10X optical objective was used for cell measurements. Cell and nucleus diameters of 37 stationary MCF-7 cells were obtained by fitting the power spectrum of backscattered ultrasound pulses and emitted PA waves, respectively, to well-established theoretical models. An Amnis ImageStreamX® IFC acquired brightfield/fluorescence images of cells and their nuclei, respectively. Masking and gating techniques were used to obtain the cell and nucleus diameters for 2004 MCF-7 cells from the IFC. For both systems, the N:C ratio was calculated as the ratio ofthe nucleus diameter to total cell diameter. The average cell and nucleus diameters from PAM were 15.2 ± 3.5 μm and 10.2 ± 3.5 μm, respectively, and were in good agreement with the average cell and nucleus diameters of 18.7 ± 2.6 μm and 12.6 ± 1.9 μm measured by the IFC. The N:C ratios were in excellent agreement, calculated to be 0.68 ± 0.19 and 0.68 ± 0.09 for PAM and IFC, respectively. This study demonstrates the ability of PAM to measure the N:C ratio of cells which in excellent agreement with IFC assessments.

The use of camera based optical ultrasound detection that records snapshot projections of the acoustic field can provide three-dimensional (3D) photoacoustic images with uniform spatial resolution. However, this is only the case if two conditions are satisfied. First, the detection angle that is covered by the acoustic field within the field of view (FOV) of the camera should be as large as possible, ideally approaching 180° in order to avoid limited view artifacts. Secondly, the extension of the region of interest (ROI) along projection direction needs to be smaller than the depth of field of the optical system for ultrasound detection. From the recorded wave pattern snapshots, photoacoustic projection images are reconstructed using a back propagation Fourier domain reconstruction algorithm. Applying the inverse Radon transform to a set of 200 projections recorded over a half rotation of the sample provides 3D photoacoustic tomography images. In this work we investigate an acoustic cavity approach to increase the detection angle, thereby reducing limited view effects due to missing information in lateral direction. The performance of the method to improve the final 3D photoacoustic image quality is investigated on a numerical phantom sample and first experimental 2D imaging results are shown.

A device for acquiring co-registered reflectance confocal microscopy (RCM) and optical-resolution photoacoustic microscopy (OR-PAM) images is presented. It uses a pulsed laser for photoacoustic excitation and a continuous laser for the confocal mode, co-aligned, focused and scanned over planes within the object. The photoacoustic part uses a probe beam deflection method, where a continuous laser beam is bent in the refractive index gradient generated by the pressure wave. First images on a carbon microfiber phantom demonstrate the capability of generating co-registered images, where the RCM mode displays back-scattering contrast, while the OR-PAM mode creates images with optical absorption contrast. In the temporal signals of both methods, typical signatures of transient cavitation were visible in the carbon fiber experiment. A lateral resolution of the setup of about 1.6 μm and a noise equivalent pressure amplitude of 90 Pa for the probe beam deflection method were measured.

Photoacoustic technique has undergone major developments in recent decades. Most of current photoacoustic systems work in the linear range, in which the photoacoustic amplitude increases proportionally to the increasing of illuminating light power. The imaging sensitivity and contrast, however, in this case are limited due to stronger background signals from intrinsic chromophores in biological tissue. The current work investigates the advantages of nonlinear photoacoustic generation compared to linear signal, by using a single-wavelength pump-probe and nanosecond-scale two-pulse excitation scheme. The results show that nonlinearity induced by this scheme yields higher detection sensitivity and contrast.

Optoacoustic tomography (OAT) is a promising modality for breast imaging that provides high resolution, detection sensitivity and diagnostic specificity for vascularized breast tumors. In OAT systems employing an arc- shaped illuminator, irregular overlaps of light beams can yield a non-uniform illumination throughout the entire volume of the breast. The imbalance in optical fluence leads to intensity loss in the reconstructed OAT images. Additionally, because optical fluence decreases with depth from breast skin surface, i.e., optical attenuation, deep breast tissues are diminished in the reconstructed images. For qualitative enhancement in 3D OAT imaging, we propose an image processing method to estimate, and compensate for, both the non-uniform incident optical fluence and the optical attenuation. We approximate the non-uniform illumination via maximum intensity extraction for polar angles in a spherical coordinate system. The location of the breast surface is estimated by detecting blood vessels nearest to the breast skin layer that appear with relatively high intensities in the reconstructed image. The breast depth is computed as the minimum distance between each voxel and the detected breast surface. The depth-dependent optical attenuation in the breast is estimated using the Beer– Lambert law down to the maximum penetration depth determined from an analysis of noise and artifacts in the reconstructed image. At each polar angle, the reciprocals of the estimated attenuation is used to compensate for the loss in intensity. The results are that previously invisible structures near the chest wall are revealed, and visible penetration depth was increased by 67% over the conventional, non-compensated volumes.

We report on the development of a preclinical 3D imaging platform integrating photoacoustic tomography and fluorescence (PAFT). The proposed multimodal imaging concept addresses known deficiencies in sensitivity, anatomical registration, and spatial resolution of the individual imaging modalities. Multi-view photoacoustic and optical projections of the studied animal are utilized to reconstruct large (27 cm3) volumes showing vascular network and blood-rich tissues, as well as regions with induced optical/fluorescence contrast with 3D resolution exceeding 150 μm. An additional 532-nm low-energy pulsed laser excitation is implemented as a separate imaging channel for registration over skin topography and superficial vasculature. PAFT technology enables functional and molecular volumetric imaging using wide range of fluorescent and luminescent biomarkers, nanoparticles, and other photosensitive constructs mapped with high fidelity over robust anatomical structures of the studied animal model. We demonstrated the PAFT performance using phantoms and by in vivo imaging of preclinical murine models.

Large number of simultaneously acquired spatially distinct pressure signals is required to improve quality of real-time photoacoustic and x-ray acoustic biomedical images [1]. In the past this approach was limited by availability of commercial multi-channel analog-to-digital converter (ADC) systems and ability to operate multiple ADC boards with synchronized clock and trigger source. The new Legion series single-board 256-channel ADC (ADC256) was designed by PhotoSound for massive parallel data acquisition utilized in photoacoustic, laser-induced ultrasound, and X-ray acoustic real-time imaging applications. ADC256 is a 12-bit ADC with a sampling rate up to 40 MHz and a USB3 computer interface. It can run at 200 Hz frame rate with 4096 points per trigger acquired by each channel. Higher trigger rates without data loss are possible with smaller number of points per trigger. ADC256 has an integrated amplifier with programmable gain up to 51 dB. Additionally, it can be equipped with a matching photoacoustic preamplifier. The system architecture is scalable to 1024 channels using four synchronized boards with a single trigger source. The clock and the trigger can be delivered from the master ADC256 board (daisy chain) or from the clock and trigger server (star topology). The data collected by each ADC board has trigger and board stamps allowing to (a) use multiple computers for data acquisition, and (b) detection of lost data events, even if the trigger rate exceeds its maximum allowed value.

Photoacoustic macroscopy uses a focused detector scanned across the tissue surface to obtain two- or three-dimensional images. Single element transducers equipped with a spherical acoustic lens suffer from the trade-off between lateral resolution and depth of field (DOF). In order to achieve a large imaging depth with constant lateral resolution over a large depth range, we investigate combinations of concentric ring arrays and acoustic lenses. The ring arrays allow dynamic focusing to a large depth range along the ring axis, and the lenses increase the transducer sensitivity. A photoacoustic sensor array is demonstrated, which consists of piezoelectric ring elements, concentrically arranged relative to a conical acoustic lens. Polymethyl-methacrylate (PMMA) is used as the lens material. A planar polyvinylidene fluoride (PVDF) film with conducting layers on both sides and 110 μm thickness was attached to the PMMA lens. The conducting layer was electrically etched to create several ring electrodes with equal detection area. Laser pulses from a near infrared optical parametric oscillator illuminated the object through the center of the ring array. The properties of the sensor array and the image formation by dynamic focusing are simulated and compared to experimental results. As demonstrated in B-scans of several phantoms, it is possible to achieve a lateral resolution in the range of 300 μm over a depth range of about two centimeter.

Photoacoustic imaging (PAI) is a promising imaging technique in preclinical study, which combines both merits of optical and ultrasound imaging. However, PA image quality is seriously suffered from various noises such as random white noise, intrinsic noise of devices and background noise from imaging environment, especially in in-vivo experiments. Regular linear filters like mean filters no longer provide a satisfactory performance. A boosted filter is necessary to degrade the noise level and enhance PA image contrast. In this paper, we applied a nonlinear de-noising filter based on mathematical morphology, which can help smooth the target boundary and well suppress impulse noise in PA images. Phantom and in-vivo experiments in this paper will show the feasibility and performance as a newly-used filter in PA image processing.

By reconstructing the optical properties such as the absorption coefficient, quantitative photoacoustic tomography (QPAT) images the micro blood vessels and the hemoglobin concentration quantitatively. QPAT is accomplished by solving the inverse problem of the photoacoustic (PA) measurement based on the phenomena of the light and PA pressure wave propagation. The light propagation is described by the radiative transfer equation, which is approximately calculated with the Monte Carlo (MC) simulation. The propagation of the PA pressure wave is described by the PA wave equation. The authors have been studying the QPAT image reconstruction algorithm using MC simulation and linearization. Near-infrared light with a wavelength of 755 nm that penetrates deep inside the biological medium was used. The absorption coefficient was reconstructed from the PA signals measured by the probe, which was the combination of the optical fiber and the focused ultrasound transducer consisting of P(VDF-TrFE) piezo electric film. The QPAT image with 10-mm depth was reconstructed in the numerical and phantom experiments. Pursuing more realistic situation of the micro vessel imaging, we conducted animal experiment to validate the QPAT image reconstruction. In the animal experiments, we tried to image the blood vessels of rabbit’s ear. The rabbit ear was placed under the tissue-mimicking scattering layer. Through the numerical, phantom, and animal experiments, the instrumental and computational conditions for QPAT for pathological imaging will be investigated by comparing the QPAT images of the phantom and animal.

Hyperosteogeny and Osteoporosis are two common bone diseases that have a high incidence in the middle-aged and elderly groups. Mild symptoms may only affect the daily life of patients, while severe ones are life-threatening. At present, detection methods based on X-ray film and ultrasound are generally applied. However, the former exist errors introduced by manual reading and a certain radiation hazard, the diagnostic results of the latter are not that satisfying as well. Photoacoustic effect combines the advantages of optics for sensitive light absorption contrast and acoustics for lower acoustic scattering in soft tissue. As a non-ionizing and non-invasive technique, its application in biomedicine is also emerging. In this paper, a classification model built on Convolutional Neural Network (CNN) was proposed to achieve automated diagnosis of hyperosteogeny, osteoporosis and normal bone. Time-domain photoacoustic signals generated by different bone types are set as the inputs of the CNN while the output results indicate the corresponding categories of the samples. The analysis results of ex vivo data demonstrated that the established model could accurately accomplish the research of classification. Thus, the proposed method has certain auxiliary value for improving the efficiency, accuracy and objectivity of clinical diagnosis of the three bone types.

Ovarian cancer has the highest mortality rate among all other cancers related to reproductive system. We have developed co-registered ultrasound (US) and photoacoustic tomography (PAT) technique (US/PAT) for non-invasive diagnosis of ultrasound identified ovarian tissue. Ultrasound images provide structural information of human ovary and PAT provides information of blood vasculature and oxygen saturation inside the ovary. However, due to the lower contrast of transvaginal US images, it is not always easy to delineate the ovaries from the surrounding tissue, especially postmenopausal ovaries. Doppler US can identify large vessels around or inside the ovary if exist and also large celiac vessels which are often in the neighborhood of ovaries. If the region of interest (ROI) includes the surrounding celiac vessels, the tumor total hemoglobin and blood oxygen saturation cannot be quantified correctly. To optimize the selection of ROI for co-registered PAT imaging, we have developed co-registered display of Doppler US, ultrasound and photoacoustic for near real-time ovarian tissue diagnosis. Doppler images provide blood flow information of large vessels and assist co-registered gray-scale US to identify ROI. PAT maps out the total hemoglobin and oxygen saturation distributions. Data for Doppler imaging is collected using our customized commercial US machine which can simultaneously save US/PAT RF data. US RF data is used to reconstruct ultrasound and Doppler images. Conventional cross correlation technique assisted by advance image processing techniques is used to compute Doppler images. We report examples of how these co-displayed images help improve PAT mapping of tumor angiogenesis and oxygen saturation using phantom data.

We developed a Photoacoustic (PA) imaging system which could be realized the fusion of PA and conventional Ultrasound (US) image. The prototype PA system for clinical study had comprised a 755 nm laser light source, and a 9 MHz US linear array probe. We successfully observed median and sural nerve bundles with US image, and PA images of blood vessels around and in that nerve bundles in the clinical study for diabetic patients. We applied this experimental design and concept to evaluate diabetic neuropathy, most common microvascular complication, to patients with type 2 diabetes. As results of the preliminary clinical trials, we found two technological challenges to overcome. The first technological challenge is to improve nerve bundle US image quality by higher frequency detection. The second challenge is to change the wavelength of laser light in order to clarify the relation between PA signal intensity and clinical staging of diabetic neuropathy. Owing to these technical challenges, the system was extended to a combination of 1064 nm and 12 MHz US linear array probe. We verified the performance of the extended prototype PA system compared with the previous system using a specially designed rabbit hypoxia model. Histologic examination was confirmed by a fusion image of PA image from sciatic nerve vessel and US morphologic image. We also performed preliminary clinical study to patients with type 2 diabetes and patient with amputation leg. The extended photoacoustic / ultrasound superposed imaging system succeed in the visualization of the nerve bundle and its internal and external blood vessels clearly. PA imaging was shown to be useful for prediction of risk of foot amputation and early detection of diabetic neuropathy.

Double-stage delay-multiply-and-sum (DS-DMAS) is one of the algorithms proposed for photoacoustic image reconstruction where a linear-array transducer is used to detect signals. This algorithm provides a higher contrast image in comparison with the conventional delay-multiply-and-sum (DMAS) and delay-and-sum (DAS), but it imposes a high computational complexity. In this paper, open accelerators (OpenACC) GPU computation parallel approach is used to lessen the computational time and address the high computational time of the DSDMAS for photoacoustic image reconstruction process. Compared with sequential execution of the DS-DMAS on CPU, a speed-up of approximately 74× is achieved (for an image having 1024 × 1024 pixels). The proposed approach provides possibility to have an accurate reconstructed photoacoustic image with a reasonable frame rate. In addition, the higher the number of the image pixels, the higher speed-up is achieved. Using the suggested GPU implementation, it is feasible to reconstruct photoacoustic images having a size of 128 × 128, and 256 × 256 with a frame rate of 3 and 2, respectively.

In photoacoustic (PA) imaging using commercial ultrasound transducers, two dimensional PA images are pro- duced by commonly used delay-and-sum (DAS) as the reconstruction method. However, the reconstructed image is affected by noise and artifacts. Here, we investigate the performance of a nonlinear (NL) beamforming method for linear-array PA image formation. The proposed algorithm uses the pth root of the recorded signals and imposes a computational complexity in the order of DAS (O(M )). We have evaluated the performance of the proposed method numerically, having a signal-to-noise ratio of 30 dB and -10 dB. It is shown that at the depth of 15 mm, the NL 3 (NL beamformer with a p=3) outperforms DAS, DMAS and NL 2 for about 26 dB, 11 dB and 11 dB, in terms of level of sidelobe, respectively. In addition, DAS, DMAS, NL 2 and NL 3 lead to a full-width-half-maximum of about 1.4 mm, 1.04 mm, 1.04 mm and 0.86 mm, respectively. The proposed method can be a great choice for sentinel lymph node imaging.

Interventional cardiac procedures often require ionizing radiation to guide cardiac catheters to the heart. To reduce the associated risks of ionizing radiation, our group is exploring photoacoustic imaging in conjunc- tion with robotic visual servoing, which requires segmentation of catheter tips. However, typical segmentation algorithms are susceptible to reflection artifacts. To address this challenge, signal sources can be identified in the presence of reflection artifacts using a deep neural network, as we previously demonstrated with a linear array ultrasound transducer. This paper extends our previous work to detect photoacoustic sources received by a phased array transducer, which is more common in cardiac applications. We trained a convolutional neural network (CNN) with simulated photoacoustic channel data to identify point sources. The network was tested with an independent simulated validation data set not included during training as well as in vivo data acquired during a pig catheterization procedure. When tested on the independent simulated validation data set, the CNN correctly classified 84.2% of sources with a misclassification rate of 0.01%, and the mean absolute location error of correctly classified sources was 0.095 mm and 0.462 mm in the axial and lateral dimensions, respectively. When applied to in vivo data, the network correctly classified 91.4% of sources with a 7.86% misclassification rate. These results indicate that a CNN is capable of identifying photoacoustic sources recorded by phased array transducers, which is promising for cardiac applications.

We had developed a real-time clinical photoacoustic (PA) and ultrasound (US) imaging system by combining a programmable ultrasound machine and a wavelength-tunable laser. The system was able to acquire real-time images of biological tissue, but the user had to restart the image acquisition software to modify parameters for optimizing the images. We have recently updated the system to adjust imaging parameters in real-time by implementing a real-time parameter control software, which is compatible with the programmable platform of the US machine. To adjust the parameters of both PA and US images, we also implemented custom functional blocks including beamforming, frequency demodulation, log compression, decimation, scan conversion, and image display. To acquire real-time images, we performed all the calculations by using parallel processing with a graphic processing unit in US machine. The updated system has the great potential to be widely applied to a variety of clinical and preclinical applications because it allows real-time optimization of imaging parameters as well as visualizing the images in real-time.

Adenocarcinoma is a type of cancers that forms in mucus-secreting glands throughout the body. For example, adenocarcinomas make up around 96% of colorectal cancers and some cervical cancers (10%). Depth of invasion for early adenocarcinoma is generally around 1-7 mm. In this report, we propose an optimal design, implementation, and evaluation of an optical fiber-based transvaginal photoacoustic/ultrasound prototype imaging probe for endo-cavity imaging of adenocarcinoma with ball-shaped fiber tips (refractive index = 1.517, radius 0.75 mm), to improve the light illumination homogeneity and increase the light delivery fluence on the central imaging area of the tissue surface. The light delivery system consists of light coupling optics, a custom-made transducer sheath, four 1-mm-core multimode optical fibers with the ball-shaped fiber tip arranged around the transducer. The probe design was optimized by simulating the light fluence distribution of the fibers with and without ball-shaped tips using a 3D model in Zemax for different design parameters such as fiber effective numerical aperture, fiber displacement from the probe base, sheath taper angle, ball-shaped fiber tips’ diameter and refractive index, etc. The laser fluence profiles were experimentally recorded through calibrated intralipid solution by camera at various imaging depths. The output power on the central imaged areas were experimentally measured by calculating the PAT signal strengths from the black threads buried inside chicken breast tissue at various depths and in-vivo photoacoustic imaging of one palmar vein proximal to the human wrist.

Quantification fatty liver is useful for early detection of liver disease. Now ultrasonic detection is used for screening fatty liver. But it lacks of quantity and objectivity. Photoacoustic (PA) imaging, a novel modality, is expected for applying to estimate fat rate of liver. To realize estimation of fat rate, we developed handheld PA system using multi wavelengths. In this study, we verified the usefulness of the system for estimate fat rate in ex vivo experiments. As a biological sample, the mixtures of chicken liver, olive oil and lard (the mixing rate of the lipid in the liver is from 0 to 0.3with an interval 0.1) was used. We acquired PA spectra of the sample by developed handheld system. Nanosecond pulses of laser light (800–1300 nm wavelength, 30 Hz repetition rate) were guided to the sample surface by an optical fiber bundle close to the linear ultrasound probe. By analyzing PA spectra, we can find liver have peak PA intensity around 900 nm and lipid have it around 1210 nm where the light absorbance is high. To estimate fat rate using photoacoustic methods, the photoacoustic signal intensity ratio between two wavelength regions was calculated as described above. Signal intensity ratios agreed well with the composition ratio between liver and lipid. From these analyses, the advantage of PA spectra for estimation of fat rate, and the feasibility of our system for early detection of fatty liver were demonstrated. Devising a technique for quantifying fatty liver and in-vivo experiments will be conducted in further studies.

Gynecologic surgery requires the clamping, cauterization, and transection of arteries that lie within mil- limeters of the ureter, posing significant potential risk for ureteral injury. By leveraging the optical absorption properties of hemoglobin and methylene blue (an FDA-approved contrast agent), we propose intraoperative pho- toacoustic imaging during hysterectomies to simultaneously visualize the uterine arteries and ureter, respectively. Three experiments were performed to test the feasibility of a spectroscopic system aimed at intraoperative visual- ization. At 690 nm, the contrast from blood and urine mixed with 200 uM methylene blue was 13.83 dB and 11.06 dB, respectively, representing a 2.77 dB contrast difference. Conversely, at 750 nm, the contrast from blood was similar (14.61 dB), and the contrast from urine mixed with 200 uM methylene blue decreased to 1.74 dB, which produced a greater contrast difference of 12.87 dB. When tissue was added, similar contrast differences were observed at these wavelengths. Finally, a laparoscopic tool was additionally visualized in real time in proximity to the ureter and uterine arteries, which supports the feasibility of a spectroscopic photoacoustic approach to differentiating the ureter from the uterine arteries in relationship to a laparoscopic tool during hysterectomies.

The standard method to treat cutaneous melanoma, which is the most prevalent skin cancer, is surgical excision of visible boundary with an additional margin. The margin is typically decided by surgeons’ experience based on the color of the lesion. It is very important to determine appropriate excision area because the operation will continue until a histological evaluation is made the there is no cancerous cells in the excised margin. Here, we demonstrate the results from our initial pilot study for detection of melanoma boundary using photoacoustic (PA) imaging. We recruited a patient who had a cutaneous melanoma lesion on the left heel. We excised the lesion with surgical operation by adding ~1 cm margin to the visible boundary, and then immediately acquired multispectral PA images of the lesion. By scanning a linear array transducer using a motorized stage, we acquired volumetric PA images of the lesion. From the multispectral analysis of PA signals, we could tell cutaneous melanoma from surrounding normal tissue and marking pen. Although much more studies are required for clinical evaluation, the initial results demonstrate that the PA imaging can provide additional information to surgeons for better selection of excision area of cutaneous melanoma.

Spinal fusion surgeries require the insertion of screws into the pedicles of vertebrae in order to connect multiple vertebrae with a metal rod and stabilize the spine after an injury or deformity. One outstanding challenge to this surgical procedure is to ensure that a drill tip maintains the correct trajectory when drilling pilot holes for screw insertion. In this work, we demonstrate a photoacoustic imaging system for drill tip tracking that will co-register photoacoustic images with pre-operative CT images. Our approach was tested with custom drill bits containing an optical fiber inside a hollow core with single-hole and multi-hole tips that were inserted in an ex vivo human vertebra. A 32 mm-deep hole was drilled in the pedicle, with the first 13 mm corresponding to the pedicle and the remaining 19 mm extending into the vertebral body. Data was acquired using a 760 nm laser with energies of 1.0 mJ, 2.2 mJ, and 3.4 mJ at the fiber tip. For the single-hole drill tip, the signal was detectable at 0-6 mm depths into the pedicle (SNR: 53.7), which represents 46% of the 13 mm pedicle length. From 6 to 14 mm, the photoacoustic signal was either no longer visualized (SNR: 26.7) or shifted from its expected location in the image due to reflection artifacts. SNR was improved to 31.14 with coherence-based beamforming methods when compared to previously reported conventional delay-and-sum beamformming methods. This enhancement provided clear visualization of low energy photoacoustic signals. Results are generally promising for photoacoustic-guided drilling during pedicle screw insertion.

Photoacoustic imaging is a hybrid medical imaging technique that combines the contrast and spectral sensitivity of optical imaging with resolution and tissue penetration of ultrasound. Due to the difference between the optical absorption spectra of deoxygenated and oxygenated hemoglobin, multispectral photoacoustic imaging holds strong potential in noninvasive local blood oxygen saturation imaging. Oxygen saturation imaging is one of the most promising applications of photoacoustic imaging and has been widely explored in studies related to tumor hypoxia, cancer therapy etc. However, clinical translation of this technology has often been limited by bulky and expensive excitation sources. Recently, we introduced a multi-wavelength real-time LED-based photoacoustic/ultrasound imaging system. In this work, potential of this LED-based system in real-time oxygen saturation imaging is demonstrated using an in vivo measurement on a human volunteer. We used ultra-fast switching two-wavelength LED arrays (750nm/850nm) along with a linear-array 7 MHz US probe for the experiments. 2D PA, US, and oxygen saturation imaging were performed on the index finger of a human volunteer. Results demonstrate that LED-based PA imaging system used in this study is promising for generating 2D/3D oxygen saturation maps along with PA and US images in real-time. We believe that these results will have profound impact in non-invasive blood oxygen saturation imaging and subsequent clinical translation of PA-based oximetry.

Thermoacoustic imaging is a hybrid technique that can provide functional and molecular information of deep tissue at lower cost using non-harmful radiation compared to competing methods. It primarily maps electro- magnetic absorption contrast at both optical and radio frequencies (RF) with ultrasonic spatial resolution. In standard practice, different simulation tools are combined to simulate the hybrid thermoacoustic imaging process, which requires communication between several software packages. A general purpose solver is critical for thermoacoustic simulations, as a variety of phenomenon must be modeled in order to represent the physical reality. Here we present ONELAB as a single simulation platform for numerically simulating thermoacoustic imaging, where optical or RF propagation inside the tissue is solved in the forward excitation path, and ultra- sound propagation is solved during the backward detection path. Our validation experiments using simulation platforms mimicking both optical and RF properties of biological tissue demonstrated that ONELAB can accurately model thermoacoustic imaging. The advantages of ONELAB include a completely open source software platform that does not depend on any specific/standard software to operate. ONELAB provides a finite element meshing utility as well as a general purpose finite element solver.

Photoacoustic imaging modality is a new biomedical imaging which provides images with high resolution and contrast from different parts of body. In this paper, we have designed a new optical system by using a fiber bundle in order to imaging of a hemorrhage inside of the infant's head. We used Monte Carlo algorithm to simulate light propagation in the infant's head, an acoustic k-space method to simulate photoacoustic signal propagation in it, and time reversal image reconstruction algorithm to get 3D image of the hemorrhage. According to our simulation, this new optical system can provides homogeneous illumination on the infant's head Leads to more accurate images. Furthermore, we have designed and optimized an optical system in order to coupling light from laser source into a fiber bundle with more than 94% efficiency.

Photoacoustic (PA) signal experiences excessive background noise when generated using cost-effective, low-energy laser diodes. A denoising technique is essential in this case. Averaging is a common approach to increase the Signal-to-Noise Ratio (SNR) of PA signals. This technique requires numbers of data acquisition in hundreds and thousands and hence, demands more hardware and time consuming at the same time. Here, an adaptive method based on Adaptive Line Enhancers (ALE) algorithm to improve the SNR of PA signals has been presented. Our results validate the feasibility of the usage of an adaptive method and also indicate excellent improvement in terms of increasing the SNR of the PA signals. Additionally, this proposed algorithm requires way less number of acquisitions as compared to the conventional averaging techniques that leads to faster PA image processing.

A co-planar, simultaneous, photoacoustic tomography guided, diffused optical tomography (CS-PAT-DOT) methodology has been presented in this paper. We detect the absorption of sub-regions with different absorption characteristics in deep tissue with a high spatial resolution. To this aim, we initially utilize compressed sensing (CS), time reversal (TR) and back projection (BP) reconstruction algorithms to reconstruct a priori information inside a heterogeneous phantom. Then the reconstructed images are used in DOT image reconstruction through the total variation method. Improvements obtained from such hybrid methodology are measured by comparing DOT and CS-PAT-DOT images. It will also show that each of the reconstructions based on the proposed method has a unique capability to accurately detect heterogeneities in the tissue at different depths; significantly improving spatial resolution in DOT images. The focus of this study is directed towards quantifying the concentrations of endogenous chromophores, e.g., oxyhemoglobin, deoxyhemoglobin and cytochrome-c-oxidase etc., which are significant indices in detecting tissue abnormalities.

Photoacoustic tomography (PAT) is an emerging modality for imaging living biological tissue. Being label free, non-invasive, and having comparable resolution to ultrasound, PAT has many medical translations. This paper demonstrates our development of a low-cost 16 element transducer array for rapid imaging (1 frame per second) of biological samples. For the first time we demonstrate quality images obtained with a completely low-cost system. A rotatable platform houses our 16 equidistant Technisonic transducers, which is rotated 22.5° to acquire a full 360° field of view. We use Ekspla NL200 series Q-switched laser at 532 nm illumination wavelength with coupled optical fiber for overhead illumination. Our transducers send data to a National Instrument data acquisition system, triggered by the previously mentioned laser for efficient detection of photoacoustic signal. We have characterized this system through the imaging of complex optical absorbing lead phantoms. Thin lead has been imaged to demonstrate the spatial resolution of the system using the point spread function. Characterization of this system will allow us to move to ex-vivo imaging. We aim to develop this system as a platform for quality small animal functional brain imaging.

Although transcranial photoacoustic imaging has been previously investigated by several groups, there are many unknowns about the distorting effects of the skull due to the impedance mismatch between the skull and underlying layers. The current computational methods based on finite-element modeling are slow, especially in the cases where fine grids are defined for a large 3-D volume. We develop a very fast modeling/simulation framework based on deterministic ray-tracing. The framework considers a multilayer model of the medium, taking into account the frequency-dependent attenuation and dispersion effects that occur in wave reflection, refraction, and mode conversion at the skull surface. The speed of the proposed framework is evaluated. We validate the accuracy of the framework using numerical phantoms and compare its results to k-Wave simulation results. Analytical validation is also performed based on the longitudinal and shear wave transmission coefficients. We then simulated, using our method, the major skull-distorting effects including amplitude attenuation, time-domain signal broadening, and time shift, and confirmed the findings by comparing them to several ex vivo experimental results. It is expected that the proposed method speeds up modeling and quantification of skull tissue and allows the development of transcranial photoacoustic brain imaging.

A photoacoustic sensor for studying the thermal properties of a single biological cell was developed. The sensor used an aluminium foil for heating the sample which enables the study of unstained samples. The PA sensor was developed to work in the heat transmission mode configuration by positioning the detector at the opposite side with respect to heated side of the sample. Rosencwaig’s theoretical model for an open PA cell configuration functional in a heat transmission mode was applied to study the thermal diffusivity of biological cells. MCF7 cells were used in this studied. We measured the thermal diffusivity of 3 cells using the sensor. The average measured thermal diffusivity of MCF7 cells was 0.05 mm²/s. The PA sensor allows the spatial mapping of the cell thermal diffusivity and can be used to measure thermal properties of cells in various phases of the cell cycle. It can also be used to measure the effective properties of cells when they have internalized various sensors (e.g. plasmonic nanoparticles).

Deep vein thrombosis (DVT) is a disorder that occurs when a blood clot (thrombus) forms in one or more of the deep veins in your body, usually in your legs. Deep vein thrombosis can cause leg pain or swelling, but also can occur with no symptoms. If the clot moves to the vital organs like lungs, heart, brain etc., it can be very fatal and can cause death to the individual. Diagnosing it at early stages is very crucial to decide the treatment strategy. The most commonly used techniques that are used for the diagnosis includes ultrasound, x-ray, CT, etc. For definitive diagnosis contrast agents are required for better visualization of the blood clots and harmful radiations are used. For label free imaging of the blood clots, photoacoustic imaging can be used. To perform in-vivo photoacoustic imaging, high framerate imaging is needed as the velocity of the blood in the veins is between 3 cm/s to 14 cm/s. In this work, we have shown high framerate photoacoustic imaging at different framerates of 5, 10, 50, 100, 500, 1000, 2000 and 3000 fps using a pulsed laser diode of 7000 Hz frequency. We have demonstrated label free imaging of blood clots at 803 nm. Blood clot has at least 1.5 times higher SNR compared to blood and can be clearly visualized against blood as background. High framerate photoacoustic imaging can be used for label free diagnosis of deep vein thrombosis.

Multi-drug-resistant bacteria, particularly Methicillin resistant, have become an ever-increasing problem. Currently, broad spectrum antibiotics are prescribed until bacteria can be cultured, a process that can take 3-4 days and is unable to deliver quantitative information about relative number of bacteria present. In order to rapidly detect, differentiate and quantify bacteria in blood samples we designed a detection system using labeled bacteriophage in conjunction with photoacoustic flow cytometry. Photoacoustic Flow cytometry is the generation of ultrasound waves created by the absorption of laser light in objects under flow. Bacteria can be tagged with dyed phage and processed through the photoacoustic flow cytometer where they are detected by the acoustic response. Here we demonstrate that E.coli can be detected and discriminated from Salmonella using this method.

In this work, we theoretically describe photoacoustic signal generation of fluorophores, for which triplet relaxation can be neglected. In the theoretical model, the excited state lifetimes, the fluorescence quantum yield, and fast vibrational relaxation are considered. Based on our findings, we propose that for fluorophores the photoacoustic signal together with knowledge about the absorption and emission spectrum allows to directly determine the fluorescence quantum yield without comparative measurements.

The liver performs important functions in our body such as protein synthesis, and blood cell degradation has been considered a major organ of the human body. Currently, ultrasound imaging and magnetic resonance imaging are frequently used to diagnose liver diseases including cancer and cirrhosis. Ultrasound imaging is commonly used for liver disease because it provides safe, easy-to-use, real-time imaging at low cost. Ultrasound uses anatomical, tissue, and blood flow information to diagnose liver disease. By adding additional information using photoacoustic (PA) imaging, the diagnosis of the liver disease can be further enhanced. In this study, we developed hyaluronate silica nanoparticles (HASi) PA contrast agents that can be targeted to liver specifically. Since the hyaluronic acid receptors such as cluster determinant 44 are overexpressed in the liver, hyaluronic acid is considered a direct marker for fibrogenesis. Therefore, hyaluronic acid conjugates have been researched for liver-specific targeting. To verify liver-specific targeting efficiency and PA contrast agent property, in vivo PA monitoring has been conducted after i.v. injection of HA-Si and silica nanoparticles. ICP-MS analysis of major organs and urine optical absorbance spectra analysis has been performed to investigate biocompatibility of HA-Si. These results demonstrate the possibility of liver-specific drug delivery of the hyaluronic acid complex and the possibility of a PA method for liver lesion monitoring.

Biomedical optoacoustic (photoacoustic) imaging is generally performed with short laser pulses with durations in the order of a few nanoseconds. This enables maximizing the conversion efficiency of optical energy into acoustic (ultrasound) energy when light is absorbed in biological tissues. The generated ultrasound waves are generally very broadband, with typical frequency spectra ranging from tens of kHz to tens of MHz. Most ultrasound transducers used for the detection of optoacoustic signals have a finite detection bandwidth, in a way that they are not optimal for the acquisition of optoacoustic signals generated with a single pulse. In this work, we analyze a narrowband excitation approach based on a tone-burst consisting of multiple equally-delayed short pulses. We compare the power spectral density of the signals generated with a tone-burst with those generated with a single pulse having the same energy under safety exposure limits. We further analyze the performance of tone-burst excitation when non-linear effects take place. Specifically, we consider non-linearities associated to temperature increase and to optical absorption saturation.

Inadvertent cuts to blood vessels and nerves poses a significant risk during percutaneous needle procedures, often leading to serious injuries and even death. We propose a computer-assisted photoacoustic imaging-based device that is able to detect these vascular structures and robotically guide the surgeons in avoiding them. A fiber-coupled pulsed laser diode capable of generating photoacoustic signals is attached through a ferrule, where a 2.5 mm diameter ultrasound ring transducer receives the corresponding photoacoustic waves. The integrated device is secured on an XYZ axis linear translational stage configuration, and robotically navigated through vessel-modelling phantoms to reach a targeted region of interest. A steering feedback algorithm calculates the relative position of the device with respect to each vessel, generates a 2D map of the navigational plane, and controls the stages to steer the device accordingly towards the target while avoiding the vessels. We first ran the algorithm in a water phantom to demonstrate feasibility, and then in a milk solution to model real tissue scattering. Our proposed device successfully avoids the phantom blood vessels in both cases through photoacoustic detection, and the corresponding 2D navigational path and plane through the phantom is mapped and recorded. Our results demonstrate that a computer-assisted photoacoustic imaging-based device is a viable method of intraoperatively guiding percutaneous needle procedures. The ability of our proposed guidance device to detect and avoid damage to blood vessels and nerves can further be used to optimize biopsies and tumor removal procedures in various parts of the body.

Photoacoustic imaging is a powerful and increasingly popular technique for tissue diagnostics. Suitable tissue- equivalent phantoms are in high demand for validating photoacoustic imaging methods and for clinical training. In this work, we describe a method of directly 3D printing a photoacoustic tissue-equivalent phantom of a kidney based on Gel Wax, which is a mix of polymer and mineral oil. A kidney phantom that is compatible with photoacoustic scanning will enable clinicians to evaluate a portable LED-based photoacoustic and ultrasound imaging system as a means of locating tumors and other abnormalities. This represents a significant step towards clinical translation of the compact system. Training using realistic phantoms reduces the risks associated with clinical procedures. Complications during procedures can arise due to the specific structure of the kidney under investigation. Thus the ability to create a 3D printed phantom based on detailed anatomical images of a specific patient enables clinicians to train on a phantom with exactly the same structure as the kidney to be treated. Recently we developed a novel 3D printer based on gel wax. The device combines native gel wax with glass microspheres and titanium dioxide (TiO₂) particles to obtain a medium with tissue-like optical and acoustic properties. 3D models created using this printer can be given a range of values of optical absorption reduced scattering coefficients. The ability to 3D patient-specific phantoms at low cost has the potential to revolutionize the production and use of tissue-equivalent phantoms in future, and can be applied to a wide range of organs and imaging modalities.

A sub-wavelength convertible Bessel-beam (BB) and Gaussian-beam (GB) photoacoustic microscope (PAM) in reflection-mode has been developed. A miniature ultrasonic transducer was placed in front of the objective with a high numerical aperture and a working distance of a few millimeters to achieve the reflection-mode and sub-wavelength resolution. For BB-PAM system, a conical lens and an achromatic doublet lens were used to achieve extended depth of field (DoF). In particular, it was designed to easily convert the system from BB to GB by removing the two lenses described above, so that the DoF of BB- and GB-PAM can be compared accurately. The 532 nm pulsed laser used in this system was coupled to a single mode fiber. The sample was scanned using X-Y direction motors and the system was controlled using Labview software. The lateral resolution of the focus regions of BB- and GB-PAM obtained were 300 and 260 nm, respectively. As a result of measuring the DoF of BB-PAM, it was about 250 ~ 300 μm, which was about 7 ~ 8 times better than the DoF of GB-PAM. In-vivo vascular structure of a mouse ear was successfully visualized using BB- and GB-PAM to compare the DoF of the two systems. Thus, the system developed in this study confirmed that BBPAM enabled high-resolution imaging at extended DoF than GB-PAM, and further believed that this system could be useful for a variety of biomedical research.

The characteristics of the transducer, such as the transducer shape, have a significant impact on the image performance in optoacoustic (photoacoustic) imaging. Several reconstruction algorithms have considered the shape of the transducer in the optoacoustic reconstruction process, showing the improvement in image quality compared to reconstruction procedures with the point detector approximation. One flexible approach assumes the surface of transducer that consists of a set of surface elements. However, this approach suffers from long computation time and excessive memory consumption, especially for model-based reconstruction strategies. Herein, we present a modified model-based reconstruction algorithm using a virtual parallel-projection method, for the optoacoustic imaging system with flat detector. In this case, the sum of the surface elements' model matrixes can be replaced by a virtual parallel-projection model matrix, in order to reduce the reconstruction time and memory consumption. The proposed method has been performed on numerical simulations, phantom experiments of microspheres with the diameter of 200 μm and in vivo experiments in mice. The reconstruction results of proposed method show the similar image quality as the results of the traditional reconstruction method setting surface elements, while the computation time and memory requirements have been efficiently decreased.

Photothermal therapy (PTT) is conducted by converting laser radiation into thermal energy due to the absorption of the photons in tissue. PTT has attracted much attention as a selective and non-invasive treatment. However, the difficulty of treating deep-lying lesions, which is due to optical scattering in biological tissues, is a major limitation of PTT. To alleviate this problem, we previously proposed dual thermal therapy (DTT) in which ultrasound and laser energies are transmitted simultaneously into the target lesion to add local temperatures increased by both energies. In DTT, the focus of ultrasound is located in the target lesion. It was demonstrated that DTT is capable of increasing treatment depth, compared to PTT. In this paper, we propose a method of further increasing treatment depth in DTT. Unlike the conventional DTT, the proposed method finds and uses an optimal ultrasound focal point to maximize the treatment depth of DTT; the focus of ultrasound is placed in the posterior portion of the target lesion during DTT and the focal depth is determined based on the results of ultrasound field simulation. By doing so, it is possible to avoid high optical scattering of the coagulation produced in the anterior segment of the target lesion. The performance of the proposed method was evaluated using a tissue-mimicking phantom. The experimental results showed that the coagulation area produced by the proposed method had a maximum size of 4 mm in the depth direction, whereas the size was about 2 mm in the case of the conventional DTT.

Traditional optical devices rely on light propagation along a straight path. However, when the light propagates through a blurred medium, its direction get scattered by microscopic particles. This inhomogeneous distortion results in a diffused focus point. Light scattering is one of the main limitations for the optical imaging. This limitation decreases the resolution in depth. Therefore, the ability of focusing light at a desired position has a huge worthwhile for applications of optical imaging. Over the past few years, it was shown that light can be focused inside an object even with strong scattering particles, just by shaping the wavefront of the incident beam. The most successful approaches for light focusing at the presence of scattering objects are feedback-based optical wavefront shaping. In this paper, an iterative feedback-based wavefront shaping is proposed. It uses the genetic algorithm. In summary, we aim to obtain a high intensity in the focus point with fewer steps in iteration while increase the signal-to-noise. The simulations results show that both the above mentioned goals are achieved using the proposed method.

Knowing the elapsed time from subcutaneous hematoma generation can suggest appropriate treatment strategies. Today, ultrasound imaging, X-ray CT, and MRI are mainly used for diagnosis. However, they have difficulty in distinguishing hematoma from surrounding tissues because of their low image contrast and large device size. Furthermore, X-ray CT entails radio exposure; MRI is contraindicated for patients with embedded metal such as pacemakers. This study evaluated the feasibility of visualizing hematoma and evaluating its characterization using the handheld photoacoustic imaging system we developed. To evaluate the feasibility of estimating the bilirubin concentration, which increases as time passes from hematoma generation, simulated hematomas containing different bilirubin concentrations embedded into optical and acoustical transparent media were measured. Measurements of simulated hematoma using biological tissues were also conducted. The samples and linear ultrasound probe were set in degassed water. Pulses of laser light were guided to the sample surface by an optical fiber bundle close to the probe. Photoacoustic signals were obtained at 700–1030 nm wavelengths. Then photoacoustic spectra at high brightness areas in reconstructed images were calculated. Spectra differed by bilirubin concentrations. The photoacoustic signal ratio between two wavelengths was calculated. For both measurements, the feasibility of estimating bilirubin concentration using wavelengths of around 700 nm and around 900 nm were indicated. These analyses demonstrated the feasibility of visualizing subcutaneous hematoma and of evaluating its characterization using our handheld photoacoustic imaging system with multiple wavelengths.

Optical-resolution photoacoustic microscopy (PAM) has been shown to enable the acquisition of high resolution (μm) functional and anatomical images. For backward-mode operation, conventional piezoelectric ultrasound transducers need to be placed far away from the signal source due to their opacity and size. This can result in reduced acoustic sensitivity. Planar Fabry-Perot polymer film interferometer (FPI) sensors have the potential to overcome this limitation since they are transparent to the excitation wavelength, can be placed immediately adjacent to the signal source for high acoustic sensitivity, and offer a broadband frequency response (0 –50 MHz). In this study, we present a high frame rate, backward-mode OR-PAM system based on a planar FPI ultrasound sensor. A ns-pulsed laser provides excitation pulses (<200 nJ, maximum pulse repetition frequency = 200 kHz, 532 nm) to generate photoacoustic waves that are detected using a planar FPI sensor interrogated at 765-781 nm. For backwardmode operation and highest acoustic sensitivity, the excitation and interrogation beams are coaxially aligned and rasterscanned. The optical transfer function of the sensor, the spatial resolution and the detection sensitivity were determined to characterise the set-up. Images of a leaf phantom and first in vivo images of zebrafish larvae were acquired. This approach will enable fast 3D OR-PAM with high resolution and high sensitivity for functional and molecular imaging applications. FPI-based ultrasound detection also has the potential to enable dual-mode optical- and acousticresolution PAM and the integration of photoacoustic imaging with purely optical modalities such as multi-photon microscopy.

In linear-array photoacoustic imaging, different types of algorithms and beamformers are used to construct the images. Delay-and-Sum (DAS), as a non-adaptive algorithm, is one of the most popular algorithms used due to its low complexity. However, the results obtained from this algorithm contain high sidelobes and wide mainlobe. The adaptive Minimum Variance (MV) beamformer can address these limitations and improve the images in terms of resolution and contrast. In this paper, it is proposed to suppress the sidelobes more efficiently compared to MV by eliminating the effect of the samples caused by noise and interference. This would be achieved by zeroing the samples corresponding to the lower values of the calculated weights. In the other words, in the proposed MV-based-sparse subarray (MVB-S) method, the subarrays are considered to be sparse. The results show that MVB-S method leads to signal-to-noise-ratio improvement about 39.72 dB and 18.92 dB in average, compared to DAS and MV, respectively, which indicates the good performance of MVB-S method in noise reduction and sidelobe suppression.

One of the most common algorithms used in Photoacoustic and ultrasound image reconstruction, is the nonadaptive Delay-and-Sum (DAS) beamformer. The results show that this algorithm suffers from low resolution and high level of sidelobes. In this paper, it is suggested to weight the DAS beamformed signals to address these limitations and improve the image quality. The new weighting factor, named Delay-Multiply-and-StandardDeviation (DMASD) is designed in the way that the standard deviation of the mutual coupled and multiplied delayed signals is calculated, normalized and multiplied to the DAS formula. Quantitative results obtained from the numerical study show that the proposed DMASD weighting factor improves the Signal-to-Noise-Ratio for about 48.62 dB and 46.53 dB, compared to DAS and the Delay-and-Standard-Deviation (DASD) weighting factor, respectively, at the depth of 35 mm. Also, the Full-Width-Half-Maximum is improved about 0.78 mm and 0.84 mm, compared to DAS and DASD weighting factor, respectively, at the same depth using the proposed DMASD weighting factor, which indicates the improvement of resolution.

One of the most common algorithms used in linear-array photoacoustic imaging, is Delay-and-Sum (DAS) beamformer due to its simple implementation. The results show that this algorithm results in a low resolution and high sidelobes. In this paper, it is proposed to use the sparse-based algorithm in order to suppress the noise level efficiently and improve the image quality. The forward problem of the beamforming is defined through a Least square (LS) method, and a ℓ₁-norm regularization term is added to the problem which forces the sparsity of the output to the existing minimization problem. The new robust method, named sparse beamforming (SB) method, significantly suppresses the sidelobes and reduces the noise level due to the sparse added term. Numerical results show that SB leads to signal-to-noise-ratio improvement about 98.69 dB and 82.26 dB, in average, compared to DAS and Delay-Multiply-and-Sum (DMAS), respectively. Also, the full-width-half-maximum is improved about 396 μm and 123 μm, in average, compared to DAS and DMAS algorithms, respectively, using the proposed SB method, which indicates the good performance of SB method in image enhancement.

UltraSound Elastography (USE) has been widely used to obtain mechanical properties of tissues. Radio frequency (RF) data is usually used in USE to estimate the displacement. However, RF data is not available in all ultrasound imaging devices. B-mode images which are basically the envelope of the RF data are the most well-known output of the common ultrasound imaging devices. In B-mode images, the phase information of RF data is lost. Consequently, USE can be more challenging and the strain image quality would be degraded. The aim of this paper is to employ Demons algorithm, which is a powerful non-rigid image registration algorithm, to estimate displacement using B-mode images. In USE, the post-compression image may have large deformations in axial direction which deteriorates the Demons algorithm performance. In order to compensate the large deformations, an optimization algorithm is proposed to find and compensate the mean value axial deformation. Experimental and numerical phantoms are used to verify the algorithm performance in normal and severe situations. The results are compared with the common normalized cross correlation (NCC) algorithm. The results confirm that Demons algorithm is an appropriate algorithm for USE for B-mode images considering the fact that phase information are not available.

We propose a new method for biochemical sensing using photoacoustic (PA) excitation of gold nanoparticles (GNPs) to achieve real-time detection of transient biomarkers. Our approach is based on the effect of a particle coating on nonlinear PA signal generation combined with our recently-developed serial PA tomographic imaging method. We have shown the ability to image the three-dimensional spatial distribution of GNPs that produce nonlinear PA signal with respect to fluence. If the occurrence of this nonlinearity is made dependent on the presence of transient biochemical markers, for example through the degradation of a nonlinearity-quenching particle coating, our serial PA tomographic imaging method can be extended for real-time three-dimensional in situ imaging of these biomarkers. Our early-stage proof-ofconcept experimental results presented here show that coating GNPs that exhibit nonlinear PA signal generation behavior is an effective method to remove the nonlinear effect. The coefficient of determination (R² ) of a linear fit to the PA signal as a function of fluence can be effectively used to differentiate the coated GNPs and the GNPs without coating. This differentiation can also be achieved using the second order coefficient to a quadratic fit to the PA signal vs. fluence data.