This PDF file contains the front matter associated with SPIE Proceedings Volume 9708, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

Quantification of vascular elasticity can help detect thrombosis and prevent life-threatening conditions such as acute myocardial infarction or stroke. Here, we propose vascular elastic photoacoustic tomography (VE-PAT) to measure vascular elasticity in humans. VE-PAT was developed by incorporating a linear-array-based photoacoustic computed tomography system with a customized compression stage. By measuring the deformation of blood vessels under uniaxial loading, VE-PAT was able to quantify the vascular compliance. We first demonstrated the feasibility of VE-PAT in blood vessel phantoms. In large vessel phantoms, VE-PAT detected a decrease in vascular compliance due to simulated thrombosis, which was validated by a standard compression test. In small blood vessel phantoms embedded 3 mm deep in gelatin, VE-PAT detected elasticity changes at depths that are difficult to image using other elasticity imaging techniques. We then applied VE-PAT to assess vascular compliance in a human subject and detected a decrease in vascular compliance when an occlusion occurred downstream from the measurement point, demonstrating the potential of VE-PAT in clinical applications such as detection of deep venous thrombosis.

Lipid deposition inside the arterial wall is a hallmark of plaque vulnerability. Overtone absorption-based intravascular photoacoustic (IVPA) catheter is a promising technology for quantifying the amount of lipid and its spatial distribution inside the arterial wall. Thus far, the clinical translation of IVPA technology is limited by its slow imaging speed due to lack of a high-power and high-repetition-rate laser source for lipid-specific excitation at 1.7 μm. Here, we demonstrate a potassium titanyl phosphate-based optical parametric oscillator (OPO) with output pulse energy up to 2 mJ at a wavelength of 1724 nm and with a repetition rate of 500 Hz. This OPO enabled IVPA imaging at 1 frame per sec, which is about 50-fold faster than previously reported IVPA systems. The IVPA imaging system was characterized by a pencil lead and a lipid-mimicking phantom for its imaging resolution, sensitivity, and specificity, respectively. Its performance was further validated by ex vivo study of an atherosclerotic human femoral artery and comparison to gold standard histology.

We have successfully developed a clinical real-time photoacoustic/ultrasound (PA/US) imaging system. The PA/US imaging system was adapted with a FDA approved commercial US imaging system and a portable pulsed laser system. All image processing and display tasks were performed in real-time by using a graphical processing unit of the US imaging system. We have tested performances of the system by measuring maximum penetration depth, noise equivalent sensitivity, and axial resolution of contrast agent deposited microtubes under chicken breast tissues. By adapting various US transducers (i.e., linear, convex, phased, and endocavity), adaptable capability of the system was verified. In addition, volumetric PA/US imaging was performed by applying a linear scanning along an elevational direction. We have successfully acquired volumetric PA/US images of human forearms in vivo. We believe that the developed clinical real-time PA/US imaging system can be utilized in various preclinical and clinical studies in the near future.

We applied a linear-array-based photoacoustic probe to detect the tumor depth and volume of melanin-containing melanoma in nude mice in vivo. We demonstrated the ability of this linear-array-based system to measure both the depth and volume of melanoma through phantom, ex vivo, and in vivo experiments. The volume detection ability also enables us to accurately calculate the rate of growth of the tumor, which is important in quantifying tumor activity. Our results show that this system can be used for clinical melanoma diagnosis and treatment at the bedside.

One of the features of photoacoustic (PA) imaging is small-vessel visualization realized without injection of a contrast agent or exposure to X-rays. For carrying out clinical studies in this field, a prototype PA imaging system has been developed. The PA imaging system utilizes a technological platform of FUJIFILM’s clinical ultrasound (US) imaging system mounting many-core MPU for enhancing the image quality of US B-mode and US Doppler mode, which can be superposed onto PA images. By evaluating the PA and US Doppler images of the prototyped system, the applicability of the prototype system to small-vessel visualization has been discussed. The light source for PA imaging was on a compact cart of a US unit and emitted 750 nm wavelength laser pulses. The laser light was transferred to illumination optics in a handheld US transducer, which was connected to the US unit. Obtained PA rf data is reconstructed into PA images in the US unit. 3D images were obtained by scanning a mechanical stage, which the transducer is attached to. Several peripheral parts such as fingers, palms and wrists were observed by PA and US Doppler imaging. As for small arteries, US Doppler images were able to visualize the bow-shaped artery in the tip of the finger. Though PA images cannot distinguish arteries and veins, it could visualize smaller vessels and showed good resolution and vascular connectivity, resulting in a complementary image for the US Doppler images. Therefore, superposed images of the PA, US B-mode and US Doppler can visualize from large to small vessels without a contrast agent, which should be a differentiating feature of US/PA combined technology from other clinical vascular imaging modalities.

Cardiovascular related diseases are ranked as the second highest cause of death in Canada. Among the most important cardiovascular diseases is atherosclerosis. Current methods of diagnosis of atherosclerosis consist of angiography, intravascular ultrasound (IVUS) and optical coherence tomography (OCT). None of these methods possesses adequate sensitivity, as the ideal technique should be capable of both depth profiling, as well as functional imaging. An alternative technique is photoacoustics (PA) which can perform deep imaging and spectroscopy. The presented study explores the application of wavelength-modulated differential photoacoustic radar (WM-DPAR) for characterizing arterial vessels. The wavelength-modulated differential photoacoustic technique was shown to be able to substantially increase the dynamic range and sensitivity of hemoglobin oxygenation level detection. In this work the differential PA technique was used with a very high frequency modulation range. To perform spectroscopic PA imaging, at least two wavelengths are required. The selected wavelengths for this work are 1210 nm and 980 nm. 1210 nm corresponds to the maximum optical absorption coefficient of cholesterol and cholesteryl esters which are the main constituents of plaques. Since water, elastin and collagen also have high absorption coefficients at 1210 nm, this wavelength alone cannot provide very high sensitivity and specificity. The additional wavelength, 980 nm corresponds to high absorption coefficient of those constituents of healthy artery tissue. The simultaneous application of the abovementioned wavelengths can provide higher sensitivity and improved specificity in detecting lipids in the arterial vessels.

We have integrated photo-acoustic imaging into an automated breast ultrasound scanner (ABUS) with the goal of simultaneously performing ultrasound (US) and multi-spectral photo-acoustic tomography (PAT). This was accomplished with minimal change to the existing automated scanner by coupling laser light into an optical fiber for flexible and robust light delivery. We present preliminary tomography data acquired with this setup, including a simple resolution-testing geometry and a tissue phantom. Integrating PAT into the ABUS such that breast imaging is possible will require illumination from below the transducer dome. To that end, we are moving towards a fiber-based, localized illumination geometry which is fixed relative to the transducer. By illuminating locally (only near the current acquisition slice), this approach reduces overall light exposure at the tissue surface, allowing higher light intensity per acquisition (which translates to higher absorber contrast), while remaining below safe exposure thresholds. We present time-domain simulations of photo-acoustic imaging under non-uniform illumination conditions, and test one potential weighting scheme which can be used to extract absorber locations.

A compact photoacoustic transrectal probe is constructed for improved imaging in brachytherapy treatment. A 192 element 5 MHz linear transducer array is mounted inside a small 3D printed casing along with an array of optical fibers. The device is fed by a pump laser and tunable NIR-optical parametric oscillator with data collected by a Verasonics ultrasound platform. This assembly demonstrates improved imaging of brachytherapy seeds in phantoms with depths up to 5 cm. The tuneable excitation in combination with standard US integration provides adjustable contrast between the brachytherapy seeds, blood filled tubes and background tissue.

Laparoscopic procedures can be an attractive treatment option for liver resection, with a shortened hospital stay and reduced morbidity compared to open surgery. One of the central challenges of this technique is visualisation of concealed structures within the liver, particularly the vasculature and tumourous tissue. As photoacoustic (PA) imaging can provide contrast for haemoglobin in real time, it may be well suited to guiding laparoscopic procedures in order to avoid inadvertent trauma to vascular structures. In this study, a clinical laparoscopic ultrasound probe was used to receive ultrasound for PA imaging and to obtain co-registered B-mode ultrasound (US) images. Pulsed excitation light was delivered to the tissue via a fibre bundle in dark-field mode. Monte Carlo simulations were performed to optimise the light delivery geometry for imaging targets at depths of 1 cm, 2 cm and 3 cm, and 3D-printed mounts were used to position the fibre bundle relative to the transducer according to the simulation results. The performance of the photoacoustic laparoscope system was evaluated with phantoms and tissue models. The clinical potential of hybrid PA/US imaging to improve the guidance of laparoscopic surgery is discussed.

Accurate identification of tissue structures such as nerves and blood vessels is critically important for interventional procedures such as nerve blocks. Ultrasound imaging is widely used as a guidance modality to visualize anatomical structures in real-time. However, identification of nerves and small blood vessels can be very challenging, and accidental intra-neural or intra-vascular injections can result in significant complications. Multi-spectral photoacoustic imaging can provide high sensitivity and specificity for discriminating hemoglobin- and lipid-rich tissues. However, conventional surface-illumination-based photoacoustic systems suffer from limited sensitivity at large depths. In this study, for the first time, an interventional multispectral photoacoustic imaging (IMPA) system was used to image nerves in a swine model in vivo. Pulsed excitation light with wavelengths in the ranges of 750 - 900 nm and 1150 - 1300 nm was delivered inside the body through an optical fiber positioned within the cannula of an injection needle. Ultrasound waves were received at the tissue surface using a clinical linear array imaging probe. Co-registered B-mode ultrasound images were acquired using the same imaging probe. Nerve identification was performed using a combination of B-mode ultrasound imaging and electrical stimulation. Using a linear model, spectral-unmixing of the photoacoustic data was performed to provide image contrast for oxygenated and de-oxygenated hemoglobin, water and lipids. Good correspondence between a known nerve location and a lipid-rich region in the photoacoustic images was observed. The results indicate that IMPA is a promising modality for guiding nerve blocks and other interventional procedures. Challenges involved with clinical translation are discussed.

Endonasal transsphenoidal surgery is an effective approach for pituitary adenoma resection, yet it poses the serious risk of internal carotid artery injury. We propose to visualize these carotid arteries, which are hidden by bone, with an optical fiber attached to a surgical tool and a transcranial ultrasound probe placed on the patient's temple (i.e. intraoperative photoacoustic imaging). To investigate energy requirements for vessel visualization, experiments were conducted with a phantom containing ex vivo sheep brain, ex vivo bovine blood, and 0.5-2.5 mm thick human cadaveric skull specimens. Photoacoustic images were acquired with 1.2-9.3 mJ laser energy, and the resulting vessel contrast was measured at each energy level. The distal vessel boundary was difficult to distinguish at the chosen contrast threshold for visibility (4.5 dB), which was used to determine the minimum energies for vessel visualization. The blood vessel was successfully visualized in the presence of the 0-2.0 mm thick sphenoid and temporal bones with up to 19.2 dB contrast. The minimum energy required ranged from 1.2-5.0 mJ, 4.2-5.9 mJ, and 4.6-5.2 mJ for the 1.0 temporal and 0-1.5 mm sphenoid bones, 1.5 mm temporal and 0-0.5 mm sphenoid bones, and 2.0 mm temporal and 0-0.5 mm sphenoid bones, respectively, which corresponds to a fluence range of 4-21 mJ/cm². These results hold promise for vessel visualization within safety limits. In a separate experiment, a mock tool tip was placed, providing satisfactory preliminary evidence that surgical tool tips can be visualized simultaneously with blood vessels.

The purpose of this work was to demonstrate that a clinical ultrasound transducer array can practically detect thermoacoustic pulses induced by irradiation by very high frequency (VHF) electromagnetic energy. This is an important step because thermoacoustic signal strength is directly proportional to the specific absorption rate (SAR), which is lower in the VHF regime than in microwave or optical regimes. A 96-channel transducer array (P4-1) providing 3 cm coverage was incorporated into a benchtop thermoacoustic imaging system for imaging fresh surgical specimens. Thermoacoustic signal was generated by 700 ns irradiation pulses with 11 kV/m electric field strength and 108 MHz carrier frequency. To improve SNR 1024 pulses were averaged at a 250 Hz repetition rate. Two sets of sinograms were acquired, separated by a 2 cm translation along the tomographic axis and reconstructed over a 6 x 6 x 5 cm3 volume. Contrast and in-plane resolution were measured by imaging a homogeneous cylindrical phantom and an 80- micron wire designed to highlight E-field polarization effects. FWHM of the in-plane point spread function varied from 250 microns to 1.1 mm, depending upon transducer used and phantom orientation relative to the electric field. Several fresh human prostates were imaged immediately after surgery. Rudimentary comparison to histology was performed and volumetric reconstruction of the multi-channel P4-1 data visualizes anatomic features that are rarely seen in ultrasound, CT, or MRI. The single element transducer provided superior image contrast, but with inferior resolution.

Chronic venous insufficiency (CVI) is one of the most common medical conditions with reported prevalence estimates as high as 30% in the adult population. Although conservative management with compression therapy may improve the symptoms associated with CVI, healing often demands invasive procedures. Besides established surgical methods like vein stripping or bypassing, endovenous laser therapy (ELT) emerged as a promising novel treatment option during the last 15 years offering multiple advantages such as less pain and faster recovery. Much of the treatment success hereby depends on monitoring of the treatment progression using clinical imaging modalities such as Doppler ultrasound. The latter however do not provide sufficient contrast, spatial resolution and three-dimensional imaging capacity which is necessary for accurate online lesion assessment during treatment. As a consequence, incidence of recanalization, lack of vessel occlusion and collateral damage remains highly variable among patients. In this study, we examined the capacity of volumetric optoacoustic tomography (VOT) for real-time monitoring of ELT using an ex-vivo ox foot model. ELT was performed on subcutaneous veins while optoacoustic signals were acquired and reconstructed in real-time and at a spatial resolution in the order of 200μm. VOT images showed spatio-temporal maps of the lesion progression, characteristics of the vessel wall, and position of the ablation fiber’s tip during the pull back. It was also possible to correlate the images with the temperature elevation measured in the area adjacent to the ablation spot. We conclude that VOT is a promising tool for providing online feedback during endovenous laser therapy.

Cryoablation of prostate cancer is an FDA approved clinical procedure, which involves repetitive rapid cooling of a lesion to lethal temperatures of -40°C and below. The major drawback of the technique is the insufficient control over the fast thermal processes that may result in severe complications (impotence, incontinence, perforation of the rectal wall) and morbidity. The developed optoacoustic imaging technique provides non-invasive real-time temperature mapping of tissue adjacent to prostate and enables more efficient control over the procedure, which is necessary to reduce side effects and accelerate the physician’s learning curve. In these studies we successfully demonstrated real-time transrectal optoacoustic imaging during prostate cryoablation in live canine model focused on optoacoustic thermography of the rectal wall within the depth of 1cm. Our method utilized previously discovered universal thermal dependence of the normalized optoacoustic response of blood. Nanosecond-pulse radiation of Ti-Sapphire laser tuned to the isosbestic point of hemoglobin (802±3 nm) was delivered via fiberoptic illuminators assembled on both sides of the linear array of the 128-channel transrectal ultrasound probe. Temperature readouts at discrete locations inside and nearby prostate were also performed using standard transperineal needle sensors. The effect of homeostasis on optoacoustic imaging in live tissue was examined during cooling and shown to be significant only within the range of ±1.5°C in respect to the body temperature. Accuracy of in vivo optoacoustic temperature measurements was determined as ±2°C for the range of temperature from +35 to -15°C, which is more than sufficient for tracking the essential isotherms in the course of clinical procedures.

Optical-resolution photoacoustic endomicroscopy (OR-PAE) allows going beyond the limited penetration depth of conventional optical-resolution photoacoustic systems. Recently, it has been shown that OR-PAE may be performed through minimally invasive multimode fibers, by raster scanning a focus spot with optical wavefront shaping [1]. Here we introduce for the first time an approach to perform OR-PAE through a multimode fiber with a full-field illumination approach. By using multiple known speckle patterns, we show that it is possible to obtain optical-diffraction limited photoacoustic images, with the same resolution as that obtained by raster scanning a focus spot, i.e that of the speckle grain size. The fluctuations patterns of the photoacoustic amplitude at each pixel in the sample plane with the series of multiple speckle illumination were used to encode each pixel. This approach with known speckle illumination requires an initial calibration stage, that consists in learn a set of fluctuation patterns pixel per pixel, which will encode patterns each pixel of the scanned area. A point-like absorber was scanned across the filed-of-view during the calibration stage to acquire the reference patterns. Image reconstruction may be carried out by cross-correlating the series of photoacoustic amplitude measured with the sample to the reference patterns obtained during the calibration stage. In this work, the approach above was carried out both theoretically with Monte-carlo simulations and experimentally through a multi-mode fiber with samples made of absorbing spheres. [1] Papadopoulos et al., " Optical-resolution photoacoustic microscopy by use of a multimode fiber", Appl. Phys. Lett., 102(21), 2013

During the past decades, prostate cancer (PCa), with an annual incident rate much higher than any other cancer, is the most commonly diagnosed cancer in American men. PCa has a relatively low progression rate yet the survival percentage decreases dramatically once the cancer has metastasized. Identifying aggressive from indolent PCa to prevent metastasis and death is critical to improving outcomes for patients with PCa. Standard procedure for assessing the aggressiveness of PCa involves the removal of tumor tissues by transrectal (TR) ultrasound (US) guided needle biopsy. The microscopic architecture of the biopsied tissue is visualized by histological or immunohistochemical staining procedures. The heterogeneity of the microscopic architecture is characterized by a Gleason score, a quantitative description of the aggressiveness of PCa. Due to the inability to identify the cancer cells, most noninvasive imaging modalities can only provide diagnosis of PCa at limited accuracy. This study investigates the feasibility of identifying PCa tumors and characterizing the aggressiveness of PCa by photoacoustic imaging assisted by cancer targeting polyacrylamide (PAA) nanoparticles (NPs). PAA is a biocompatible material used in clinics for the past 20 years. PAA NPs can protect capsulated optical contrast agents from interference by enzymes and enable prolonged systematic circulation in the living biological environment. The cancer targeting mechanism is achieved by conjugating the NPs to F3 peptides, which trace nucleolin overexpressed on the surface of cancer cells. Preliminary studies have shown that the NPs are capable of staining the PCa cells in vivo.

Optoacoustic technique has been shown to resolve anatomical, functional and molecular features at depths that go beyond the reach of epi-illumination optical microscopy offering new opportunities for endoscopic imaging. Herein, we interrogate the merits of optoacoustic endoscopy implemented by translating a sound detector in linear or curved geometries. The linear and curved detection geometries are achieved by employing an intravascular ultrasound transducer (IVUS) within a plastic guide shaped to a line or a curve. This concept could be used together with optical endoscopes to yield hybrid optical and optoacoustic imaging.

Crohn’s disease (CD) is an autoimmune disease, which may cause obstructing intestinal strictures due to inflammation, fibrosis (deposition of collagen), or a combination of both. Identifying the different stages of the disease progression is still challenging. In this work, we indicated the feasibility of non-invasively characterizing intestinal strictures using photoacoustic imaging (PAI), utilizing the uniquely optical absorption of hemoglobin and collagen. Surgically removed human intestinal stricture specimens were investigated with a prototype PAI system. 2D PA images with acoustic resolution at wavelength 532, 1210 and 1310 nm were formulated, and furthermore, the PA histochemical components images which show the microscopic distributions of histochemical components were solved. Imaging experiments on surgically removed human intestinal specimens has demonstrated the solved PA images were significantly different associated with the presence of fibrosis, which could be applied to characterize the intestinal strictures for given specimens.

There is considerable interest in the development of photoacoustic endoscopy (PAE) probes for the clinical assessment of pathologies in the gastrointestinal (GI) tract, guiding minimally invasive laparoscopic surgeries and applications in foetal medicine. However, most previous PAE probes integrate mechanical scanners and piezoelectric transducers at the distal end which can be technically complex, expensive and pose challenges in achieving the necessary level of miniaturisation. We present two novel all-optical forward-viewing endoscopic probes operating in widefield tomography mode that have the potential to overcome these limitations. In one configuration, the probe comprises a transparent 40 MHz Fabry-Pérot ultrasound sensor deposited at the tip of a rigid, 3 mm diameter coherent fibre-optic bundle. In this way, the distal end of coherent fibre bundle acts as a 2D array of wideband ultrasound detectors. In another configuration, an optical relay is used between the distal end face of flexible fibre bundle and the Fabry-Pérot sensor to enlarge the lateral field of view to 6 mm x 6 mm. In both configurations, the pulsed excitation laser beam is full-field coupled into the fibre bundle at the proximal end for uniform backward-mode illumination of the tissue at the probe tip. In order to record the photoacoustic waves arriving at the probe tip, the proximal end of the fibre bundle is optically scanned in 2D with a CW wavelength-tunable interrogation laser beam thereby interrogating different spatial points on the sensor. A time-reversal image reconstruction algorithm was used to reconstruct a 3D image from the detected signals. The 3D field of view of the flexible PAE probe is 6 mm x 6 mm x 6 mm and the axial and lateral spatial resolution is 30 µm and 90 µm, respectively. 3D imaging capability is demonstrated using tissue phantoms, ex vivo tissues and in vivo. To the best of our knowledge, this is the first forward-viewing implementation of a photoacoustic endoscopy probe, and it offers several advantages over previous distal-end scanning probes. These include a high degree of miniaturisation, no moving parts at the distal end and simple and inexpensive fabrication with the potential to realise disposable probes for clinical imaging of the GI tract and other minimally invasive applications.

The acoustically-mismatched skull bone poses significant challenges for the application of ultrasonic and optical techniques in neuroimaging, still typically requiring invasive approaches using craniotomy or skull thinning. Optoacoustic imaging partially circumvents the acoustic distortions due to the skull because the induced wave is transmitted only once as opposed to the round trip in pulse-echo ultrasonography. To this end, the mouse brain has been successfully imaged transcranially by optoacoustic scanning microscopy. Yet, the skull may adversely affect the lateral and axial resolution of transcranial brain images. In order to accurately characterize the complex behavior of the optoacoustic signal as it traverses through the skull, one needs to consider the ultrawideband nature of the optoacoustic signals.

Here the insertion loss of murine skull has been measured by means of a hybrid optoacoustic-ultrasound scanning microscope having a spherically focused PVDF transducer and pulsed laser excitation at 532 nm of a 20 μm diameter absorbing microsphere acting as an optoacoustic point source. Accurate modeling of the acoustic transmission through the skull is further performed using a Fourier-domain expansion of a solid-plate model, based on the simultaneously acquired pulse-echo ultrasound image providing precise information about the skull's position and its orientation relative to the optoacoustic source. Good qualitative agreement has been found between the a solid-plate model and experimental measurements.

The presented strategy might pave the way for modeling skull effects and deriving efficient correction schemes to account for acoustic distortions introduced by an adult murine skull, thus improving the spatial resolution, effective penetration depth and overall image quality of transcranial optoacoustic brain microscopy.

Adequate oxygenation is essential for normal embryogenesis and fetal growth. Perturbations in the intrauterine oxidative environment during pregnancy are associated with several pathophysiological disorders such as pregnancy loss, preeclampsia, and intrauterine growth restriction. We proposed to use optoacoustic technology for monitoring placental and fetal umbilical blood oxygenation. In this work, we studied optoacoustic monitoring of oxygenation in placenta and umbilical cord blood ex vivo using technique of placenta perfusion. We used a medical grade, nearinfrared, tunable, optoacoustic system developed and built for oxygenation monitoring in blood vessels and in tissues. First, we calibrated the system for cord blood oxygenation measurements by using a CO-Oximeter (gold standard). Then we performed validation in cord blood circulating through the catheters localized on the fetal side of an isolated placental lobule. Finally, the oxygenation measurements were performed in the perfused placental tissue. To increase or decrease blood oxygenation, we used infusion of a gas mixture of 95% O₂ + 5% CO₂ and 95% N₂ + 5% CO₂, respectively. In placental tissue, up to four cycles of changes in oxygenation were performed. The optoacoustically measured oxygenation in circulating cord blood and in placental lobule closely correlated with the actual oxygenation data measured by CO-Oximeter. We plan to further test the placental and cord blood oxygenation monitoring with optoacoustics in animal and clinical studies.

Many types of cancer therapies target the tumor microenvironment, causing biochemical and morphological changes in tissues. In therapies using ultrasound activated microbubbles, vascular collapse is typically reported. Red blood cells (RBCs) that leak out of the vasculature become exposed to the ceramide that is released from damaged endothelial cells. Ceramide can induce programmed cell death in RBCs (eryptosis), and is characterized by cell shrinkage, membrane blebbing and scrambling. Since the effect of eryptotic cells on generated photoacoustics (PA) signals has not been reported, we investigated the potential PA may have for cancer treatment monitoring by using PA spectral analysis to sense eryptosis. To induce eryptosis, C2-ceramide was added to RBC suspensions and that were incubated for 24 hours at 37°C. A control and ceramide-induced sample was imaged in a vessel phantom using a high frequency PA system (VevoLAZR, 10 – 45 MHz bandwidth) irradiated with multiple wavelengths ranging from 680 to 900 nm. PA spectral parameters were measured and linked to changes in RBCs as it underwent eryptosis. These samples were examined using optical microscopy, a blood gas analyzer and an integrating sphere setup to measure optical properties (wavelengths 600 – 900 nm). The results of the experiment demonstrate how PA spectral analysis can be used to identify eryptosis at a depth of more than 1 cm into the phantom using ultrasound derived the y-intercept and mid bandfit (MBF) parameters at optical wavelengths of 800 – 900 nm. These parameters were correlated to the morphological and biochemical changes that eryptotic RBCs display. The results establish the potential of PA in cancer treatment monitoring through sensing treatment induced eryptosis.

Cavitation therapy based on high-amplitude focused ultrasound (e.g., Histotripsy) has shown great promise in clinical trials. The technique realizes localized treatments of tissues and diseased cells by controlling cavitation zones, which can be even smaller than its acoustic spot sizes. Also, the short pressure pulse used in the technique can minimize the unwanted heat accumulation, which the conventional piezoelectric transducers suffer from due to low operating frequencies and relatively long acoustic pulses. However, this modality requires bulky system composed of array of piezoelectric elements and electric amplifiers in order to obtain high pressure amplitude. Moreover, especially when treating an area much smaller than the acoustic spot size, this approach may be vulnerable to nucleation sites within the focal volume, which can potentially induce cavitation and thus enlarge the total treatment area. Here, we show targeted cell-level therapy by using laser generated ultrasound. By employing a concave lens coated by a carbon nanotube (CNT)-polymer composite, high-amplitude acoustic pressure can be obtained at a tight focal spot (<100 um). The small focal spot, comparable to cavitation zone, lead to controlled cavitation treatment. Such feature can be exploited for treating intraocular tumors but without harming other parts of the eye (e.g. healthy retina and choroid) and therefore preserve the vision of the patients. We demonstrate that the localized disruption effects can be used for cell-level surgery to remove cells and to kill cells. Some experimental examples are shown using animal eyeballs.

Exogenous insulin administration is the mainstay treatment therapy for patients with Type-1 diabetes mellitus (T1DM). However, for select patients, clinical islet transplantation is an alternative therapeutic treatment. In this procedure, islets are transplanted into the hepatic portal vein, and despite improved success within the last decade, obstacles are still associated with this approach. It has been discovered that the subcutaneous space may be an effective alternative site for islet transplantation, and may provide advantages of easy access and potential for simple monitoring. The ability to monitor islet viability and the transplant microenvironment may be key to future success in islet transplantation. A subcutaneous device-less technique has been developed to facilitate angiogenesis in the islet transplant site, however, a method for monitoring the potential engraftment site have yet to be explored fully. Here we demonstrate the ability to track angiogenesis in mice with 1, 2, 3 and 4 weeks post-catheter implant on both sides of the abdomen using a FujiFilm VisualSonics Vevo-LAZR system. Quantitative analysis on vessel densities exhibited gradual vessel growth successfully induced by catheter implantation. Our study demonstrates the ability of employing photoacoustic and micro-ultrasound imaging to track angiogenesis around the catheter site prior to islet transplantation.

Acoustic resolution photoacoustic Doppler flowmetry (AR-PAF) is a technique that has the potential to overcome the spatial resolution and depth penetration limitations of current blood flow measuring methods. Previous work has shown the potential of the technique using blood-mimicking phantoms, but it has proved difficult to make accurate measurements in blood, and thus in vivo application has not yet been possible. One explanation for this difficulty is that whole blood is insufficiently heterogeneous. Through experimental measurements in red blood cell suspensions of different concentrations, as well as in whole blood, we provide new insight and evidence that refutes this assertion. We show that the velocity measurement accuracy is influenced by bandlimiting not only due to the detector frequency response, but also due to spatial averaging of absorbers within the detector field-of-view. In addition, there is a detrimental effect of limited light penetration, but this can be mitigated by selecting less attenuated wavelengths of light, and also by employing range-gating signal processing. By careful choice of these parameters as well as the detector centre frequency, bandwidth and field-of-view, it is possible to make AR-PAF measurements in whole blood using transducers with bandwidths in the tens of MHz range. These findings have profound implications for the prospects of making deep tissue measurements of blood flow relevant to the study of microcirculatory abnormalities associated with cancer, diabetes, atherosclerosis and other conditions.

We used raster-scan optoacoustic mesoscopy (RSOM) at 50 MHz, and at 100 MHz, to monitor tumor growth, and tumor angiogenesis, which is a central hallmark of cancer, in-vivo. In this study we compared the performance, and the effect of the 50 MHz, and the 100 MHz frequencies on the quality of the final image.
The system is based on a reflection-mode implementation of RSOM. The detectors used are custom made, ultrawideband, and spherically focused. The use of such detectors enables light coupling from the same side as the detector, thus reflection-mode. Light is in turn coupled using a fiber bundle, and the detector is raster scanned in the xy-plane. Subsequently, to retrieve small features, the raw data are reconstructed using a multi-bandwidth, beamforming reconstruction algorithm.
Comparison of the system performance at the different frequencies shows as expected a higher resolution in case of the 100 MHz detector compared to the 50 MHz. On the other hand the 50 MHz has a better SNR, can detect features from deeper layers, and has higher angular acceptance. Based on these characteristics the 50 MHz detector was mostly used. After comparing the performance we monitored the growth of B16F10 cells, melanin tumor, over the course of 9 days. We see correspondence between the optoacoustic measurements and the cryoslice validations. Additionally, in areas close to the tumor we see sprouting of new vessels, starting at day 4-5, which corresponds to tumor angiogenesis.

Intraocular tumors are life-threatening conditions. Long-term mortality from uveal melanoma, which accounts for 80% of primary intraocular tumors, could be as high as 25% depending on the size, ciliary body involvement and extraocular extension. The treatments of intraocular tumors include eye-sparing approaches such as radiotherapy and thermotherapy, and the more aggressive enucleation. The accurate diagnosis of intraocular tumors is thereby critical in the management and follow-up of the patients. The diagnosis of intraocular tumors is usually based on clinical examination with acoustic backscattering based ultrasonography. By analyzing the high frequency fluctuations within the ultrasound (US) signals, microarchitecture information inside the tumor can be characterized. However, US cannot interrogate the histochemical components formulating the microarchitecture. One representative example is the inability of US imaging (and other contemporary imaging modalities as well) in differentiating nevoid and melanoma cells as the two types of cells possesses similar acoustic backscattering properties. Combining optical and US imaging, photoacoustic (PA) measurements encode both the microarchitecture and histochemical component information in biological tissue. This study attempts to characterize ocular tumors by analyzing the high frequency signal components in the multispectral PA images. Ex vivo human eye globes with melanoma and retinoblastoma tumors were scanned using less than 6 mJ per square centimeters laser energy with tunable range of 600-1700 nm. A PA-US parallel imaging system with US probes CL15-7 and L22-14 were used to acquire the high frequency PA signals in real time. Preliminary results show that the proposed method can identify uveal melanoma against retinoblastoma tumors.

Microwave-based thermoacoustic tomography (TAT), based on the measurement of ultrasonic waves induced by microwave pulses, can reveal tissue dielectric properties that may be closely related to the physiological and pathological status of the tissues. Using microwaves as the excitation source improved imaging depth because of their deep penetration into biological tissues. We demonstrate, for the first time, in vivo microwave-based thermoacoustic imaging in rats. The transducer is rotated around the rat in a full circle, providing a full two-dimensional view. Instead of a flat ultrasonic transducer, we used a virtual line detector based on a cylindrically focused transducer. A 3 GHz microwave source with 0.6 µs pulse width and an electromagnetically shielded transducer with 2.25 MHz central frequency provided clear cross-sectional images of the rat’s body. The high imaging contrast, based on the tissue’s rate of absorption, and the ultrasonically defined spatial resolution combine to reveal the spine, kidney, muscle, and other deeply seated anatomical features in the rat’s abdominal cavity. This non-invasive and non-ionizing imaging modality achieved an imaging depth beyond 6 cm in the rat’s tissue. Cancer diagnosis based on information about tissue properties from microwave band TAT can potentially be more accurate than has previously been achievable.

Photoacoustics is a novel medical imaging technique with high potential for early detection of different diseases such as skin cancer or rheumatology. It is a hybrid modality with pulsed laser light for excitation of the tissue, and ultrasound as response. One of the hurdles for its introduction into the clinic, or even in clinical pilot studies and larger trials, is the bulkiness and price of existing photoacoustic systems. This presentation describes how recent developments in diode laser technology lead to a compact ultrasound scanner with built-in photoacoustic functionality. This is a key for the introduction of photoacoustic technology in the clinic and future point of care systems. We have developed a diode laser system and driver that deliver pulse energies which up to now were only achievable with Nd:YAG lasers. The efficiency and compactness allows integration in a handheld probe. The paper will highlights the laser technology and its radical integration with a medical ultrasound scanner, leading to a first prototype for clinical pilot studies.

Two custom fibre lasers have been developed. One is designed for widefield photoacoustic tomography (PAT) and uses a custom drawn large core diameter fibre (100μm) to provide high pulse energies (5mJ). It also provides a variable pulse repetition frequency (100Hz-400Hz) and pulse duration (10-150ns) and is compact (of comparable dimensions to a desktop PC) and does not require external water cooling. This system was used to acquire in vivo images of the subcutaneous microvasculature in the human palm. The second laser is designed for Optical Resolution Photoacoustic Microscopy (OR-PAM) and provides a high quality beam (M2<1.1), pulse energies >1μJ with a pulse repetition frequency (PRF) up to 2MHz, and a 532nm emission wavelength. The high PRF of this laser was exploited for ultra-fast image acquisition. The compact size and enhanced functionality of these lasers offers a major opportunity to facilitate the translation of photoacoustic imaging to practical applications in medicine and biology.

Quantitative photoacoustic tomography (QPAT) is a hybrid imaging modality that simultaneously reconstructs absorption and scattering coefficients with multi-source or multi-wavelength setting. In contrast with PAT, QPAT eliminates the artifacts due to photon transfer in depth and characterizes the intrinsic biological attributes. However, data acquisition for QPAT is time consuming because for each optical source (or wavelength), a 360-degree acoustic measurement on the boundary of imaging region is required. In this work, we investigate a novel limited-view multi-source (LVMS) QPAT scheme and reconstruction algorithm based on coupled opto-acousto model. A unique setting that binds optical source and acoustic detector together is presented. Under each illumination, only a limited-view measurement is acquired, which is incomplete for PAT reconstruction but sufficient for direct QPAT reconstruction; then the source and detector rotate to next data acquisition position synchronously. In terms of reconstruction, a sparsity-regularized formulation based on total variation norm is adopted here and optimized through alternating direction method of multipliers with LBFGS solver, during which the adjoint method is used for rapid computation of numerical gradient of objective function. However, the aperture effect i.e. anisotropic angular sensitivity of a finite-dimension transducer would cause temporal distortion of receiving acoustic signal and further resolution reduction. Therefore, we numerically integrate ideal PAT system spatial impulse response (SIR) with the angular response from circular transducer to improve the modeling accuracy. In summary, the proposed LVMS-QPAT has the potential to shorten the data acquisition time, enhance system SNR and improve image resolution.

Photoacoustic (PA) conversion of metal film absorbers is known to be inefficient because of their low thermal expansion and high light reflectance, as compared to polymeric materials containing light absorbing fillers. Specifically, the PA signal for metal films is typically an order of magnitude lower than those for PDMS-based composites consisting of carbon materials such as carbon blacks, carbon nanotubes, and carbon fibers. However, the carbon-PDMS composites have several disadvantages, e.g., difficulty in controlling film thickness, aggregation of the carbon fillers, and poor patternablility. To overcome these issues and achieve comparable PA amplitudes, a polymer-metal film composite was developed consisting of a thin metal absorber and adjacent transparent polymer layers. The proposed structure shows efficient PA conversion. The measured PA amplitude of the metal film composite is an order of magnitude higher than that of metal-only samples, and comparable to those of the carbon-PDMS composites. The enhanced PA conversion is accomplished by using metal film of a few tens of nanometers, which greatly facilitates heat transfer from the metal film to the surrounding polymers. Moreover, integrating the metal film composite with a photonic cavity can compensate light absorption loss of the thinner metal film. Theoretical and experimental analysis is conducted for understanding the mechanism behind such improvement. This strategy could be implemented for spatial PA signal patterns, especially for deep tissue PA imaging of implants or image-guiding tools. Furthermore, this approach also provides a guideline for designing photoacoustic transmitters and contrast agents.

We present a multimodal optical setup, allowing non-contact photoacoustic imaging, optical coherence tomography (OCT), and non-contact mid-infrared photoacoustic spectroscopy. Photoacoustic signals are generated using a Nd:YAG laser and a tunable quantum cascade laser for photoacoustic imaging and spectroscopy, respectively. Photoacoustic signals are acquired by measuring the surface displacement of a specimen using a fiber-optic Mach-Zehnder interferometer. In the same fiber-optic network a spectral-domain OCT system is realized. Light from the photoacoustic detection laser and the OCT source are multiplexed into one fiber and the same objective is used for both imaging modalities. Light reflected from specimens is demultiplexed and guided to the respective imaging systems. To allow fast non-contact PAI and OCT imaging the detection spot is scanned across the specimens’ surface using a galvanometer scanner. As the same fiber-network and optical components are used for photoacoustic and OCT imaging the obtained images are co-registered intrinsically. Imaging is demonstrated on tissue mimicking and biological samples; spectral information is obtained for polystyrene and hemoglobin.

Photoacoustic spectrum analysis (PASA) has been found to have the ability to characterize microstructures in phantoms. The ability of PASA technique which is subjected to the ultrasound detector has been reported in identify tissue with hundreds of microns in size. This paper demonstrated the feasibility of micro-ring, an ultrasonic detector with ultra-broad bandwidth, in characterizing microspheres’ sizes ranging from few microns to 100 microns using PASA technique and the results were compared with a commercial hydrophone. In order to further verify the capability of micro-ring, spectrum of single micro-spheres with sizes of tens of microns were measured and compared to simulation result. Our work proves that micro-ring based PASA technique has the ability of differentiating the particles with different size in phantoms.

Polymer optical fibre (POF) is a relatively new and novel technology that presents an innovative approach for ultrasonic endoscopic applications. Currently, piezo electric transducers are the typical detectors of choice, albeit possessing a limited bandwidth due to their resonant nature and a sensitivity that decreases proportionally to their size. Optical fibres provide immunity from electromagnetic interference and POF in particular boasts more suitable physical characteristics than silica optical fibre. The most important of these are lower acoustic impedance, a reduced Young’s Modulus and a higher acoustic sensitivity than single-mode silica fibre at both 1 MHz and 10 MHz. POF therefore offers an interesting alternative to existing technology. Intrinsic fibre structures such as Bragg gratings and Fabry-Perot cavities may be inscribed into the fibre core using UV lasers. These gratings are a modulation of the refractive index of the fibre core and provide the advantages of high reflectivity, customisable bandwidth and point detection. We present a compact in fibre ultrasonic point detector based upon a POF Bragg grating (POFBG) sensor. We demonstrate that the detector is capable of leaving a laboratory environment by using connectorised fibre sensors and make a case for endoscopic ultrasonic detection through use of a mounting structure that better mimics the environment of an endoscopic probe. We measure the effects of water immersion upon POFBGs and analyse the ultrasonic response for 1, 5 and 10 MHz.

Conventional contacting transducers are highly sensitive and readily available for ultrasonic and photoacoustic imaging. On the other hand, optical detection can be advantageous when a small sensor footprint, large bandwidth and no contact are essential. However, most optical methods utilizing interferometry or Doppler vibrometry rely on the reflection of light from the object. We present a non-contact detection method for photoacoustic and ultrasound imaging--termed Gas-Coupled Laser Acoustic Detection (GCLAD)--that does not involve surface reflectivity. GCLAD measures the displacement along a line in the air parallel to the object. Information about point displacements along the line is lost with this method, but resolution is increased over techniques that utilize finite point-detectors when used as an integrating line detector. In this proceeding, we present a formula for quantifying surface displacement remotely with GCLAD. We will validate this result by comparison with a commercial vibrometer. Finally, we will present two-dimensional imaging results using GCLAD as a line detector for photoacoustic and laser-ultrasound imaging.

We present a novel akinetic optical ultrasound sensor, consisting of a rigid, fiber-coupled Fabry-P´erot interferometer (FPI) with a central opening. The sensing principle relies exclusively on the detection of pressure-induced changes of the refractive index in the liquid located between the cavity mirrors. This enables resonance-free, inherently linear signal detection over a large bandwidth. We demonstrate that the sensor allows to realize exceedingly low noise equivalent pressure (NEP) values of 2 Pa over a 20 MHz measurement bandwidth, while maintaining a large full field of view of 2,7mm × 1,3mm as well as a flat frequency response. Imaging tests on phantoms and biological tissue show the suitability of the XARION-sensor for optical resolution photoacoustic microscopy (OR-PAM) applications. Transparent in axial direction, the sensor facilitates the implementation of highly sensitive fast-scanning reflection-mode OR-PAM setups, as well as easy integration with other imaging modalities such as confocal microscopy or OCT.

We develop an air-couple ultrasound detector based on an optical fluidic ring resonator (OFRR) suspended on a Ushaped holder. The OFRR is a glass capillary with an outer diameter of approximately 130 μm and a wall thickness in the order of 1~10 μm. The circular cross section of the OFRR supports the high-Q whispering gallery mode (WGM) that circulates along the circumference. Incoming ultrasound pressure results in a small refractive index change in the glass wall and geometrical change in the OFRR shape, both of which in turn lead to a spectral shift in the WGM that can be sensitively detected owing to WGM with high optical Q-factors (>10⁷). Due to the suspension nature of the OFRR, the ultrasound detection can be carried out in air, which is advantageous in comparison with other ultrasound detections that require acoustic coupling media such water, gel or solid. The sensitivity can be tuned and optimized by changing the diameter and wall thickness. Besides the optical detection, we also demonstrate optomechanical ultrasound mixing, in which optomechanical vibration is first excited within the OFRR that subsequently modulates the ultrasound wave. Our work will lead to the development of a new type of air-coupled ultrasound detector that can be used for photo-acoustic imaging, non-invasive ultrasound detection of external objects, and ultrasound detection/characterization of internal objects (such as particles and liquids) flowing inside the capillary.

The combination of photoacoustic tomography (PAT) with optical coherence tomography (OCT) has seen steady progress over the past few years. With the benchtop and semi-benchtop configurations, preclinical and clinical results have been demonstrated, paving the way for wider applications using dual modality PAT/OCT systems. However, as for the most updated semi-benchtop PAT/OCT system which employs a Fabry-Perot polymer film sensor, it is restricted to only human palm imaging due to the limited flexibility of the probe. The passband limit of the polymer film sensor further restricts the OCT source selection and reduces the sensitivity of the combined OCT system. To tackle these issues, we developed an articulated PAT/OCT probe for both preclinical and clinical applications. In the probe design, the sample arm of OCT sub-system and the interrogation part of the PAT sub-system are integrated into one compact unit. The polymer film sensor has a quick release function so that before each OCT scan, the sensor can be taken off to avoid the sensitivity drop and artefacts in OCT. The holding mechanism of the sensor is also more compact compared to previous designs, permitting access to uneven surfaces of the subjects. With the help of the articulated probe and a patient chair, we are able to perform co-registered imaging on human subjects on both upper and lower extremities while they are at rest positions. An increase in performance characteristics is also achieved. Patients with skin diseases are currently being recruited to test its clinical feasibility.

It is difficult to achieve sub-micron resolution in backward mode OA microscopy using conventional piezoelectric detectors, because of wavefront distortions caused by components placed in the optical path, between the sample and the objective lens, that are required to separate the acoustic wave from the optical beam. As an alternate approach, an optoacoustic microscope (OAM) was constructed using the probe beam deflection technique (PBDT) to detect laserinduced acoustic signals. The all-optical OAM detects laser-generated pressure waves using a probe beam passing through a coupling medium, such as water, filling the space between the microscope objective lens and sample. The acoustic waves generated in the sample propagate through the coupling medium, causing transient changes in the refractive index that deflect the probe beam. These deflections are measured with a high-speed, balanced photodiode position detector. The deflection amplitude is directly proportional to the magnitude of the acoustic pressure wave, and provides the data required for image reconstruction. The sensitivity of the PBDT detector expressed as noise equivalent pressure was 12 Pa, comparable to that of existing high-performance ultrasound detectors. Because of the unimpeded working distance, a high numerical aperture objective lens, i.e. NA = 1, was employed in the OAM to achieve near diffraction-limited lateral resolution of 0.5 μm at 532nm. The all-optical OAM provides several benefits over current piezoelectric detector-based systems, such as increased lateral and axial resolution, higher sensitivity, robustness, and potentially more compatibility with multimodal instruments.

Gold and silver plasmonic nanoparticles (NPs) are widely used as a contrast agent for photoacoustic (PA) imaging, taking advantage of the strong optical absorption cross-section of these particles due to their localized surface-plasmon resonance. Inspired by recent developments in ultra-high frequency wide-bandwidth transducers, we propose utilizing off-resonance ultrashort laser sources with a pulse width in the femtosecond (fs) and picosecond (ps) range to increase the efficiency of PA imaging. Also, from the fact that the laser pulse duration is shorter than the heat diffusion time of the materials, we expect practically no collateral damage of the laser irradiated biological tissues. Our preliminary studies show that irradiating the NPs with an ultrashort-pulsed laser has the potential to achieve substantially higher efficiency at generating the PA signal. Enhanced by the presence of NPs, the laser field causes a highly localized plasma nucleation around the vicinity of the NPs. Plasma relaxes through electron-ion interaction and releases a pressure wave in the surrounding medium. However, in this process, it is crucial to precisely control the heat energy absorption in the NPs to avoid their fragmentation. In this talk we present a model to simulate an optimized plasma–mediated PA signal dynamics generated from off-resonance ultrashort laser excitation (λ =800 nm, τ = 70 fs – 2 ps) of a variety of plasmonic NPs with sizes ranging from 50 nm to 100 nm.

A flow cytometer that uses sound waves to determine the size of biological cells is presented. In this system, a microfluidic device made of polydimethylsiloxane (PDMS) was developed to hydrodynamically flow focus cells in a single file through a target area. Integrated into the microfluidic device was an ultrasound transducer with a 375 MHz center frequency, aligned opposite the transducer was a pulsed 532 nm laser focused into the device by a 10x objective. Each passing cell was insonfied with a high frequency ultrasound pulse, and irradiated with the laser. The resulting ultrasound and photoacoustic waves from each cell were analyzed using signal processing methods, where features in the power spectra were compared to theoretical models to calculate the cell size. Two cell lines with different size distributions were used to test the system: acute myeloid leukemia cells (AML) and melanoma cells. Over 200 cells were measured using this system. The average calculated diameter of the AML cells was 10.4 ± 2.5 μm using ultrasound, and 11.4 ± 2.3 μm using photoacoustics. The average diameter of the melanoma cells was 16.2 ± 2.9 μm using ultrasound, and 18.9 ± 3.5 μm using photoacoustics. The cell sizes calculated using ultrasound and photoacoustic methods agreed with measurements using a Coulter Counter, where the AML cells were 9.8 ± 1.8 μm and the melanoma cells were 16.0 ± 2.5 μm. These results demonstrate a high speed method of assessing cell size using sound waves, which is an alternative method to traditional flow cytometry techniques.

The destruction of blood vessels is a commonly used cancer therapeutic strategy. Bleeding consequently follows and leads to the accumulation of blood in the interstitium. Photoacoustic (PA) imaging is well positioned to detect bleeding due to its sensitivity to hemoglobin. After treatment vascular disruption can occur within just a few hours, which leads to bleeding which might be detected using PA to assess therapeutic effectiveness. Deep micro-vessels cannot typically be resolved using acoustic-resolution PA. However, spectral analysis of PA signals may still permit assessment of bleeding. This paper introduces a theoretical model to simulate the PA signals from disrupted vessels using a fractal model. The fractal model uses bifurcated-cylinder bases to represent vascular trees. Vessels have circular absorption cross-sections. To mimic bleeding from blood vessels, the diffusion of hemoglobin from micro-vessels was simulated. The PA signals were computed and in the simulations were detected using a linear array transducer (30 MHz center frequency) for four different vascular trees (at 256 axial spatial locations/tree). The Fourier Transform of each beam-formed PA signal was computed and the power spectra were fitted to a straight line within the -6 dB bandwidth of the receiving transducer. When comparing the power spectra before and after simulated bleeding, the spectral slope and mid-band fit (MBF) parameters decreased by 0.12 dB/MHz and 2.12 dB, while the y-intercept did not change after 1 hour of simulated bleeding. The results suggest that spectral PA analysis is sensitive to changes in the concentration and spatial distribution of hemoglobin in tissue, and changes due to bleeding can be detected without the need to resolve individual vessels. The simulations support the applicability of PA imaging in cancer treatment monitoring by detecting micro-vessel disruption.

Ultrasound flow imaging is widely used for quantification of blood flow in vivo, but estimating low flow velocities remains challenging. Pulsed photoacoustic flowmetry could be an alternative – but has not been shown capable of deep in vivo imaging so far. A new photoacoustic system is proposed, that has more potential for deep in-vivo applications. In this work the system is tested in vitro. In contrast to earlier research, 1064 nm NIR laser irradiation is used, that would allow deeper in-vivo light penetration. For detection, a 15 MHz transducer with lower in-vivo tissue ultrasound attenuation is used. Both changes are not trivial, as they reduce the overall visibility of the photoacoustic signals from red blood cells. This work shows the flow estimation performance of this system in vitro, and aims to serve as a point of reference when moving to an in-vivo application. Using this 15 MHz transducer, 1D flow profiles of flowing blood were extracted. The jitter (standard error) in the velocity estimation over the profiles was 10% when estimating flow of particles and 20% with whole blood; the bias in flow estimation was roughly 30%.

Microcirculation may be characterized by the vascular pressure as it is influenced by pressure-driven perfusion. Crosssections of blood vessels can be visualized by photoacoustic imaging and compressing on vessels causes deformation. The photoacoustic signals of blood, when compressed to the point of vessel collapse, may or may not vanish depending on the buckling process it undergoes. We form relative pressure images of microvessels by tracking vessel collapse as a function of externally applied pressure using photoacoustic imaging.

In this study we show that the spectral distortion of OA signals, caused by wavelength-dependent optical attenuation inside the bulk tissue, can be corrected based on OA imaging, when using multiple-irradiation sensing. The tissue is modeled as a strongly scattering background, in which a discrete number of blood vessels, characterized by a higher absorption than the background, are sparsely distributed. OA signals generated by these vessels, which serve as intrinsic “fluence detectors”, are recorded as a function of irradiation position. In order to account for realistic situations, we have developed a semi-empirical light diffusion model that is fitted to the recorded signals, so as to determine the background’s optical effective attenuation coefficient for arbitrarily shaped tissues. The experimental validation of this model was performed on tissue-mimicking phantoms. The results demonstrate a successful correction of the measured OA spectrum of the embedded vessel-like inclusions, in the presence of lateral geometrical boundaries and when vessel-like absorbing structures influence the light propagation.

The ability to evaluate tumor oxygenation in the clinic could indicate prognosis and enable treatment monitoring, since oxygen deficient cancer cells are more resistant to chemotherapy and radiotherapy. MultiSpectral Optoacoustic Tomography (MSOT) is a hybrid technique combining the high contrast of optical imaging with the spatial resolution and penetration depth similar to ultrasound. We aim to demonstrate that MSOT can be used to monitor the development of tumor vasculature.

To establish the relationship between MSOT derived imaging biomarkers and biological changes during tumor development, we performed MSOT on nude mice (n=10) bearing subcutaneous xenograft U87 glioblastoma tumors using a small animal optoacoustic tomography system. The mice were maintained under inhalation anesthesia during imaging and respired oxygen content was modified between 21% and 100%. The measurements from early (week 4) and late (week 7) stages of tumor development were compared. To further explore the functionality of the blood vessels, we examined the evolution of changes in the abundance of oxy- and deoxyhemoglobin in the tumors in response to a gas challenge. We found that the kinetics of the change in oxygen saturation (SO₂) were significantly different between small tumors and the healthy blood vessels in nearby normal tissue (p=0.0054). Furthermore, we showed that there was a significant difference in the kinetics of the gas challenge between small and large tumors (p=0.0015). We also found that the tumor SO₂ was significantly correlated (p=0.0057) with the tumor necrotic fraction as assessed by H&E staining in histology. In the future, this approach may be of use in the clinic as a method for tumor staging and assessment of treatment response.

Photoacoustic tomography (PAT) is an imaging technique combining strong contrast of optical imaging to high spatial resolution of ultrasound imaging. These strengths are achieved via photoacoustic effect, where a spatial absorption of light pulse is converted into a measurable propagating ultrasound wave. The method is seen as a potential tool for small animal imaging, pre-clinical investigations, study of blood vessels and vasculature, as well as for cancer imaging.

The goal in PAT is to form an image of the absorbed optical energy density field via acoustic inverse problem approaches from the measured ultrasound data. Quantitative PAT (QPAT) proceeds from these images and forms quantitative estimates of the optical properties of the target. This optical inverse problem of QPAT is illposed. To alleviate the issue, spectral QPAT (SQPAT) utilizes PAT data formed at multiple optical wavelengths simultaneously with optical parameter models of tissue to form quantitative estimates of the parameters of interest.

In this work, the inverse problem of SQPAT is investigated. Light propagation is modelled using the diffusion equation. Optical absorption is described with chromophore concentration weighted sum of known chromophore absorption spectra. Scattering is described by Mie scattering theory with an exponential power law. In the inverse problem, the spatially varying unknown parameters of interest are the chromophore concentrations, the Mie scattering parameters (power law factor and the exponent), and Gruneisen parameter. The inverse problem is approached with a Bayesian method. It is numerically demonstrated, that estimation of all parameters of interest is possible with the approach.

Molecular imaging for breast cancer detection, infectious disease diagnostics and preclinical animal research may be achievable through combined use of targeted exogenous agents – such as nanoparticles – and spectral Photoacoustic Tomography (PAT). However, tissue heterogeneity can alter fluence distributions and acoustic propagation, corrupting measured PAT absorption spectra and complicating in vivo nanoparticle detection and quantitation. Highly absorptive vascular structures represent a common confounding factor, and variations in vessel hemoglobin saturation (SO2) may alter spectral content of signals from adjacent/deeper regions. To evaluate the impact of this effect on PAT nanoparticle detectability, we constructed heterogeneous phantoms with well-characterized channel-inclusion geometries and biologically relevant optical and acoustic properties. Phantoms contained an array of tubes at several depths filled with hemoglobin solutions doped with varying concentrations of gold nanorods with an absorption peak at 780 nm. Both overlying and target network SO2 was tuned using sodium dithionite. Phantoms were imaged from 700 to 900 nm using a custom PAT system comprised of a tunable pulsed laser and a research-grade ultrasound system. Recovered nanoparticle spectra were analyzed and compared with results from both spectrophotometry and PAT data from waterimmersed tubes containing blood and nanoparticle solutions. Results suggested that nanoparticle selection for a given PAT application should take into account expected oxygenation states of both target blood vessel and background tissue oxygenation to achieve optimal performance.

In this study, we present some examples of waveform engineering applications in frequency-domain photoacoustics (PA). An example of using linear frequency modulation (LFM) for PA spectroscopy is the capability of simultaneous probing/imaging with multiple wavelengths. Use of mismatched coded waveforms enables encoding the signal sources and, therefore, facilitates simultaneous probing and imaging. This method enables high frame rate functional imaging with reduced motion artifacts. Furthermore, it is shown than that phase of the PA cross-correlation induced with a LFM can yield the absolute absorption coefficient of the chromophores. This method is not affected by fluence attenuation or variation due to the absorption and scattering of the overlayer material.

Non-alcoholic fatty liver disease (NAFLD) is a common liver disease affecting 30% of the population in the United States. Biopsy is the gold standard for diagnosing NAFLD. Liver histology assesses the amount of fat, and determines type and extent of cell injury, inflammation and fibrosis. However, liver biopsy is invasive and is limited by sampling error. Current radiological diagnostic modalities can evaluate the 'physical' morphology in liver by quantifying the backscattered US signals, but cannot interrogate the 'histochemical' components forming these backscatterers. For example, ultrasound (US) imaging can detect the presence of fat but cannot differentiate steatosis alone from steatohepatitis. Our previous study of photoacoustic physiochemical analysis (PAPCA) has demonstrated that this method can characterize the histological changes in livers during the progression of NAFLD in animal models. In this study, we will further validate PAPCA with human livers. Ex vivo human liver samples with steatosis, fibrosis and cirrhosis will be scanned using optical illumination at wavelengths of 680-1700 nm and compared to histology results. In vivo study on human subjects with confirmed steatosis is planned using our PA-ultrasound (US) parallel imaging system based on Verasonic US imaging flatform with an L7-4 probe. 10 mJ/cm² per pulse optical energy at 755 nm will be delivered to the skin surface, which is under the safety limit of American National Standard Institute. Preliminary study with ex vivo human tissue has demonstrated the potential of the proposed approach in differentiating human liver conditions.

The Methylene Blue loaded Polyacrylamide Nanoparticles (MB-PAA NPs) are used for oxygen sensing and Photodynamic therapy (PDT), a promising therapeutic modality employed for various tumors, with distinct advantages of delivery of biomedical agents and protection from other bio-molecules overcoming inherent limitations of molecular dyes. Lifetime-resolved photoacoustic spectroscopy using quenched-phosphorescence method is applied with MB-PAA NPs so as to sense oxygen, while the same light source is used for PDT. The dye is excited by absorbing 650 nm wavelength light from a pump laser to reach triplet state. The probe laser at 810 nm wavelength is used to excite the first triplet state at certain delayed time to measure the dye lifetime which indicates oxygen concentration. The 9L cells (106 cells/ml) incubated with MB-PAA NP solution are used for monitoring oxygen level change during PDT in situ test. The oxygen level and PDT efficacy are confirmed with a commercial oximeter, and fluorescence microscope imaging and flow cytometry results. This technique with the MB-PAA NPs allowed us to demonstrate a potential non-invasive theragnostic operation, by monitoring oxygen depletion during PDT in situ, without the addition of secondary probes. Here, we demonstrate this theragnostic operation, in vitro, performing PDT while monitoring oxygen depletion. We also show the correlation between O2 depletion and cell death.

This paper attempts to experimentally and analytically quantify the aggregation-induced changes in the photoacoustic amplitude (PAA) by simultaneously examining the effect of red blood cell (RBC) aggregate size and optical illumination wavelength. In experiments, the pulsatile flow of human whole blood at 60 bpm was imaged using the VevoLAZR system equipped with a 40-MHz-linear-array probe. The samples were illuminated every 10 nm from 700 to 900 nm. For the analytical model, the PAA from both a collection of randomly distributed RBCs of 5, 10, 15, 20, 25, and 30 cells and a single absorber as a spherical aggregate of RBCs formed by the corresponding number of RBCs. The oxygen saturation (sO2) was measured as 74% and 80% for the non-aggregated RBCs and the RBC aggregation. These values were assigned to the analytical RBC aggregates containing between 5 and 30 cells. The normalized PAA (nPAA) for the experimental results was compared to that generated by the theoretical calculations. At a given wavelength, the analytical nPAA for the collection of RBCs were identical for all numbers of RBCs, but that for the RBC aggregate increased with the number of RBCs forming the aggregate due to the increase in the effective photoacoustic absorber size. The experimental as well as analytical nPAA for both RBC aggregation and non-aggregation increased with the wavelength at a given absorber size. This was due to the fact that the PAA is mainly determined by the optical absorption coefficient (μa) which increases due to the relationship between εHbO and wavelength. In addition, the difference of PAA between RBC aggregation and nonaggregation also increased with the wavelength due to the increase in the μa induced by the hypothesized enhanced sO2 resulting from the increased size of RBC aggregates. These results can be used as a means of estimating the oxygen loading and unloading during blood flow. This investigation elucidates the quantitative relationship between the RBC aggregate size and the optical illumination wavelength for probing the physiology of flowing blood.

Three-dimensional (3D) in vitro models bridge the gap between typical two-dimensional cultures and in vivo conditions. However, conventional optical imaging methods such as confocal microscopy and two-photon microscopy cannot accurately depict cellular processing in 3D models due to limited penetration of photons. We developed a dualwavelength optical-resolution photoacoustic microscopy (OR-PAM), which provides sufficient penetration depth and spatial resolution, for studying CD8+ cytotoxic T lymphocytes (CTLs) trafficking in an in vitro 3D tumor microenvironment. CTLs play a cardinal role in host defense against tumor. Efficient trafficking of CTLs to the tumor microenvironment is a critical step for cancer immunotherapy. For the proposed system, gold nanospheres and indocyanine green (ICG) have been remarkable choices for contrast agents for photoacoustic signals due to their excellent biocompatibility and high optical absorption. With distinct absorption spectrums, targeted cells with gold nanospheres and ICG respectively can be identified by switching 523-nm and 800-nm laser irradiation. Moreover, we use an x–y galvanometer scanner to obtain high scanning rate. In the developed system, lateral and axial resolutions were designed at 1.6 μm and 5 μm, respectively. We successfully showed that dual-spectral OR-PAM can map either the distribution of CTLs with gold nanospheres at a visible wavelength of 523 nm or the 3D structure of tumor spheres with ICG in an in vitro 3D microenvironment. Our OR-PAM can provide better biological relevant information in cellular interaction and is potential for preclinical screening of anti-cancer drugs.

A method for concurrent photoacoustic (PA) and ultrasound (US) imaging with single laser pulses was previously demonstrated. An optical-absorbing multilayer film that can generate a US pulse based on the thermoelastic effect is used. With such a film, the generated US can be adjusted so that it does not overlap with the spectrum of the PA signal generated by the light transmitting through the layer. Thus, the US signal and the PA signal can be generated and separated by using a single laser pulse with spectral filtering. In this study, we continue with the same concurrent imaging approach and propose a cost-effective and portable design. The design consists of a pulsed laser diode with the repetition rate up to 25 kHz and energy of 2 μJ/pulse. A multilayer film is employed to generate narrow band US signals under laser excitation for US imaging. With simple spectral filtering, the PA signals and the US signals can be separated. With optical resolution, the system has a theoretical lateral resolution of 2 μm in PA imaging and 200 μm in US imaging. One of the applications of the proposed microscope is for tumor biology, where angiogenesis is an essential topic for understanding tumor growth and tumor metastasis. We will demonstrate performance of the proposed system by imaging vasculature networks.

In deep photoacoustic imaging, resolution is inherently limited by acoustic diffraction, and ultrasonic frequencies cannot be arbitrarily increased because of attenuation in tissue. Here we report on the use of multiple speckle illumination to perform super resolution photoacoustic imaging. We show that the analysis of speckle-induced second-order fluctuations of the photoacoustic signal combined with deconvolution enables to resolve optically absorbing structures below the acoustic diffraction limit.

We have designed and implemented a novel acoustic lens based focusing technology into a prototype photoacoustic imaging camera. All photoacoustically generated waves from laser exposed absorbers within a small volume get focused simultaneously by the lens onto an image plane. We use a multi-element ultrasound transducer array to capture the focused photoacoustic signals. Acoustic lens eliminates the need for expensive data acquisition hardware systems, is faster compared to electronic focusing and enables real-time image reconstruction. Using this photoacoustic imaging camera, we have imaged more than 150 several centimeter size ex-vivo human prostate, kidney and thyroid specimens with a millimeter resolution for cancer detection. In this paper, we share our lens design strategy and how we evaluate the resulting quality metrics (on and off axis point spread function, depth of field and modulation transfer function) through simulation. An advanced toolbox in MATLAB was adapted and used for simulating a two-dimensional gridded model that incorporates realistic photoacoustic signal generation and acoustic wave propagation through the lens with medium properties defined on each grid point. Two dimensional point spread functions have been generated and compared with experiments to demonstrate the utility of our design strategy. Finally we present results from work in progress on the use of two lens system aimed at further improving some of the quality metrics of our system.

Reflection artifacts caused by acoustic inhomogeneities are a main challenge to deep-tissue photoacoustic imaging. Photoacoustic transients generated by the skin surface and superficial vasculature will propagate into the tissue and reflect back from echogenic structures to generate reflection artifacts. These artifacts can cause problems in image interpretation and limit imaging depth. In its basic version, PAFUSion mimics the inward travelling wave-field from blood vessel-like PA sources by applying focused ultrasound pulses, and thus provides a way to identify reflection artifacts. In this work, we demonstrate reflection artifact correction in addition to identification, towards obtaining an artifact-free photoacoustic image. In view of clinical applications, we implemented an improved version of PAFUSion in which photoacoustic data is backpropagated to imitate the inward travelling wave-field and thus the reflection artifacts of a more arbitrary distribution of PA sources that also includes the skin melanin layer. The backpropagation is performed in a synthetic way based on the pulse-echo acquisitions after transmission on each single element of the transducer array. We present a phantom experiment and initial in vivo measurements on human volunteers where we demonstrate significant reflection artifact reduction using our technique. The results provide a direct confirmation that reflection artifacts are prominent in clinical epi-photoacoustic imaging, and that PAFUSion can reduce these artifacts significantly to improve the deep-tissue photoacoustic imaging.

We developed a new and improved Laser Optoacoustic Imaging System, LOIS-3D for preclinical research applications in small animal models. The advancements include (i) a new stabilized imaging module with a more homogeneous illumination of the mouse yielding a better spatial resolution (<0.2 mm) and (ii) a new low noise amplifier incorporated into the ultrasonic probe and providing the noise equivalent pressure around 2 Pa resulting in increased signal-to-noise ratio and the optical absorption sensitivity of about 0.15 cm-1. We also improved scan time and the image reconstruction times. This prototype has been commercialized for a number of biomedical research applications, such as imaging vascularization and measuring hemoglobin / oxyhemoglobin distribution in the organs as well as imaging exogenous or endogenous optoacoustic contrast agents. As examples, we present in vivo experiments using phantoms and mice with and without tumor injected with contrast agents with indocyanine green (ICG). LOIS-3D was capable of detecting ~1-2 pmole of the ICG, in tissues with relatively low blood content. With its high sensitivity and excellent spatial resolution LOIS-3D is an advanced alternative to fluorescence and bioluminescence based modalities for molecular imaging in live mice.

Optical imaging of brain voltage signals is significantly limited in depth due to optical scattering and the absorptive property of brain tissue. Photoacoustic (PA) imaging promises to break this hard limit by utilizing both ballistic and diffused photons. To demonstrate the feasibility of PA, we used an in vivo mouse model. The brain cortex tissue was stained with dipicrylamine dye, electrically stimulated, and imaged with a customized dual-isosbestic-wavelength PA microscope (DIW-PAM). DIW-PAM separates voltage-induced PA signals from blood-induced PA signals and thereby allows recording the voltage response of mouse cortex tissue without interference from hemoglobin responses. The resting state PA voltage response signal exhibited a noise-like signal in the frequency domain. Upon 3 Hz electrical stimulation, the PA voltage response signal showed frequency peaks of 3.2 Hz and 6.3 Hz (Fig. 1). Although dipicrylamine dye is not fast enough for recording neuron action potentials, it served well for the purpose of this feasibility study. In conclusion, we successfully demonstrated in vivo photoacoustic imaging of mouse brain voltage signals for the first time. If a fast voltage-sensitive dye is available, using photoacoustic computed tomography (PACT) instead of PA microscopy could allow acquiring full-field PA action potential images at a speed limited only by the laser pulse repetition rate.

Optoacoustic techniques rely on ultrasound transmission between optical absorbers within tissues and the measurement location. Much like in echography, commonly used piezoelectric transducers require either direct contact with the tissue or through a liquid coupling medium. The contact nature of this detection approach then represents a disadvantage of standard optoacoustic systems with respect to other imaging modalities (including optical techniques) in applications where non-contact imaging is needed, e.g. in open surgeries or when burns or other lesions are present in the skin. Herein, non-contact optoacoustic imaging using raster-scanning of a spherically-focused piezoelectric air-coupled ultrasound transducer is demonstrated. When employing laser fluence levels not exceeding the maximal permissible human exposure, it is shown possible to attain detectable signals from objects as small as 1 mm having absorption properties representative of blood at near-infrared wavelengths with a relatively low number of averages. Optoacoustic images from vessel-mimicking tubes embedded in an agar phantom are further showcased. The initial results indicate that the air-coupled ultrasound detection approach can be potentially made suitable for non-contact biomedical imaging with optoacoustics.

This paper reports the development of a new two-axis micromechanical scanning transducer for handheld 3D ultrasound imaging. It consists of a miniaturized single-element ultrasound transducer driven by a unique 2-axis liquid-immersible electromagnetic microactuator. With a mechanical scanning frequency of 19.532 Hz and an ultrasound pulse repetition rate of 5 kHz, the scanning transducer was scanned along 60 concentric paths with 256 detection points on each to simulate a physical 2D ultrasound transducer array of 60 × 256 elements. Using the scanning transducer, 3D pulse-echo ultrasound imaging of two silicon discs immersed in water as the imaging target was successfully conducted. The lateral resolution of the 3D ultrasound image was further improved with the synthetic aperture focusing technique (SAFT). The new two-axis micromechanical scanning transducer doesn’t require complex and expensive multi-channel data acquisition (DAQ) electronics. Therefore, it could provide a new approach to achieve compact and low-cost 3D ultrasound and photoacoustic imaging systems, especially for handheld operations.

This study explores wavelength-modulated differential photo-acoustic (WM-DPA) imaging for non-invasive early cancer detection via sensitive characterization of functional information such as hemoglobin oxygenation (sO₂) levels. Well-known benchmarks of tumor formation such as angiogenesis and hypoxia can be addressed this way. While most conventional photo-acoustic imaging has almost entirely employed high-power pulsed lasers, frequency-domain photo-acoustic radar (FD-PAR) has seen significant development as an alternative technique. It employs a continuous wave laser source intensity-modulated and driven by frequency-swept waveforms. WM-DPA imaging utilizes chirp modulated laser beams at two distinct wavelengths for which absorption differences between oxy- and deoxygenated hemoglobin are minimum (isosbestic point, 805 nm) and maximum (680 nm) to simultaneously generate two signals detected using a standard commercial array transducer as well as a single-element transducer that scans the sample. Signal processing is performed using Lab View and Matlab software developed in-house.

Minute changes in total hemoglobin concentration (tHb) and oxygenation levels are detectable using this method since background absorption is suppressed due to the out-of-phase modulation of the laser sources while the difference between the two signals is amplified, thus allowing pre-malignant tumors to become identifiable. By regulating the signal amplitude ratio and phase shift the system can be tuned to applications like cancer screening, sO₂ quantification and hypoxia monitoring in stroke patients. Experimental results presented demonstrate WM-DPA imaging of sheep blood phantoms in comparison to single-wavelength FD-PAR imaging. Future work includes the functional PA imaging of small animals in vivo.

An optimal genetically-encoded probe for photoacoustic (PA) imaging should exhibit high optical absorption, low fluorescence quantum yield, and an absorption maxima within the near-infrared (NIR) window. One promising candidate is a newly engineered chromoprotein (CP), designated dark small ultra-red fluorescent protein (dsmURFP), which is based on a cyanobacterial phycobiliprotein. To optimize dsmURFP characteristics for PA imaging, we have developed a directed evolution method to iteratively screen libraries of protein variants with three different screening systems. Firstly, we took inspiration from dark-acceptor (also known as dark-quencher)-based Förster resonance energy transfer (FRET) constructs, and used dsmURFP as a dark acceptor from a mCardinal fluorescent donor. The rationale for this design was that the higher the extinction coefficient of the dsmURFP, the more the emission of the donor would be quenched. In addition, more energy transferred to the dark acceptor would lead to more thermoelastic expansion and a stronger PA signal. Three rounds of evolution using this first strategy resulted in dsmURFP1.3 that quenched the emission of mCardinal ~2-fold more efficiently than dsmURFP. Secondly, an absorption-based screening based on visual inspection of plates led to identification of the variant dsmURFP1.4, which exhibited a 2-fold higher absorbance and a 5 nm red shift. Thirdly, we developed a colony-based photoacoustic screening method. To demonstrate the utility of our optimized variants, we used photoacoustic imaging to visualize dsmURFP and its variants in phantom and in vivo experiments using chicken embryo models and murine bacterial bladder infection models.

Photoacoustic (PA) imaging has been shown to provide detailed 3-D images of genetically expressed reporters, such as fluorescent proteins and tyrosinase-induced melanin. Their unambiguous detection in vivo is a vital prerequisite for molecular imaging of biological processes at a cellular and molecular level. This typically requires multiwavelength imaging and spectral unmixing techniques, which can be computationally expensive. In addition, fluorescent proteins often exhibit fluence-dependent ground state depopulation and photobleaching which can adversely affect the specificity of unmixing methods. To overcome these problems, a phytochrome-based reporter protein and a dual-wavelength excitation method have been developed to obtain reporter-specific PA contrast. Phytochromes are non-fluorescent proteins that exhibit two isomeric states with different absorption spectra. Using dual-wavelength excitation pulses in the red and near-infrared wavelength region, these states can be switched, resulting in a modulation of the total absorption coefficient, and hence the PA signal amplitude. Since this is not observed in endogenous chromophores, signals acquired using simultaneous pulses can be subtracted from the sum of signals obtained from separate pulses to provide a reporterspecific contrast mechanism and elimination of the tissue background. PA signals measured in protein solutions using separate and simultaneous excitation pulses at 670 nm and 755 nm (< 6 mJ cm^-2) showed a difference in amplitude of a factor of five. Photobleaching was not observed. To demonstrate suitability for in vivo applications, mammalian cells were transduced virally to express phytochrome, and imaged in tissue phantoms and in mice in an initial preclinical study. The results show that this method has the potential to enable deep-tissue PA reporter gene imaging with high specificity.

In fluorophores, such as exogenous dyes and genetically expressed proteins, the excited state lifetime can be modulated using pump-probe excitation at wavelengths corresponding to the absorption and fluorescence spectra. Simultaneous pump-probe pulses induce stimulated emission (SE) which, in turn, modulates the thermalized energy, and hence the photoacoustic (PA) signal amplitude. For time-delayed pulses, by contrast, SE is suppressed. Since this is not observed in endogenous chromophores, the location of the fluorophore can be determined by subtracting images acquired using simultaneous and time-delayed pump-probe excitation. This simple experimental approach exploits a fluorophorespecific contrast mechanism, and has the potential to enable deep-tissue molecular imaging at fluences below the MPE. In this study, some of the challenges to its in vivo implementation are addressed. First, the PA signal amplitude generated in fluorophores in vivo is often much smaller than that in blood. Second, tissue motion can give rise to artifacts that correspond to endogenous chromophores in the difference image. This would not allow the unambiguous detection of fluorophores. A method to suppress motion artifacts based on fast switching between simultaneous and time-delayed pump-probe excitation was developed. This enables the acquisition of PA signals using the two excitation modes with minimal time delay (20 ms), thus minimizing the effects of tissue motion. The feasibility of this method is demonstrated by visualizing a fluorophore (Atto680) in tissue phantoms, which were moved during the image acquisition to mimic tissue motion.

Imaging plays an increasingly important role in clinical management and preclinical studies of cancer. Application of optical molecular imaging technologies, in combination with highly specific contrast agent approaches, eminently contributed to understanding of functional and histological properties of tumors and anticancer therapies. Yet, optical imaging exhibits deterioration in spatial resolution and other performance metrics due to light scattering in deep living tissues. High resolution molecular imaging at the whole-organ or whole-body scale may therefore bring additional understanding of vascular networks, blood perfusion and microenvironment gradients of malignancies. In this work, we constructed a volumetric multispectral optoacoustic tomography (vMSOT) scanner for cancer imaging in preclinical models and explored its capacity for real-time 3D intravital imaging of whole breast cancer allografts in mice. Intrinsic tissue properties, such as blood oxygenation gradients, along with the distribution of externally administered liposomes carrying clinically-approved indocyanine green dye (lipo-ICG) were visualized in order to study vascularization, probe penetration and extravasation kinetics in different regions of interest within solid tumors. The use of v-MSOT along with the application of volumetric image analysis and perfusion tracking tools for studies of pathophysiological processes within microenvironment gradients of solid tumors demonstrated superior volumetric imaging system performance with sustained competitive resolution and imaging depth suitable for investigations in preclinical cancer models.

At clinically-relevant depths, the frequency content of photoacoustic signals encodes information about the size, concentration and spatial distribution of non-resolvable blood vessels. This study evaluates whether photoacoustics can detect cancer therapy-induced vascular perturbations. Photoacoustic/ultrasound (PA/US) spectral analysis was combined with functional, PA-based oxygenation and power Doppler (PD) perfusion estimates to assess treatment response. Co-registered, in-vivo US/PA/PD imaging of mice bearing breast cancer tumors was performed pre-treatment and 30m/2h/5h/24h/7d post-treatment (VevoLAZR, Fujifilm VisualSonics). Hyperthermia treatment (1h, 43C) was performed after systemic injections of doxorubicin-loaded thermosensitive liposomes (TSL, n=13) or free doxorubicin (DOX, n=11). Response was classified according to 2h, PA-based oxygenation drop and endpoint (>9d), caliper-based volume reduction. At all time-points/wavelengths (750/850nm), the spectral-slope (SS) was computed from the normalized US/PA power spectra using depth-matched reference phantoms. The percent-vascularity (PV) was estimated for the animal with the largest oxygenation-drop at 2h. TLS-treated responders decreased their PA-SS by 1.9x @750nm and 5.8x @850nm 30m post-treatment and remained constant for 24h; tumor oxygenation followed the same trend. Non-responding SS remained unchanged for 24h. The 750nm SS was 18.7x lower than 850nm suggesting the TSL is sensitive vessel oxygenation. Responder PV decreased 100% when the 30m oxygenation dropped 15% and increased 7x when the 7d oxygenation increased 20%. DOX-responders exhibited similar trends to TSL-responders although the 750nm PA-SS was 1.6x smaller and post-treatment PV was 50% higher. The US-SS remained unchanged until 7d post-treatment suggesting its sensitivity to tumor cell-death. These findings suggest that PA spectral analysis has potential in monitoring cancer treatment response.

Synthetic and genetically encoded chromo- and fluorophores have become indispensable tools for biomedical research enabling a myriad of applications in imaging modalities based on biomedical optics. The versatility offered by the optoacoustic (photoacoustic) contrast mechanism enables to detect signals from any substance absorbing light, and hence these probes can be used as optoacoustic contrast agents. While contrast versatility generally represents an advantage of optoacoustics, the strong background signal generated by light absorption in endogeneous chromophores hampers the optoacoustic capacity to detect a photo-absorbing agent of interest. Increasing the optoacoustic sensitivity is then determined by the capability to differentiate specific features of such agent. For example, multispectral optoacoustic tomography (MSOT) exploits illuminating the tissue at multiple optical wavelengths to spectrally resolve (unmix) the contribution of different chromophores. Herein, we present an alternative approach to enhance the sensitivity and specificity in the detection of optoacoustic contrast agents. This is achieved with photoswitchable probes that change optical absorption upon illumination with specific optical wavelengths. Thereby, temporally unmixed MSOT (tuMSOT) is based on photoswitching the compounds according to defined schedules to elicit specific time-varying optoacoustic signals, and then use temporal unmixing algorithms to locate the contrast agent based on their particular temporal profile. The photoswitching kinetics is further affected by light intensity, so that tuMSOT can be employed to estimate the light fluence distribution in a biological sample. The performance of the method is demonstrated herein with the reversibly switchable fluorescent protein Dronpa and its fast-switching fatigue resistant variant Dronpa-M159T.

In this study, multifunctional theranostic agents for photoacoustic (PA), ultrasound (US), fluorescent imaging, and for therapeutic drug delivery were developed and tested. These agents consisted of a shell made from a biodegradable Poly(lactide-co-glycolic acid) (PLGA) polymer, loaded with perfluorohexane (PFH) liquid and gold nanoparticles (GNPs) in the core, and lipophilic carbocyanines fluorescent dye DiD and therapeutic drug Paclitaxel (PAC) in the shell. Their multifunctional capacity was investigated in an in vitro study. The PLGA/PFH/DiD-GNPs particles were synthesized by a double emulsion technique. The average PLGA particle diameter was 560 nm, with 50 nm diameter silica-coated gold nano-spheres in the shell. MCF7 human breast cancer cells were incubated with PLGA/PFH/DiDGNPs for 24 hours. Fluorescent and PA images were recorded using a fluorescent/PA microscope using a 1000 MHz transducer and a 532 nm pulsed laser. For the particle vaporization and drug delivery test, MCF7 cells were incubated with the PLGA/PFH-GNPs-PAC or PLGA/PFH-GNPs particles for 6, 12 and 24 hours. The effects of particle vaporization and drug delivery inside the cells were examined by irradiating the cells with a laser fluence of 100 mJ/cm², and cell viability quantified using the MTT assay. The PA images of MCF7 cells containing PLGA/PFH/DiD-GNPs were spatially coincident with the fluorescent images, and confirmed particle uptake. After exposure to the PLGA/PFHGNP- PAC for 6, 12 and 24 hours, the cell survival rate was 43%, 38%, and 36% respectively compared with the control group, confirming drug delivery and release inside the cells. Upon vaporization, cell viability decreased to 20%. The particles show potential as imaging agents and drug delivery vehicles.

We propose a novel multispectral reconstruction-classification method for simultaneously recovering absorption and scattering coefficients from images of absorbed optical energy. In contrast with pre-existing chromophore reconstruction methods, this approach does not require prior knowledge of the characteristic spectra of the absorbers, which is not always available. Numerical experiments performed on anatomically realistic 3D phantoms show that this approach allows for improved recovery of both the optical absorption and scattering with respect to reconstruction-only methods, and accurate classification of chromophores of clinical interest.

Compressed sensing allows performing much fewer measurements than advised by the Shannon sampling theory. This is surprising because it requires the solution of a system of equations with much fewer equations than unknowns. This is possible if one can assume sparsity of the solution, which means that only a few components of the solution are significantly different from zero. An important ingredient for compressed sensing is the restricted isometry property (RIP) of the sensing matrix, which is satisfied for certain types of random measurement ensembles. Then a sparse solution can be found by minimizing the ℓ¹-norm. Using standard approaches, photoacoustic imaging generally neither satisfies sparsity of the data nor the RIP. Therefore, no theoretical recovery guarantees could be given. Despite ℓ¹- minimization has been used for photoacoustic image reconstruction, only marginal improvements in comparison to classical photoacoustic reconstruction have been observed. We propose the application of a sparsifying temporal transformation to the detected pressure signals, which allows obtaining theoretical recovery guarantees for our compressed sensing scheme. Such a sparsifying transform can be found because spatial and temporal evolution of the pressure wave are not independent, but connected by the wave equation. We give an example of a sparsifying transform and apply our compressed sensing scheme to reconstruct images from simulated data.

Optoacoustic Tomography is a fast developing imaging modality, combining the high resolution and penetration depth of ultrasound detection with the high contrast available from optical absorption in tissue. The spectral profile of near infrared excitation light used in optoacoustic tomography instruments is modified by absorption and scattering as it propagates deep into biological tissue. The resulting images therefore provide only qualitative insight into the distribution of tissue chromophores. Knowledge of the spectral profile of excitation light across the mouse is needed for accurate determination of the absorption coefficient in vivo. Under the conditions of constant Grueneisen parameter and accurate knowledge of the light fluence, a linear relationship should exist between the initial optoacoustic pressure amplitude and the tissue absorption coefficient. Using data from a commercial optoacoustic tomography system, we implemented an iterative optimization based on the σ-Eddington approximation to the Radiative Transfer Equation to derive a light fluence map within a given object. We segmented the images based on the positions of phantom inclusions, or mouse organs, and used known scattering coefficients for initialization. Performing the fluence correction in simple phantoms allowed the expected linear relationship between recorded and independently measured absorption coefficients to be retrieved and spectral coloring to be compensated. For in vivo data, the correction resulted in an enhancement of signal intensities in deep tissues. This improved our ability to visualize organs at depth (> 5mm). Future work will aim to perform the optimization without data normalization and explore the need for methodology that enables routine implementation for in vivo imaging.

In optoacoustic imaging, the resolution and image quality in a certain imaging position usually cannot be enhanced without changing the imaging configuration. Post-reconstruction image processing methods offer a new possibility to improve image quality and resolution. We have developed a geometrical super-resolution (GSR) method which uses information from spatially separated frames to enhance resolution and contrast in optoacoustic images. The proposed method acquires several low resolution images from the same object located at different positions inside the imaging plane. Thereafter, it applies an iterative registration algorithm to integrate the information in the acquired set of images to generate a single high resolution image. Herein, we present the method and evaluate its performance in simulation and phantom experiments, and results show that geometrical super-resolution techniques can be a promising alternative to enhance resolution in optoacoustic imaging.

Photoacoustic offers promising perspectives in probing and imaging subsurface optically absorbing structures in biological tissues. The optical uence absorbed is partly dissipated into heat accompanied with microdilatations that generate acoustic pressure waves, the intensity which is related to the amount of fluuence absorbed. Hence the photoacoustic signal measured offers access, at least potentially, to a local monitoring of the absorption coefficient, in 3D if tomographic measurements are considered. However, due to both the diffusing and absorbing nature of the surrounding tissues, the major part of the uence is deposited locally at the periphery of the tissue, generating an intense acoustic pressure wave that may hide relevant photoacoustic signals. Experimental strategies have been developed in order to measure exclusively the photoacoustic waves generated by the structure of interest (orthogonal illumination and detection). Temporal or more sophisticated filters (wavelets) can also be applied. However, the measurement of this primary acoustic wave carries a lot of information about the acoustically inhomogeneous nature of the medium. We propose a protocol that includes the processing of this primary intense acoustic wave, leading to the quantification of the surrounding medium sound speed, and, if appropriate to an acoustical parametric image of the heterogeneities. This information is then included as prior knowledge in the photoacoustic reconstruction scheme to improve the localization and quantification.

Photoacoustic tomography (PAT) exploits optical contrast and ultrasonic detection principles to form images of absorbed optical energy density within tissue. Based on the photoacoustic effect, PAT directly and quantitatively measures specific optical absorption. A full-ring ultrasonic transducer array based photoacoustic computed tomography (PACT) system was recently developed for small animal whole-body imaging with a full-view detection angle and high in-plane resolution (100 µm). However, due to the band-pass frequency response of the piezoelectric transducer elements, the reconstructed images present bipolar (both positive and negative) pixel values, which is artificial and counterintuitive for physicians and biologists seeking to interpret the image. Moreover, bipolar pixel values hinder quantification of physiological parameters, such as oxygen saturation and blood flow speed. Unipolar images can be obtained by deconvolving the raw channel data with the transducer’s electrical impulse response and applying non-negativity during iteration, but this process requires complex transducer modeling and time-consuming computation. Here, we present a multi-view Hilbert transformation method to recover the unipolar initial pressure for full-ring PACT. Multi-view Hilbert transformation along the acoustic wave propagation direction minimizes reconstruction artifacts during envelope extraction and maintains the signal-to-noise ratio of the reconstructed images. The in-plane isotropic spatial resolution of this method was quantified to 168 μm within a 20 × 20 mm2 field of view. The effectiveness of the proposed algorithm was first validated by numerical simulations and then demonstrated with ex-vivo mouse brain structural imaging and in-vivo mouse wholebody imaging.

We define a deconvolution based photoacoustic reconstruction with sparsity regularization (DPARS) algorithm for image restoration from projections. The proposed method is capable of visualizing tissue in the presence of constraints such as the specific directivity of sensors and limited-view Photoacoustic Tomography (PAT). The directivity effect means that our algorithm treats the optically-generated ultrasonic waves based on which direction they arrive at the transducer. Most PA image reconstruction methods assume that sensors have omni-directional response; however, in practice, the sensors show higher sensitivity to the ultrasonic waves coming from one specific direction. In DPARS, the sensitivity of the transducer to incoming waves from different directions are considered. Thus, the DPARS algorithm takes into account the relative location of the absorbers with respect to the transducers, and generates a linear system of equations to solve for the distribution of absorbers. The numerical conditioning and computing times are improved by the use of a sparse Discrete Fourier Transform (DCT) representation of the distribution of absorption coefficients. Our simulation results show that DPARS outperforms the conventional Delay-and-Sum reconstruction method in terms of CNR and RMS errors. Experimental results confirm that DPARS provides images with higher resolution than DAS.

Photoacoustic (or optoacoustic) microscopy has great potential as a diagnostic tool in biomedical research. For in vivo imaging, an important requirement is to keep the measurement time as short as possible. In light-sheet photoacoustic microscopy (LIS-PAM) a cylindrical lens illuminates a thin section perpendicular to the sample surface with a short laser pulse and a projection of the excited acoustic wave pattern leaving the sample is recorded with a camera. From the recorded data, a B-scan photoacoustic image is obtained by applying a two-dimensional reconstruction algorithm, without requiring any mechanical scanning. Hence, LIS-PAM is capable of real-time B-scan imaging with acoustical resolution within the individual B-scans and optical out of plane resolution up to a depth limited by optical diffusion. A 3D image is composed of reconstructed B-scan images recorded while scanning the excitation line along the sample surface. Using a camera with 200 Hz frame rate a C-scan image (5x5 mm² field of view) can be recorded in less than 5 seconds (without averaging). The achievable sensitivity and resolution of the optical phase contrast detection system were estimated theoretically with 0.34 kPa mm without averaging and 30 μm, respectively. A first experiment on a phantom that mimics tissue properties shows the applicability of this technique for in-vivo imaging.

We introduce a novel multi-scale photoacoustic remote sensing (PARS) imaging system. Our system can provide optical resolution details for superficial structures as well as acoustic resolution for deep-tissue imaging down to 5 cm, in a non-contact setting. PARS system does not require any contact with the sample or ultrasound coupling medium. The optical resolution PARS (OR-OARS) system uses optically focused pulsed excitation with optical detection of photoacoustic signatures using a long-coherence interrogation beam co-focused and co-scanned with the excitation spot. In the OR-PARS initial pressures are sampled right at their subsurface origin where acoustic pressures are largest. The Acoustic resolution PARS (AR-PARS) picks up the surface oscillation of the tissue caused by generated photoacoustic signal using a modified version of Michelson interferometry. By taking advantage of 4-meters polarization maintaining single-mode fiber and a green fiber laser we have generated a multi-wavelength source using stimulated Raman scattering. Remote functional imaging using this multi-wavelength excitation source and PARS detection mechanism has been demonstrated. The oxygen saturation estimations are shown for both phantom and in vivo studies. Images of blood vessel structures for an In vivo chicken embryo model is demonstrated. The Phantom studies indicates ~3µm and ~300µm lateral resolution for OR-PARS and AR-PARS respectively. To the best of our knowledge this is the first dual modality non-contact optical and acoustic resolution system used for in vivo imaging.

An acoustic/photoacoustic microscope was used to create micrometer resolution images of stained cells from a blood smear. Pulse echo ultrasound images were made using a 1000 MHz transducer with 1 μm resolution. Photoacoustic images were made using a fiber coupled 532 nm laser, where energy losses through stimulated Raman scattering enabled output wavelengths from 532 nm to 620 nm. The laser was focused onto the sample using a 20x objective, and the laser spot co-aligned with the 1000 MHz transducer opposite the laser. The blood smear was stained with Wright-Giemsa, a common metachromatic dye that differentially stains the cellular components for visual identification. A neutrophil, lymphocyte and a monocyte were imaged using acoustic and photoacoustic microscopy at two different wavelengths, 532 nm and 600 nm. Unique features in each imaging modality enabled identification of the different cell types. This imaging method provides a new way of imaging stained leukocytes, with applications towards identifying and differentiating cell types, and detecting disease at the single cell level.

Using a water-immersion optical objective in conjunction with a miniature 40-MHz ultrasonic transducer, we developed reflection-mode photoacoustic microscopy with a transverse resolution as high as 320 nm. Here, we further integrated two-photon microscopy capability into the system to enable multimodality in vivo biomedical imaging at submicron resolution. As a result, the system is capable of tri-modality label-free imaging of microvasculature, collagen, and cell morphology, based on the contrast of optical absorption, second-harmonic generation, and autofluorescence, respectively. In addition, we demonstrated simultaneous microscopic imaging of neuron and microvasculature in the brain cortex of a living mouse, which may offer new opportunities for studying the mechanisms of neurovascular coupling.

We recently introduced photoacoustic remote sensing (PARS) microscopy as an all-optical non-contact optical-resolution modality with absorption-based photoacoustic contrast. A pulsed excitation beam is optically focused into a sample then the resulting photoacoustic signal is sensed using a confocal long-coherence probe beam right at the source of the large pressures generated. Several mechanisms are proposed to explain the source of these large signals, including surface-displacements, local refractive-index step-modulation, scatterer displacements, and photothermal mechanisms. We carefully model each of these mechanisms and predict the fraction of modulated light from each. Experimental measurements detect ~0.1% of the incident interrogation light is modulated and this is confirmed with theoretical modulation calculations. We provide experimental evidence that pressure-induced refractive-index step modulation and scatter position modulation may be highly significant modulation mechanisms. We also model theoretical limitations of signal-to-noise and discuss future system optimization opportunities.

Photoacoustic imaging (PAI) is an emerging hybrid imaging modality that can provide a strong optical absorption contrast using the photoacoustic (PA) effect, and breaks through the fundamental imaging depth limit of existing optical microscopy such as optical coherence tomography (OCT), confocal or two-photon microscopy. In PAI, a short-pulsed laser is illuminated to the tissue, and the PA waves are generated by thermoelastic expansion. Despite the high lateral resolution of optical-resolution photoacoustic microscopy (OR-PAM) thanks to the tight optical focus, the lateral resolution of OR-PAM is limited to the optical diffraction limit, which is approximately a half of the excitation wavelength. Here, we demonstrate a new super-resolution photoacoustic microscopy (SR-PAM) system by breaking the optical diffraction limit. The conventional microscopes with nanoscale resolutions such as a scanning electron microscope (SEM) and transmission electron microscope (TEM) are typically used to image the structures of nanomaterials, but these systems should work in a high vacuum environment and cannot provide the optical properties of the materials. Our newly developed SR-PAM system provides the optical properties with a nanoscale resolution in a normal atmosphere. We have photoacoustically imaged single gold nanoparticles with an average size of 80 nm in diameter and shown their PA expansion properties individually. The lateral resolution of this system was approximately 20 nm. Therefore, this tool will provide an unprecedented optical absorption property with an accurate nanoscale resolution and greatly impact on materials science and nanotechnology field.

We present simultaneous photoacoustic and fluorescence microscopy using a modulated laser diode. Photoacoustic waves and modulated fluorescence are generated by using a laser diode with a wavelength of 405nm at modulation frequencies in the Megahertz range. Additionally, a continuous wave offset radiation can be superposed. The excitation light is focused to the sample using a high NA objective. Luminescence is collected by the same objective in a confocal configuration and measured by an avalanche photo diode. Photoacoustic waves are recorded on the opposite site of the sample using a hydrophone. Acoustic and luminescence signals are recorded using a lock-in technique. We present photoacoustic imaging on red blood cells and multimodal imaging on fluorescent microspheres. Nonlinear photoacoustic and luminescence effects were introduced with the help of a superposed continuous wave laser.

The planar Fabry Perot (FP) photoacoustic scanner has been shown to provide exquisite high resolution 3D images of soft tissue structures in vivo to depths up to approximately 10mm. However a significant limitation of current embodiments of the concept is low image acquisition speed. To increase acquisition speed, a novel multi-beam scanner architecture has been developed. This enables a line of equally spaced 8 interrogation beams to be scanned simultaneously across the FP sensor and the photoacoustic signals detected in parallel. In addition, an excitation laser operating at 200Hz was used. The combination of parallelising the detection and the high pulse repetition frequency (PRF) of the excitation laser has enabled dramatic reductions in image acquisition time to be achieved. A 3D image can now be acquired in 10 seconds and 2D images at video rates are now possible.

Most photoacoustic scanners use piezoelectric detectors but these have two key limitations. Firstly, they are optically opaque, inhibiting backward mode operation. Secondly, it is difficult to achieve adequate detection sensitivity with the small element sizes needed to provide near-omnidirectional response as required for tomographic imaging. Planar Fabry-Perot (FP) ultrasound sensing etalons can overcome both of these limitations and have proved extremely effective for superficial (<1cm) imaging applications. To achieve small element sizes (<100μm), the etalon is illuminated with a focused laser beam. However, this has the disadvantage that beam walk-off due to the divergence of the beam fundamentally limits the etalon finesse and thus sensitivity - in essence, the problem is one of insufficient optical confinement. To overcome this, novel planoconcave micro-resonator sensors have been fabricated using precision ink-jet printed polymer domes with curvatures matching that of the laser wavefront. By providing near-perfect beam confinement, we show that it is possible to approach the maximum theoretical limit for finesse (f) imposed by the etalon mirror reflectivities (e.g. f=400 for R=99.2% in contrast to a typical planar sensor value of f<50). This yields an order of magnitude increase in sensitivity over a planar FP sensor with the same acoustic bandwidth. Furthermore by eliminating beam walk-off, viable sensors can be made with significantly greater thickness than planar FP sensors. This provides an additional sensitivity gain for deep tissue imaging applications such as breast imaging where detection bandwidths in the low MHz can be tolerated. For example, for a 250 μm thick planoconcave sensor with a -3dB bandwidth of 5MHz, the measured NEP was 4 Pa. This NEP is comparable to that provided by mm scale piezoelectric detectors used for breast imaging applications but with more uniform frequency response characteristics and an order-of-magnitude smaller element size. Following previous proof-of-concept work, several important advances towards practical application have been made. A family of sensors with bandwidths ranging from 3MHz to 20MHz have been fabricated and characterised. A novel interrogation scheme based on rapid wavelength sweeping has been implemented in order to avoid previously encountered instability problems due to self-heating. Finally, a prototype microresonator based photoacoustic scanner has been developed and applied to the problem of deep-tissue (>1cm) photoacoustic imaging in vivo. Imaging results for second generation microresonator sensors (with R = 99.5% and thickness up to ~800um) are compared to the best achievable with the planar FP sensors and piezoelectric receivers.

Fabry-Pérot (FP) sensors have been used to produce in-vivo photoacoustic images of exquisite quality. However, for simplicity of construction FP sensors are produced in a planar form. Planar sensors suffer from a limited detection aperture, due to their planarity. We present a novel sensor geometry that allowed a greater field of view by placing a second sensor orthogonal to the first. This captured data from the deeper lying regions of interest and mitigated the limited view.

Reconstructing images from measured time domain signals is an essential step in tomography-mode photoacoustic imaging. However, in practice, there are many complicating factors that make it difficult to obtain high-resolution images. These include incomplete or undersampled data, filtering effects, acoustic and optical attenuation, and uncertainties in the material parameters. Here, the processing and image reconstruction steps routinely used by the Photoacoustic Imaging Group at University College London are discussed. These include correction for acoustic and optical attenuation, spatial resampling, material parameter selection, image reconstruction, and log compression. The effect of each of these steps is demonstrated using a representative in vivo dataset. All of the algorithms discussed form part of the open-source k-Wave toolbox (available from http://www.k-wave.org).

Optical-resolution photoacoustic microscopy (OR-PAM) can achieve submicron lateral resolution by tightly focusing the excitation light, while the axial resolution is still limited by the frequency bandwidth of the ultrasonic transducer. The Grueneisen relaxation effect, in which the Grueneisen parameter changes within the thermal relaxation time following a laser impulse heating, can provide excellent axial resolution due to its optical sectioning property. Based on this effect, Grueneisen relaxation photoacoustic microscopy (GR-PAM) was developed and demonstrated ex vivo. Here, we present for the first time in vivo imaging of mouse brains with improved axial resolution based on GR-PAM. An intensity-modulated continuous-wave (CW) 532 nm laser thermally heated the in-focus absorber. Another 532 nm pulsed laser, which is aligned confocally with the CW laser, generated the photoacoustic (PA) signal from the absorber. The difference between the amplitudes of the photoacoustic signals with and without heating was used for image reconstruction. The achieved axial resolution is ~12.5 µm, which is fivefold better than the acoustically determined value for a 20 MHz-bandwidth ultrasound transducer. The system was demonstrated by imaging a blood-filled tube ex vivo and blood vessels of mouse brains in vivo. The blood-filled tube diameter obtained from the PA image by GR-PAM is 105 µm, which is much closer to its actual diameter (100 µm) than the value from conventional OR-PAM (160 µm). This axial resolution improvement was further validated in imaging mouse brains in vivo, and yielded significantly narrower axial profiles of the vessels. This in vivo demonstration of imaging by GR-PAM might inspire more applications in PA biomedical imaging and sensing.

A miniature (175 μm) all-optical photoacoustic probe has been developed for minimally invasive sensing and imaging applications. The probe comprises a single optical fibre which delivers the excitation light and a broadband 50 MHz Fabry-Pérot (F-P) ultrasound sensor at the distal end for detecting the photoacoustic waves. A graded index lens proximal to the F-P sensor is used to reduce beam walk-off and thus increase sensitivity as well as confine the excitation beam in order to increase lateral spatial resolution. The probe was evaluated in non-scattering media and found to provide lateral and axial resolutions of < 100 μm and < 150 μm respectively for distances up to 1 cm from the tip of the probe. The ability of the probe to detect a blood vessel mimicking phantom at distances up to 7 mm from the tip was demonstrated in order to illustrate its potential suitability for needle guidance applications.

The melanin in the retinal pigment epithelium (RPE) protects retina and other ocular tissues by photo-screening and acting as antioxidant and free radical scavenger. It helps maintain normal visual functions since human eye is subjected to lifelong high oxygen stress and photon exposure. Loss of the RPE melanin weakens the protection mechanism and jeopardizes ocular health. Local decrease in the RPE melanin concentration is believed to be both a cause and a sign of early-stage age-related macular degeneration (AMD), the leading blinding disease in developed world. Current technology cannot quantitatively measure the RPE melanin concentration which might be a promising marker in early AMD screening. Photoacoustic ophthalmoscopy (PAOM), as an emerging optical absorption-based imaging technology, can potentially be applied to measure the RPE melanin concentration if the dependence of the detectable photoacoustic (PA) signal amplitudes on the RPE melanin concentrations is verified. In this study, we tested the feasibility of using PA signal ratio from RPE melanin and the nearby retinal blood vessels as an indicator of the RPE melanin variation. A novel whole eye optical model was designed and Monte Carlo modeling of light (MCML) was employed. We examined the influences on quantification from PAOM axial resolution, the depth and diameter of the retinal blood vessel, and the RPE thickness. The results show that the scheme is robust to individual histological and illumination variations. This study suggests that PAOM is capable of quantitatively measuring the RPE melanin concentration in vivo.

Acute cardiovascular events are mostly due to a blood clot or thrombus induced by the sudden rupture of vulnerable atherosclerotic plaques within coronary artery walls. Based on the high optical absorption contrast of the lipid rich plaques within the vessel wall, intravascular photoacoustic (IVPA) imaging at 1.7 μm spectral band has shown promising capabilities for detecting of lipid composition, but the translation of the technology for in vivo application is limited by the slow imaging speed. In this work, we will present a high speed integrated IVPA/US imaging system with a 500 Hz optical parametric oscillator laser at 1725 nm (5 nm linewidth). A miniature catheter with 1.0 mm outer diameter was designed with a polished 200 μm multimode fiber and an ultrasound transducer with 45 MHz center frequency. Two optical illumination methods by gradient-index (GRIN) lens and ball lens are introduced and compared for higher spatial resolution. At 1725 nm, atherosclerotic rabbit abdominal aorta was imaged at two frame per second, which is more than one order of magnitude faster than previous reported IVPA imaging. Furthermore, by wide tuning range of the laser wavelength from 1680 nm to 1770 nm, spectroscopic photoacoustic analysis of lipid-mimicking phantom and an human atherosclerotic artery was performed ex vivo.

Optical imaging of genetically encoded probes has revolutionized biomedical studies by providing valuable information about targeted biological processes. Here, we report a novel imaging technique, termed reversibly switchable photoacoustic tomography (RS-PAT), which exhibits large penetration depth, high detection sensitivity, and super-resolution. RS-PAT combines advanced photoacoustic imaging techniques with, for the first time, a nonfluorescent photoswitchable bacterial phytochrome. This bacterial phytochrome is the most near-infrared shifted genetically encoded probe reported so far. Moreover, this bacterial phytochrome is reversibly photoconvertible between its far-red and near-infrared light absorption states. Taking maximum advantage of the powerful imaging capability of PAT and the unique photochemical properties of the phytochrome, RS-PAT has broken through both the optical diffusion limit for deep-tissue imaging and the optical diffraction limit for super-resolution photoacoustic microscopy. Specifically, with RS-PAT we have achieved an unprecedented detection sensitivity of ~2 μM, or as few as ~20 tumor cells, at a centimeter depth. Such high sensitivity is fully demonstrated in our study by monitoring tumor growth and metastasis at whole-body level with ~100 μm resolution. Moreover, our microscopic implementation of RS-PAT is capable of imaging mammalian cells with a sub-diffraction lateral resolution of ~140 nm and axial resolution of ~400 nm, which are respectively ~2-fold and ~75-fold finer than those of our conventional photoacoustic microscopy. Overall, RS-PAT is a new and promising imaging technology for studying biological processes at different length scales.

Neural scientists can benefit greatly from imaging tools that can penetrate thick brain tissue. Compared with traditional optical microscopy methods, photoacoustic imaging can beat the optical diffusion limit and achieve such deep tissue imaging with high spatial resolution. In this study, we used an optical-resolution photoacoustic microscope to image the odor-evoked neuronal activities in a drosophila model. Drosophila brain neurons stably express GCaMP5G, a calcium-sensitive fluorescent protein whose optical absorption coefficient changes with calcium influx during action potentials. We recorded an ~20% odor-evoked fractional photoacoustic signal increase at all depths of the drosophila brain in vivo, with and without removal of the brain cuticle, at a recording rate of 1 kHz. Our results were confirmed by concurrent fluorescent recordings. Furthermore, by performing fast 2D scanning, we imaged the antenna lobe region, which is of particular interest in neuroscience, at a volumetric rate of ~1 Hz with a sub-neuron resolution of 3 m. Unlike optical imaging, which requires surgical removal of the scattering brain cuticle, our photoacoustic system can image through the cuticle and measure neuronal signals of the whole drosophila brain without invasive surgery, enabling minimal disturbance to the animal’s behaviors. In conclusion, we have demonstrated photoacoustic imaging of calcium signals in drosophila brains for the first time. Utilizing the deep imaging capability of photoacoustic tomography, our methods could potentially be extended to in vivo imaging of neuronal activities from deep brains in other animal models.

The bandwidth limitation and aperture size of the transducer limits the resolution of a photoacoustic computed tomography system. If the separation between two sources is smaller than the point spread function width of the imaging system, they will appear as a single object at different wavelengths. It was shown previously in ultrasound motion imaging that phase difference between two consecutive frames can be used to detect lateral or axial motion with submicron resolution. We tested this method in the context of static PA imaging of two unresolved PA sources. We set up an experiment where we imaged a green and a yellow wire of 40 μm width with known relative absorption coefficients, separated by 355μm. Imaging was performed at 650nm and 460nm. The PA signal is recorded by a single element flat 1MHz transducer (Panametrics 0.5’’ V303) in the plane of the wires, so the targets are axially spaced seen from the transducer. We reconstructed the signals originating from both unresolved sources and measured the separation between them to be 350 µm. Similar performance was obtained using an array transducer, viewing the wires from the top so they were laterally separated in the imaging plane. The signal at two different wavelengths was recorded using a commercial imaging system. The two-wavelength phase difference at every pair of channels provides an estimate of the distance between the two absorbers, determined to be 350 µm by the median of the two-channel estimates.

In photoacoustic imaging, the angular reception performance of ultrasonic transducers is a critical parameter to be considered for system designers. The quantitative comparison between cMUT and PZT emphasizes the difference between the transducer requirements and specifications between conventional ultrasound and photoacoustic imaging. In this present work, we show significant benefits of cMUT based array transducers over conventional PZT arrays for the improvement of quality in photoacoustic imaging systems.

A method based on photoacoustic analysis is proposed to diagnose dental pulp vitality. Photoacoustic analysis enables to get signal from deeper tissues than other optical analyses and therefore, signal detection from root canal of thick dental tissues such as molar teeth is expected. As a light source for excitation of photoacoustic waves, a microchip Q-switched YAG laser with a wavelength of 1064 nm was used and owing to large penetration depth of the near infrared laser, photoacoustic signals from dental root were successfully obtained. It was found that the photoacoustic signals from the teeth containing hemoglobin solution in the pulp cavity provide vibration in high frequency region. It was also shown that the intensities of the high frequency component have correlation with the hemoglobin concentration of solution. We applied short-time Fourier transform for evaluation of photoacoustic signals and this analysis clearly showed photoacoustic signals from dental root.

Multispectral photoacoustic (MS-PA) imaging has been researched to image molecular probes in the presence of strong background signals produced from intrinsic optical absorbers. Spectral fitting method (SFM) discriminates probe signals from background signals by fitting the PA spectra that are calculated from MS-PA images to reference spectra of the probe and background, respectively. Because hemoglobin is a dominant optical absorber in visible to near-infrared wavelength range, absorption spectra of hemoglobin have been widely used as reference background spectra. However, the spectra of background signals produced from heterogeneous biological tissue differ from the reference background spectra due to presence of other intrinsic optical absorbers and effect of optical scattering. Due to the difference, the background signals partly remain in the probe images. To image the probe injected in subcutaneous tumors of mice clearly, we added the melanosome absorption spectrum to the reference background spectra because skin contains nonnegligible concentration of melanosome and the spectrum is very similar to the scattering spectrum of biological tissue. The probe injected in the subcutaneous tumor of mice was an enzyme-activatable probe which show their original colors only in the presence of γ-glutamyltranspeptidase, an enzyme associated with cancer. The probes have been successfully used for rapid fluorescence imaging of cancer. As a result of MS-PA imaging, by considering the melanosome absorption spectrum, the background signals were successfully suppressed and then clearer probe image was obtained. Our MS-PA imaging method afforded successful imaging of tumors in mice injected with activatable PA probes.

Optical-resolution photoacoustic microscopy (OR-PAM) is a novel microscopic tool to provide in vivo optically sensitive images in biomedical research. Conventional OR-PAM systems are typically slow and bulky because of the linear scanning stages with stepping motors. For practical purposes, however, fast imaging speed and small footprint are crucial. To address these issues, we have developed a real-time compact OR-PAM system equipped with a waterproof two-axis MEMS scanner. The OR-PAM system consists of key components such as an ultrasonic transducer with a bandwidth of 50 MHz, an opto-acoustic beam combiner (BC), and an MEMS scanner. These are all installed inside a small water tank, with dimensions of 30 mm × 90 mm × 30 mm along the x-, y-, and z-axes, respectively. A pulsed laser with a repetition rate of 50 kHz is confocally aligned with the photoacoustic (PA) waves in the BC to maximize the SNRs. The fast scanning ability of the MEMS scanner fully utilizes the A-scan speed of 50 kHz. For instance, the B- and C-scan imaging speeds are 125 Hz and 0.625 Hz, respectively, when the acquired PA maximum amplitude projection image has 200 × 200 pixels along the x- and y-axes, respectively. The measured lateral resolution of 3.6 μm and axial resolution of 27 μm are sufficient to resolve the small capillaries. Finally, we have successfully obtained in vivo PA images of iris microvasculatures in mice. This real-time and compact OR-PAM system is optimized to examine small animals in clinical studies.

The effects of the optical properties of gold nanoparticle (AuNP) on the photoacoustic (PA) signals were investigated with the numerical simulation and experiment using various spherical and polyhedral AuNPs. In the numerical simulation, the absorption and scattering cross sections were calculated by the discrete dipole approximation. The absorbed light energy was calculated by the Monte Carlo method. Then PA pressure wave was simulated based on the PA wave equation. In the experiment, the PA signals from the AuNP suspensions were measured by use of a P(VDFTrFE) piezoelectric film and a Q-switched Nd:YAG laser operated at 532 nm. It was demonstrated that the PA signals were characterized by both of the absorption cross sections and the scattering cross section. The scattering medium containing the AuNPs enhanced the amplitude of the PA signals.

A requirement to reconstruct photoacoustic (PA) image is to have a synchronized channel data acquisition with laser firing. Unfortunately, most clinical ultrasound (US) systems don’t offer an interface to obtain synchronized channel data. To broaden the impact of clinical PA imaging, we propose a PA image reconstruction algorithm utilizing US B-mode image, which is readily available from clinical scanners. US B-mode image involves a series of signal processing including beamforming, followed by envelope detection, and end with log compression. Yet, it will be defocused when PA signals are input due to incorrect delay function. Our approach is to reverse the order of image processing steps and recover the original US post-beamformed radio-frequency (RF) data, in which a synthetic aperture based PA rebeamforming algorithm can be further applied. Taking B-mode image as the input, we firstly recovered US postbeamformed RF data by applying log decompression and convoluting an acoustic impulse response to combine carrier frequency information. Then, the US post-beamformed RF data is utilized as pre-beamformed RF data for the adaptive PA beamforming algorithm, and the new delay function is applied by taking into account that the focus depth in US beamforming is at the half depth of the PA case. The feasibility of the proposed method was validated through simulation, and was experimentally demonstrated using an acoustic point source. The point source was successfully beamformed from a US B-mode image, and the full with at the half maximum of the point improved 3.97 times. Comparing this result to the ground-truth reconstruction using channel data, the FWHM was slightly degraded with 1.28 times caused by information loss during envelope detection and convolution of the RF information.

Recently, photoacoustic tomography (PAT) has emerged as a remarkable non-invasive imaging modality that provides a strong optical absorption contrast, high ultrasonic resolution, and great penetration depth. Thus, PAT has been widely used as an in vivo preclinical imaging tool. Surface-enhanced Raman spectroscopy (SERS) is another attractive sensing technology in biological research because it offers highly sensitive chemical analyses and multiplexed detection. By performing dual-modal imaging of SERS and PAT, high-resolution structural PAT imaging and high-sensitivity SERS sensing can be achieved. At the same time, it is equally important to develop a dual modal contrast agent for this purpose. To perform both PAT and SERS, we synthesized PEGylated silver bumpy nanoshells (AgBSs). The AgBSs generate strong PA signals owing to their strong optical absorption properties as well as sensitive SERS signals because of the surface plasmon resonance effect. Then, multiplexed Raman chemicals were synthesized to enhance the sensitivity of Raman. We have photoacoustically imaged the sentinel lymph nodes of small animals after intradermal injection of multiplexed agents. Furthermore, the chemical composition of each agent has been distinguished through SERS.

We present a multi-scale imaging system that integrates five optoacoustic and multi-photon modalities into the same device. The hybrid microscope offers a unique zoom-in ability by allowing for optoacoustic microscopy and mesoscopy scans of the sample within the same imaging framework. Furthermore, by combining several label-free modalities, we are able to visualize a broad range of anatomical features, taking advantage of their complementary contrast mechanisms. We characterize the spatial resolution and relative orientation of the different sub-modalities and demonstrate the system’s performance by the imaging of several model organisms ex vivo. The presented ability to dynamically vary scanning volume and resolution together with its multi-contrast and label-free imaging capabilities make the hybrid microscope a promising tool for comprehensive biological imaging.

Chemotherapy and radiation-therapy are conventional treatment procedure of cancer. Though radiation therapy is very common practice for cancer treatment, it has limitations including incomplete and non specific destruction. Heating characteristics of magnetic nanoparticle (MNP) is modelled using molecular dynamics simulation setup. This model would give an understanding for the treatment of cancer cell through MNP associated radiation-therapy. In this paper, alternating magnetic field driven heat generation of MNP is studied using classical molecular dynamics. Temperature is measured as an ensemble average of velocity of the atoms. Temperature stabilization is achieved. Under this simulation setting with certain parameters, 45°C temperature was obtained in our simulations. Simulation data would be helpful for experimental analysis to treat cancerous cell in presence of MNP under exposure to radiofrequency. The in vitro thermal characteristics of magnetite nanoparticles using magnetic coil of various frequencies (5, 7.5, 10 and 15 kHz), the saturation temperature was found at 0.5 mg/mL concentration. At frequency 50 kHz the live/dead and MTT assay was performed on magnetite nanoparticles using MC3T3 cells for 10 min duration. Low radio frequency (RF) radiation induced localized heat into the metallic nanoparticles which is clearly understood using the molecular dynamics simulation setup. Heating of nanoparticle trigger the killing of the tumor cells, acts as a local therapy, as it generates less side effects in comparison to other treatments like chemotherapy and radiation therapy.

Sound velocity measurement is of great importance to the application of biomedical especially in the research of acoustic detection and acoustic tomography. Using correct sound velocities in each medium other than one unified sound propagation speed, we can effectively enhance sound based imaging resolution. Photoacoustic tomography (PAT), is defined as cross-sectional or three-dimensional (3D) imaging of a material based on the photoacoustic effect and it is a developing, non-invasive imaging method in biomedical research. This contribution proposes a method to concurrently calculate multiple acoustic speeds in different mediums. Firstly, we get the size of infra-structure of the target by B-mode ultrasonic imaging method. Then we build the photoacoustic (PA) image of the same target with different acoustic speed in different medium. By repeatedly evaluate the quality of reconstruct PA image, we dynamically calibrate the acoustic speeds in different medium to build a finest PA image. Thus, we take these speeds of sound as the correct acoustic propagation velocities in according mediums. Experiments show that our non-invasive method can yield correct speed of sound with less than 0.3% error which might benefit future research in biomedical science.

We present a photoacoustic microscopy (PAM) system based on a Fabry-Perot Interferometer (FPI) consisting of a transparent Polydimethylsiloxane (PDMS) thin film. Most of the PAM systems have limitations with the system alignment because the ultrasound transducers for detection are not transparent. Therefore, the excitation laser source should avoid the opaque transducer to illuminate the sample, which makes the system difficult to build-up. Especially, the system volume is highly limited to be compact. In our experiment, to solve these difficulties, a FPI based on the PDMS film has been implemented and applied to measure the acoustic wave signal. The system uses a FPI as an acoustic wave detector instead of a conventional ultrasound transducer. A tunable laser was used to choose the quadrature-point at which the signal has the highly sensitve and linear response to the acoustic wave. Also a 20Hz pulsed Nd:YAG laser was used to generate acoustic waves from a sample. When the acoustic waves arrive at the PDMS film, one of the surfaces of the film is modulated at the detecting point, which gives the tuned FPI interference signal. From the signal arriving time, the depth location of the sample is calculated. As a primary experiment using the PDMS thin film as an ultrasound transducer, a couple of narrow black friction tapes located in a water container were used as the samples. This proposed imaging method can be used in various applications for the detection and measurement of acoustic waves.

Monitoring cerebral blood flow (CBF) is crucial, as inadequate perfusion, even for relatively short periods of time, may lead to brain damage or even death. Thus, significant research efforts are directed at developing reliable monitoring tools that will enable continuous, bed side, simple and cost-effective monitoring of CBF. All existing non invasive bed side monitoring methods, which are mostly NIRS based, such as Laser Doppler or DCS, tend to underestimate CBF in adults, due to the indefinite effect of extra-cerebral tissues on the obtained signal. If those are to find place in day to day clinical practice, the contribution of extra-cerebral tissues must be eliminated and data from the depth (brain) should be extracted and discriminated. Recently, a novel technique, based on ultrasound modulation of light was developed for non-invasive, continuous CBF monitoring (termed ultrasound-tagged light (UTL or UT-NIRS)), and shown to correlate with readings of 133Xe SPECT and laser Doppler. We have assembled a comprehensive computerized simulation, modeling this acousto-optic technique in a highly scattering media. Using the combination of light and ultrasound, we show how depth information may be extracted, thus distinguishing between flow patterns taking place at different depths. Our algorithm, based on the analysis of light modulated by ultrasound, is presented and examined in a computerized simulation. Distinct depth discrimination ability is presented, suggesting that using such method one can effectively nullify the extra-cerebral tissues influence on the obtained signals, and specifically extract cerebral flow data.

Photo-acoustic tomography (PAT) is capable of imaging optical absorption in depths beyond the diffusion limit. As blood is one of the main absorbers in tissue, one important application is the visualization of vasculature, which can provide important clues for diagnosing diseases like cancer. While the state-of-the-art work in photo-acoustic 3D angiography has focused on computed tomography systems involving complex setups, we propose an approach based on optically tracking a freehand linear ultrasound probe that can be smoothly integrated into the clinical workflow. To this end, we present a method for calibration of a PAT system using an N-wire phantom specifically designed for PAT and show how to use local gradient information in the 3D reconstructed volume to significantly enhance the signal. According to experiments performed with a tissue mimicking intra-lipid phantom, the signal-to-noise ratio, contrast and contrast-to-noise ratio measured in the full field of view of the linear probe can be improved by factors of 1.7±0.7, 14.6±5.8 and 2.8±1.2 respectively, when comparing the post envelope detection reconstructed 3D volume with the processed one. Qualitative validation performed in tissue mimicking gelatin phantoms further showed good agreement of the reconstructed vasculature with corresponding structures extracted from X-ray computed tomographies. As our method provides high contrast 3D images of the vasculature despite a low hardware complexity its potential for clinical application is high.

The short focal depth of a Gaussian beam limits the volumetric imaging speed of optical resolution photoacoustic microscopy (OR-PAM). A Bessel beam, which is diffraction-free, provides a long focal depth, but its side-lobes may deteriorate image quality when the Bessel beam is directly employed to excite photoacoustic signals in ORPAM. Here, we present a nonlinear approach based on the Grueneisen relaxation effect to suppress the side-lobe artifacts in photoacoustic imaging. This method extends the focal depth of OR-PAM and speeds up volumetric imaging. We experimentally demonstrated a 1-mm focal depth with a 7-μm lateral resolution and volumetrically imaged a carbon fiber and red blood cell samples.

We present a laser diode-based photoacoustic microscopy (PAM) system with a minimized light intensity loss for ovarian tissue imaging. A 905 nm, 650 W output peak power pulsed laser diode (PLD) is utilized as the light source. The intrinsic properties and the construction of this PLD typically make it challenging to focus its beam to a small spot size with a lowloss optical system. An optical system comprising a combination of aspheric and cylindrical lenses is presented that allows a low-loss collimation and tight focusing of the light beam. The lateral resolution of this PAM system is measured to be 40 μm using edge spread function estimation. Images of black human hairs, polyethylene tubes filled with rat blood, ex vivo mouse ear and ex vivo porcine ovary are presented.

During the last two decades, optoacoustic imaging has been developed as a novel biomedical imaging technique based on the generation of ultrasound waves by means of laser light. In this work, we investigate the optoacoustic response from graphene-based solutions by using a compact and cost-effective system based on an assembly of several 905-nm pulsed high-power diode lasers coupled to a bundle of 200-μm diameter- core optical fibers. The coupled light is conveyed into a lens system and focused on an absorber consisting of graphene-based nanomaterials (graphene oxide, reduced graphene oxide, and reduced graphene-oxide/gold-nanoparticle hybrid, respectively) diluted in ethanol and hosted in slightly scattering optical phantoms. The high absorption of these graphene-based solutions suggests their potential future use in optoacoustic applications as contrast agents.

Prior research in photoacoustic tomography has consistently demonstrated its ability to image structures near the surface of tissue with a high degree of optical contrast. However, despite significant advancements in the field, there has been little to no development of clinical applications for photoacoustic tomography, principally due to the requirement for backwardmode operation, i.e., it must detect the photoacoustic signal on the same side of the tissue as the incident laser light. This results in the standard ultrasonic transducer occluding the path of the inciting laser beam. Therefore, developing a technique to deliver light into the tissue, while incorporating commonly available ultrasonic detection equipment without occluding the beam propagation or modifying the equipment in any way, would provide a significant benefit to the field, and potentially improve its clinical applicability. Here, we propose a new method to accomplish this aim, using planar optical waveguides that employ the optical tunneling phenomenon to transmit light directly into tissue (pig skin) through physical contact with the sample. A commercially available, 10MHz, unfocused ultrasonic transducer was positioned on the rear face of the waveguide and was used to detect photoacoustic signals generated within the tissue as the signals propagated perpendicularly through the waveguide substrate. Unlike alternative solutions to the occlusion problem, this modality does not necessitate the use of custom manufactured transducers, expensive dichroics, or additional laser systems, and thereby represents a viable approach for the easy implementation of photoacoustic tomography in a clinical setting.

Quantitative photoacoustic tomography is an emerging imaging technique aimed at estimating the optical parameters inside tissue from photoacoustic images. The method proceeds from photoacoustic tomography by taking the estimated initial pressure distributions as data and estimating the absolute values of the optical parameters. Therefore, both the data and the noise of the second (optical) inverse problem are affected by the method applied to solve the first (acoustic) inverse problem. In this work, the Bayesian approach for quantitative photoacoustic tomography is taken. Modelling of noise and errors and incorporating their statistics into the solution of the inverse problem are investigated.

We have achieved penetration depth of 30mm by photoacoustic imaging system using LED light source integrated transducer to image a clinical metal needle inserted into a tissue mimicking phantom. We developed the transducer that integrated near-infrared LED array light source, which was connected to a photoacoustic imaging system which drove LED array light source and controlled photoacoustic data acquisition process. Conventionally solid-state laser has been used as the light source for photoacoustic imaging system. Because LED is diffused light source, laser safety glasses is not necessary, also inflexible fibers are not used to guide light close to a transducer, and we integrated LED light source inside the transducer, which became compact and practical size for conventional ultrasound equipment users. We made LED light source unit as detachable to the transducer easily, so wave-length of light can be selectable by changing the LED light source unit.

Photoacoustic computed tomography (PACT) is an emerging computed imaging modality that exploits optical contrast and ultrasonic detection principles to form images of the absorbed optical energy density within tissue. If the object possesses spatially variant acoustic properties that are unaccounted for by the reconstruction algorithm, the estimated image can contain distortions. While reconstruction algorithms have recently been developed for compensating for this effect, they generally require the objects acoustic properties to be known a priori. To circumvent the need for detailed information regarding an objects acoustic properties, we have previously proposed a half-time reconstruction method for PACT. A half-time reconstruction method estimates the PACT image from a data set that has been temporally truncated to exclude the data components that have been strongly aberrated. In this approach, the degree of temporal truncation is the same for all measurements. However, this strategy can be improved upon it when the approximate sizes and locations of strongly heterogeneous structures such as gas voids or bones are known. In this work, we investigate PACT reconstruction algorithms that are based on a variable temporal data truncation (VTDT) approach that represents a generalization of the half-time reconstruction approach. In the VTDT approach, the degree of temporal truncation for each measurement is determined by the distance between the corresponding transducer location and the nearest known bone or gas void location. Reconstructed images from a numerical phantom is employed to demonstrate the feasibility and effectiveness of the approach.

The hybrid nature of optoacoustic tomography (OAT) brings together the advantages of both optical imaging and ultrasound imaging, making it a promising tool for breast cancer imaging. It is advocated in the modern imaging science literature to utilize objective, or task-based, measures of system performance to guide the optimization of hardware design and image reconstruction algorithms. In this work, we investigate this approach to assess the performance of OAT breast imaging systems. In particular, we apply principles from signal detection theory to compute the detectability of a simulated tumor at different depths within a breast, for two different system designs. The signal-to-noise ratio of the test statistic computed by a numerical observer is employed as the task-specific summary measure of system performance. A numerical breast model is employed that contains both slowly varying background and vessel structures as the background model, and superimpose a deterministic signal to emulate a tumor. This study demonstrates how signal detection performance of a numerical observer will vary as a function of signal depth and imaging system characteristics. The described methodology can be employed readily to systematically optimize other OAT imaging systems for tumor detection tasks.

Photoacoustic-computed microscopy (PACM) differs from conventional photoacoustic microscopy (PAM) imaging techniques in a way that thousands of optical foci are generated simultaneously using a two-dimensional microlens array, and raster-scanning these optical foci provides wide-field images. A major limitation of PACM is the slow imaging speed caused by the high power pulsed lasers and large amount of acoustic detectors. Here, we addressed this problem through compressed sensing and image inpainting. Compressed sensing minimizes the number of transducer elements used to acquire each frame, while inpainting minimizes the scanning steps. Combining these two approaches, we improved the imaging speed by sixteen times.

Most image reconstruction methods in photoacoustic computed tomography (PACT) assume that the acoustic properties of the object and the surrounding medium are homogeneous. This can lead to strong artifacts in the reconstructed images when there are significant variations in sound speed or density. Air voids represent a particular challenge due to the severity of the differences between the acoustic properties of air and water. In whole-body small animal imaging, the presence of air voids in the lungs, stomach, and gastrointestinal system can limit image quality over large regions of the object. Iterative reconstruction methods based on the photoacoustic wave equation can account for these acoustic variations, leading to improved resolution, improved contrast, and a reduction in the number of imaging artifacts. However, the strong acoustic heterogeneities can lead to instability or errors in the numerical wave solver. Here, the impact of air voids on PACT image reconstruction is investigated, and procedures for their compensation are proposed. The contributions of sound speed and density variations to the numerical stability of the wave solver are considered, and a novel approach for mitigating the impact of air voids while reducing the computational burden of image reconstruction is identified. These results are verified by application to an experimental phantom.

Optoacoustic imaging (OAI) is a hybrid biomedical imaging modality based on the generation and detection of ultrasound by illuminating the target tissue by laser light. Typically, laser light in visible or near infrared spectrum is used as an excitation source. OAI is based on the implementation of image reconstruction algorithms using the spatial distribution of optical absorption in tissues. In this work, we apply a time-domain back-projection (BP) reconstruction algorithm and a wavelet filtering for point and line detection, respectively. A comparative study between point detection and integrated line detection has been carried out by evaluating their effects on the image reconstructed. Our results demonstrate that the back-projection algorithm proposed is efficient for reconstructing high-resolution images of absorbing spheres embedded in a non-absorbing medium when it is combined with the wavelet filtering.

Photoacoustic microscopy (PAM) provides high resolution and large penetration depth by utilizing the high optical sensitivity and low scattering of ultrasound. Hybrid PAM systems can be classified into two categories: opticalresolution photoacoustic microscopy (OR-PAM) and acoustic-resolution photoacoustic microscopy (AR-PAM). ORPAM provides a very high lateral resolution with a strong optical focus, but the penetration depth is limited to one optical transport mean free path. AR-PAM provides a relatively greater penetration depth using diffused light in biological tissues. The resolution of AR-PAM is determined by its ultrasonic parameters. In this study, we performed an in vivo testing of a switchable OR-/AR-PAM system. In this system, two modes can be switched by changing its collimator lens and optical fiber. The lateral resolution of OR-PAM was measured using a resolution test target, and the full width at half maximum (FWHM) of the edge spread function was 2.5 μm. To calculate the lateral resolution of ARPAM, a 6-μm-diameter carbon fiber was used, and the FWHM of the line spread function was 80.2 μm. We successfully demonstrated the multiscale imaging capability of the switchable OR-/AR-PAM system by visualizing microvascular networks in mouse ears, brain, legs, skin, and eyes.

Using a handheld photoacoustic probe, we proposed a cuffing-based method to quantify blood flow speed in humans. By cuffing and releasing the blood vessel, we can measure the blood flow speed downstream. In phantom experiments, we demonstrated that the minimum and maximum measurable flow speeds were 0.035 mm/s and 42 mm/s, respectively. In human experiments, flow speeds were measured in three different blood vessels: a radial artery in the right forearm, a radial artery in the index finger of the right hand, and a radial vein in the right forearm.

Photoacoustic imaging (PAI) and microsurgery are attracting interest for cancer treatment. The absorption of light triggers thermoelastic processes that cause ultrasound emission and even cavitation. The ultrasounds emission is exploited to reconstruct images, the cavitation may be used to destroy malignant cells. Gold nanorods (GNRs) have been investigated as contrast agents for PAI, but still little is known about the trigger of cavitation processes. Here we study the influence of GNRs parameters, such as their size, coating and environment, on the cavitation threshold. We expect these results will provide useful indications to develop new theranostics techniques based on light-ultrasound interaction

We demonstrate a noncontact photoacoustic imaging (PAI) system in which an optical interferometer is used for ultrasound detection. The system is based on a modified optical-fiber Michelson interferometer that measures the surface displacement caused by photoacoustic pressure. A synchronization method is utilized to keep its high sensitivity to reduce the influence of ambient vibrations. The system is experimentally verified by imaging of a phantom. The experimental results indicate that the proposed system can be used for noncontact PAI with high resolution and high bandwidth.

In contrast to the well-established and widely used theory of photoacoustic signal generation by single delta-like pulses, the field of multiple pulse excitation is not yet studied well. Using double-pulse excitation can be beneficial, but as ultrasound transducers have a certain waveform duration, the inter-pulse delays used might be limited.

In order to assess the strength of the transducer influence at short delay times and develop data analysis procedure, we investigate the photoacoustic responses of a phantom sample to double-pulse excitation measured with different transducers. Both focused and flat surface single element transducers are used in the study. The central frequencies are chosen in the low-frequency band as they are most widely used in clinical ultrasound and one higher frequency transducer is taken for comparison.

Despite not observing signal amplification due to Grueneisen relaxation effect, we show that transducer influence is not exceeding measurement error. Additionally we prove that single pulse subtraction procedure can be used to restore the second pulse waveform in double pulse excitation scheme. We believe using this procedure can be beneficial when transducer’s waveform duration is longer than used inter-pulse delays.

We present an all optical fiber combined-imaging system that integrates non-contact photoacoustic tomography (NPAT) and optical coherence tomography (OCT) to simultaneously provide PA and OCT images. The fiber-based PAT system utilizing a Mach-Zehnder interferometer with a fiber laser of 1550 nm measures the photoacoustic signal at the sample surface. For the generation of a PA signal, a pulse train from a bulk type Nd:YAG laser illuminates the sample via a large core multimode optical fiber. The fiber-based OCT operating at a center wavelength of 1310 nm allowed is combined with the fiber-based PAT system by sharing the same optical fiber probe. The two lights from the fiber laser and the OCT source are guided into the probe through each port of a 2 by 2 optical fiber coupler. The back-reflected lights from the sample are guided to respective imaging systems by the same coupler. With these both NPAT and OCT images could be co-registered without physical contact with the sample. To demonstrate the feasibility of the proposed system, a phantom experiment has been carried out with a phantom composed of a black PET fiber and a fishing wire. The proposed all fiber-optic combined-imaging system has the potential for minimally invasive and improved diagnosis.

We have successfully imaged photoacoustic differences of light absorbance between two images acquired by different wave-length LED array light source. Compared to photoacoustic imaging system using conventional solid-state laser light source, LED light source can be driven at higher frequency pulses, so we were able to get the subtraction image at higher frame rate that calculated from two images which were captured at each wave-length LED light pulse timing. We developed LED array light source which is composed to have two different wave-length chips, so each wave-length light pulse can be controlled and emitted freely. Thus LED array light source can be composed as multiple selectable wavelength more than two, and a various combination of subtraction image may become available at high frame rate.

The field of Optical Fiber Sensors (OFS) is gaining tremendous popularity in recent years. OFS natural immunity to electromagnetic disturbances, inherent biocompatibility and compactness making them highly attractive for ultrasound sensing. Moreover, their compatibility with photoacoustics can make them useful in situations where traditional piezoelectric probes are inadequate. However, the issue of multiplexing individual OFS into an array remains a challenging and costly task. In this work, we demonstrate a straightforward approach for multiplexing multiple broadband OFS for ultrasound sensing by exploiting most of the photoreceiver's bandwidth. The design is based on a recently developed system in which all sensing elements are connected to a single interrogator and to a single digitizing circuit. To mitigate aliasing, the system employs I/Q coherent detection. Synchronization of the sensor interrogation with the excitation enables very high repetition rates (kHz) making it ideal for applications where imaging of dynamic processes is desired.

Previously we described the potential for multiple illumination photoacoustic tomography to provide quantitative reconstructions, however this work used only simulated data. We have developed a custom photoacoustic-ultrasound tomography system capable of multiple illuminations and parallel acquisition from a 256 element 5 MHz transducer ring array with 8-cm diameter. The multiple illumination scheme uses a free-space light delivery geometry where a rotational stage scans a pulsed laser beam onto different incident locations around the sample. For each illumination location a photoacoustic image is reconstructed using a modified backprojection algorithm. Images from different source locations have the potential to be combined to form an improved deep-tissue image using our previously developed iterative algorithms. We complement the photoacoustic imaging data with unique ultrasound imaging data. Most previous ultrasound tomography methods have used migration algorithms, iterative ray-based analysis, wave-equation modeling, or frequency-based algorithms that all demand large amounts of data and computational power. We propose a new UST method that offers isotropic resolution, provides scattering contrast, as well as the potential for measuring ultrasound scattering anisotropy and decoupling density and compressibility contributions. The imaging system is driven by a Verasonics scan engine and programmed for both ultrasound and photoacoustic imaging modes. Resolution has been measured to be 150 μm for ultrasound and 200 μm for photoacoustic images. Imaging capabilities are demonstrated on phantoms with custom-tailored ultrasound scattering and optical properties, as well as in murine models.

In traditional photoacoustic tomography, external illumination is used to excite acoustic waves. However, with the assistance of fibre-transmitted light, multidirectional illumination or internal illumination can be achieved which can obtain a better image at a deeper depth. Laser pulses delivered by fibre are energy-limited by the fibre core size and damage threshold. To increase the amplitude of photoacoustic waves and their penetration, it is necessary to improve the fibre coupling energy and efficiency. To improve the coupling performance of single fibres, we use a cylindrical lens array to homogenize the incident beam before a coupling lens. Simulation in Zemax shows that this approach flattens the beam profile on the front surface of the fibre, decreasing the risk of fibre damage. Experimental results with fibre core diameters of 1mm and 1.5mm show that both types of fibre can output more than 50mJ energy per pulse at 700nm wavelength. The coupling efficiency is measured to be above 70% and even reaches 90% as the wavelength changes from 675nm to 900nm. This improvement of coupling energy in single fibres will benefit photoacoustic tomography applications using internal illumination.

Photoacoustic (PA) imaging is a non-invasive, non-ionizing imaging technology with high optical contrast between blood and tissue, and with high sensitivity of hemoglobin concentration and oxygen saturation due to different optical absorption spectra resulting from different oxygenation of hemoglobin. Most PA imaging systems implement a nanosecond pulsed laser source as excitation source to induce PA signal, and rely on broadband amplifiers to record time-domain PA signals [1-6]. Some groups, however, have reported using modulated continuous-wave lasers as an excitation source for frequency-domain imaging [7-9]. Frequency-domain imaging offers the potential of lock-in amplification which has sensitivities as low as nV even in noise orders of magnitude higher than the signal. However, although modulated CW sources works for low cost and compact PA imaging, it does not satisfy thermal and stress confinement conditions required for optimal PA signal strength. Here, we investigate a PA methodology using pulsed fiber lasers as excitation laser source combined with lock-in amplification technology. For comparison, we also studied time-domain PA methodology. Phantom studies show that signal-to-noise ratio (SNR) obtained with frequency domain PA imaging is significantly more sensitive than that obtained using time-domain PA imaging when the laser pulse repetition rate (PRR) matches the bandwidth of ultrasound transducer. Therefore, high sensitive PA imaging technology using pulsed fiber laser sources with lock-in amplification may potentially greatly extend the depth of PA imaging.

We have developed an intraoperative multimodal surgical microscopy system that provides simultaneous real-time enlarged surface views and subsurface anatomic information during surgeries by integrating spectral domain optical coherence tomography (SD-OCT), optical-resolution photoacoustic microscopy (OR-PAM), and conventional surgical microscopy. By sharing the same optical path, both OCT and PAM images were simultaneously acquired. Additionally, the custom-made needle-type transducer received the generated PA signals enabling convenient surgical operation without using a water bath. Using a simple augmented device, the OCT and PAM images were projected on the view plane of the surgical microscope. To quantify the performance of our system, we measured spatial resolutions of our system. Then, three microsurgery simulation and analysis were processed: (1) ex vivo needle tracking and monitoring injection of carbon particles in biological tissues, (2) in vivo needle tracking and monitoring injection of carbon particles in tumor-bearing mice, and (3) in vivo guiding of melanoma removal in melanoma-bearing mice. The results indicate that this triple modal system is useful for intraoperative purposes, and can potentially be a vital tool in microsurgeries.

Photoacoustic tomography (PAT) provides a unique tool to diagnose inflammatory arthritis. However, the specificity and sensitivity of PAT based on endogenous contrasts is limited. The development of contrast enhanced PAT imaging modalities in combination with small molecule contrast agents could lead to improvements in diagnosis and treatment of joint disease. Accordingly, we adapted and tested a PAT clinical imaging system for imaging the human joints, in combination with a novel PAT contrast agent derived from an FDA-approved small molecule drug. Imaging results based on a photoacoustic and ultrasound (PA/US) dual-modality system revealed that this contrast-enhanced PAT imaging system may offer additional information beyond single-modality PA or US imaging system, for the imaging, diagnosis and assessment of inflammatory arthritis.

Melanoma is a very malicious type of cancer as it metastasizes early and hence its late diagnosis leads to death. Consequently, early diagnosis of melanoma and its removal is considered the most effective way of treatment. We present a design of a high frequency acoustic microscopy and infrared reflectance system for the early detection of melanoma. Specifically, the identification of morphological changes related to carcinogenesis is required. In this work, we simulate of the propagation of the ultrasonic waves of the order of 100 MHz as well as of electromagnetic waves of the order of 100 THz in melanoma structures targeting to the estimation and optimization of the basic characteristics of the systems. The simulation results of the acoustic microscopy subsystem aim to provide information such as the geometry of the transducer, the center frequency of operation, the focal length where the power transmittance is optimum and the spot size in focal length. As far as the infrared is concerned the optimal frequency range and the spot illumination size of the external probe is provided. This information is next used to assemble a properly designed system which is applied to melanoma phantoms as well as real skin lesions. Finally, the measurement data are visualized to reveal the information of the experimented structures, proving noteworthy accuracy.

Because optoacoustic tomography (OAT) can provide functional information based on hemoglobin contrast, it is a promising imaging modality for breast cancer diagnosis. Developing an effective OAT breast imaging system requires balancing multiple design constraints, which can be expensive and time-consuming. Therefore, computer- simulation studies are often conducted to facilitate this task. However, most existing computer-simulation studies of OAT breast imaging employ simple phantoms such as spheres or cylinders that over-simplify the complex anatomical structures in breasts, thus limiting the value of these studies in guiding real-world system design. In this work, we propose a method to generate realistic numerical breast phantoms for OAT research based on clinical magnetic resonance imaging (MRI) data. The phantoms include a skin layer that defines breast-air boundary, major vessel branches that affect light absorption in the breast, and fatty tissue and fibroglandular tissue whose acoustical heterogeneity perturbs acoustic wave propagation. By assigning realistic optical and acoustic parameters to different tissue types, we establish both optic and acoustic breast phantoms, which will be exported into standard data formats for cross-platform usage.

We proposed to use transmission (forward) mode for cerebral, noninvasive, transcranial optoacoustic monitoring, imaging, and sensing in humans. In the transmission mode, the irradiation of the tissue of interest and detection of optoacoustic signals are performed from opposite hemispheres, while in the reflection (backward) mode the irradiation of the tissue of interest and detection of optoacoustic signals are performed from the same hemisphere. Recently, we developed new, transmission-mode optoacoustic probes for patients with traumatic brain injury (TBI) and for neonatal patients. The transmission mode probes have two major parts: a fiber-optic delivery system and an acoustic transducer (sensor). To obtain optoacoustic signals in the transmission mode, in this study we placed the sensor on the forehead, while light was delivered to the opposite side of the head. Using a medical grade, multi-wavelength, OPObased optoacoustic system tunable in the near infrared spectral range (680-950 nm) and a novel, compact, fiber-coupled, multi-wavelength, pulsed laser diode-based system, we recorded optoacoustic signals generated in the posterior part of the head of adults with TBI and neonates. The optoacoustic signals had two distinct peaks: the first peak from the intracranial space and the second peak from the scalp. The first peak generated by cerebral blood was used to measure cerebral blood oxygenation. Moreover, the transmission mode measurements provided detection of intracranial hematomas in the TBI patients. The obtained results suggest that the transmission mode can be used for optoacoustic brain imaging, tomography, and mapping in humans.

Photoacoustic tomography (PAT) is a potential hybrid imaging modality which is gaining attention in the field of medical imaging. Typically a Q-switched Nd:YAG laser is used to excite the tissue and generate photoacoustic signals. But, they are not suitable for clinical applications owing to their high cost, large size. Also, their low pulse repetition rate (PRR) of few tens of hertz prevents them from being used in real-time PAT. So, there is a growing need for an imaging system capable of real-time imaging for various clinical applications. In this work, we are using a nanosecond pulsed laser diode as an excitation source and a clinical ultrasound imaging system to obtain the photoacoustic imaging. The excitation laser is ~803 nm in wavelength with energy of ~1.4 mJ per pulse. So far, the reported frame rate for photoacoustic imaging is only a few hundred Hertz. We have demonstrated up to 7000 frames per second framerate in photoacoustic imaging (B-mode) and measured the flow rate of fast moving obje ct. Phantom experiments were performed to test the fast imaging capability and measure the flow rate of ink solution inside a tube. This fast photoacoustic imaging can be used for various clinical applications including cardiac related problems, where the blood flow rate is quite high, or other dynamic studies.

Photoacoustic tomography (PAT) is a promising biomedical imaging modality for small animal imaging, breast cancer imaging, monitoring of vascularisation, tumor angiogenesis, blood oxygenation, total haemoglobin concentration etc. The existing PAT systems that uses Q-switched Nd:YAG and OPO nanosecond lasers have limitations in clinical applications because they are expensive, non-potable and not suitable for real-time imaging due to their low pulse repetition rate. Low-energy pulsed near-infrared diode laser which are low-cost, compact, and light-weight (<200 grams), can be used as an alternate. In this work, we present a photoacoustic tomography system with a pulsed laser diode (PLD) that can nanosecond pulses with pulse energy 1.3 mJ/pulse at ~803 nm wavelength and 7000 Hz repetition rate. The PLD is integrated inside a single-detector circular scanning geometric system. To verify the high speed imaging capabilities of the PLD-PAT system, we performed in vivo experimental results on small animal brain imaging using this system. The proposed system is portable, low-cost and can provide real-time imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technique for microvasculature imaging at high spatial resolution and contrast. In this work, we present a practical visible laser-diode-based OR-PAM (LD-OR-PAM) prototype for vasculature imaging, which has the desirable properties of being portable, low-cost, and label-free. The prototype employs a 300 mW pulsed laser diode in a 3.8 mm diameter package, emitting 174 ns pulses at 405 ± 5 nm wavelength and a pulse energy of 52 nJ. An aspheric objective with a numerical aperture of 0.60 is used to achieve microscope optical illumination. The laser diode excitation has a compact size of 4.5 × 1.8 × 1.8 cm³ assembled with a cooling block. The lateral resolution was tested to be 0.95 μm on ~7 μm carbon fibers. The subcutaneous microvasculature on a mouse back was label-free imaged ex vivo, which demonstrates the potential of the LD-OR-PAM prototype for in vivo imaging skin chromosphores such as hemoglobin. Our ultimate aim is to provide a practical and affordable OR-PAM system as a routine instrument for standard clinical applications.

We propose a saline-injection-based method to quantify blood flow velocity in vivo with acoustic-resolution photoacoustic tomography. By monitoring the saline-blood-interface propagating in the blood vessel, we can resolve the flow velocity. In phantom experiments, a root-mean-squared error of prediction of 0.29 mm/s was achieved. By injecting saline into a mouse tail vein covered with 1 mm chicken tissue, we showed that the flow velocity in the tail vein could be measured at depth, which is especially pertinent to monitoring blood flow velocity in patients undergoing intravenous infusion.

The ability to determine plaque cap thickness during catheterization is thought to be of clinical importance for plaque vulnerability assessment. While methods to compositionally assess cap integrity are in development, a method utilizing currently available tools to measure cap thickness is highly desirable. NIRS-IVUS is a commercially available dual imaging method in current clinical use that may provide cap thickness information to the skilled reader; however, this is as yet unproven.

Ten autopsy hearts (n=15 arterial segments) were scanned with the multimodality NIRS-IVUS catheter (TVC Imaging System, Infraredx, Inc.) to identify lipid core plaques (LCPs). Skilled readers made predictions of cap thickness over regions of chemogram LCP, using NIRS-IVUS. Artery segments were perfusion fixed and cut into 2 mm serial blocks. Thin sections stained with Movat’s pentachrome were analyzed for cap thickness at LCP regions. Block level predictions were compared to histology, as classified by a blinded pathologist.

Within 15 arterial segments, 117 chemogram blocks were found by NIRS to contain LCP. Utilizing NIRSIVUS, chemogram blocks were divided into 4 categories: thin capped fibroatheromas (TCFA), thick capped fibroatheromas (ThCFA), pathological intimal thickening (PIT)/lipid pool (no defined cap), and calcified/unable to determine cap thickness. Sensitivities/specificities for thin cap fibroatheromas, thick cap fibroatheromas, and PIT/lipid pools were 0.54/0.99, 0.68/0.88, and 0.80/0.97, respectively. The overall accuracy rate was 70.1% (including 22 blocks unable to predict, p = 0.075). In the absence of calcium, NIRS-IVUS imaging provided predictions of cap thickness over LCP with moderate accuracy. The ability of this multimodality imaging method to identify vulnerable coronary plaques requires further assessment in both larger autopsy studies, and clinical studies in patients undergoing NIRS-IVUS imaging.

The ability to accurately quantify chromophore concentration from photoacoustic images would have a major impact on pre-clinical and clinical imaging. Recent years have seen significant advances in the theoretical understanding of quantitative photoacoustic imaging and in the development of model-based inversion strategies that overcome issues such as non-uniqueness and non-linearity. Nevertheless, their full in vivo implementation has not successfully been achieved, partially because experimental uncertainties complicate the transition. In this study, a sensitivity analysis is performed to assess the impact on accuracy of having uncertainty in critical experimental parameters such as scattering, beam diameter, beam position and calibration factor. This study was performed using two virtual phantoms, at one illumination and four optical wavelengths. The model-based inversion was applied in 3 variants - one just inverting for chromophores and two others further inverting for either a scaling factor or the scatterer concentration. The performance of these model-based inversions is also compared to linear unmixing strategies - with and without fluence correction. The results show that experimental uncertainties in a priori fixed parameters - especially calibration factor and scatterer concentration - significantly affect accuracy of model-based inversions and therefore measures to ameliorate this uncertainty should be considered. Including a scaling parameter in the inversion appears to improve quantification estimates. Furthermore, even with realistic levels of experimental uncertainty in model-based input parameters, they outperform linear unmixing approaches. If parameter uncertainty is large and has significant impact on accuracy, the parameter can be included as an unknown in model-based schemes.

Patients affected by diseases caused by arteriosclerosis are increasing. Atherosclerosis, which is becoming an especially difficult health problem, forms plaques from lipids such as cholesterol located in walls of the aorta, cerebral artery, and coronary artery. Because lipid-rich plaques are vulnerable and because arterial rupture causes acute vascular occlusion, early detection is crucially important to prevent plaque growth and rupture. Ultrasound systems can detect plaques but cannot discriminate between vulnerable and equable plaques. To evaluate plaques non-invasively and easily, we developed a handheld photoacoustic imaging device. Its usefulness was verified in phantom experiments with a bovine aorta in which mimic plaque had been embedded. Photoacoustic images taken at wavelengths that produce high light absorbance by lipids show strong photoacoustic signals from the boundary of the mimic plaque. Results confirmed that our system can evaluate plaque properties by analysis with the photoacoustic spectrum. The effects of surrounding tissues and tissue components on plaque evaluation were investigated using a layered phantom. The mimic plaque located under a 6 mm blood layer was also evaluated. Results of these analyses demonstrate the system’s usefulness.

A new technique for simultaneously acquiring photoacoustic images as well as images based on the optical attenuation of single cells in a human blood smear was developed. An ultra-high frequency photoacoustic microscope equipped with a 1 GHz transducer and a pulsed 532 nm laser was used to generate the images. The transducer and 20X optical objective used for laser focusing were aligned coaxially on opposing sides of the sample. Absorption of laser photons by the sample yielded conventional photoacoustic (PA) signals, while incident photons which were not attenuated by the sample were absorbed by the transducer, resulting in the formation of a photoacoustic signal (tPA) within the transducer itself. Both PA and tPA signals, which are separated in time, were recorded by the system in a single RF-line. Areas of strong signal in the PA images corresponded to dark regions in the tPA images. Additional details, including the clear delineation of the cell cytoplasm and features in red blood cells, were visible in the tPA image but not the corresponding PA image. This imaging method has applications in probing the optical absorption and attenuation characteristics of biological cells with sub-cellular resolution.

Independent component analysis (ICA) is a blind source unmixing method that may be used under certain circumstances to decompose multi-wavelength photoacoustic (PA) images into separate components representing individual chromophores. It has the advantages of being fast, easy to implement and computationally inexpensive. This study uses simulated multi-wavelength PA images to investigate the conditions required for ICA to be an accurate unmixing method and compares its performance to linear inversion. An approximate fluence adjustment based on spatially homogeneous optical properties equal to that of the background region was applied to the PA images before unmixing with ICA or LI. ICA is shown to provide accurate separation of the chromophores in cases where the absorption coefficients are lower than certain thresholds, some of which are comparable to physiologically relevant values. However, the results also show that the performance of ICA abruptly deteriorates when the absorption is increased beyond these thresholds. In addition, the accuracy of ICA decreases in the presence of spatially inhomogeneous absorption in the background.

Transrectal ultrasonography (TRUS) is the most popular imaging modality for diagnosing and treating prostate cancer. TRUS-guided prostate biopsy is mandatory for the histological diagnosis of patients with elevated serum prostatespecific antigen (PSA), but its diagnostic accuracy is not satisfactory due to TRUS’s low resolution. As a result, a considerable number of patients are required to undergo an unnecessary repeated biopsy. Photoacoustic imaging (PAI) can be used to provide microvascular network imaging using hemoglobin as an intrinsic, optical absorption molecule. We developed an original TRUS-type PAI probe consisting of a micro-convex array transducer with an optical illumination system to provide superimposed PAI and ultrasound images. TRUS-type PAI has the advantage of having much higher resolution and greater contrast than does Doppler TRUS. The purpose of this study was to demonstrate the clinical feasibility of the transrectal PAI system. We performed a clinical trial to compare the image of the cancerous area obtained by transrectal PAI with that obtained by TRUS Doppler during prostate biopsy. The obtained prostate biopsy cores were stained with anti-CD34 antibodies to provide a microvascular distribution map. We also confirmed its consistency with PAI and pre-biopsy MRI findings. Our study demonstrated that transrectal identification of tumor angiogenesis under superimposed photoacoustic and ultrasound images was easier than that under TRUS alone. We recognized a consistent relationship between PAI and MRI findings in most cases. However, there were no correspondences in some cases.

Stem cell based treatments hold great potential and promise to address many unmet clinical needs. The importance of non-invasive imaging techniques to monitor transplanted stem cells qualitatively and quantitatively is crucial. The objective of this study was to create a critical size bone defect in the rat femur and then assess the ability of the differentiated mesenchymal stem cells (MSCs) to repair the defect using high frequency co-registered photoacoustic(PA)/ultrasound(US) imaging and micro computed tomography (μCT) over an 8 week period. Combined PA and US imaging was performed using 256 elements, 21 MHz frequency linear-array transducer combined with multichannel collecting system. In vivo 3D PA and US images of the defect bone in the rat femur were acquired after 4 and 8 weeks of the surgery. 3D co-registered structural such as microvasculature and the functional images such as total concentration of haemoglobin (HbT) and the haemoglobin oxygen saturation (sO2) were obtained using PA and US imaging. Bone formation was assessed after 4 and 8 weeks of the surgery by μCT. High frequency linear-array based coregistered PA/US imaging has been found promising in terms of non-invasiveness, sensitivity, adaptability, high spatial and temporal resolution at sufficient depths for the assessment of the reparative ability of MSCs in a rat critical size bone repair defect model.

In cardiology, a vulnerable plaque is considered to be a key subject because it is strongly related to atherosclerosis and acute myocardial infarction. Because conventional intravascular imaging devices exhibit several limitations with regard to vulnerable plaque detection, the need for an effective lipid imaging modality has been continuously suggested. Photoacoustic (PA) imaging is a medical imaging technique with a high level of ultrasound (US) resolution and strong optical contrast. In this study, we successfully developed an integrated intravascular photoacoustic/ultrasound (IV-PAUS) imaging system with a catheter diameter of 1.2 mm for lipid-rich atherosclerosis imaging. An Nd:YAG pulsed laser with an excitation wavelength of 1064 nm was utilized. IV-PAUS offers 5-mm depth penetration and axial and lateral PA imaging resolutions of 94 μm and 203 μm, respectively, as determined by imaging a 6-μm carbon fiber. We initially obtained 3-dimensional (3D) co-registered PA/US images of metal stents. Subsequently, we successfully obtained 3D coregistered PA/US ex vivo images using an iliac artery from a rabbit atherosclerosis model. Accordingly, lipid-rich plaques were sufficiently differentiated from normal tissue in the ex vivo experiment. We validated these findings histologically to confirm the lipid content.

The absorption of visible light by human skin is governed by a number of natural chromophores: Eumelanin, pheomelanin, oxyhemoglobin, and deoxyhemoglobin are the major absorbers in the visible range in cutaneous tissue. Label-free quantification of these tissue chromophores is an important step of optoacoustic (photoacoustic) imaging towards clinical application, since it provides relevant information in diseases. In tumor cells, for instance, there are metabolic changes (Warburg effect) compared to healthy cells, leading to changes in oxygenation in the environment of tumors. In malignant melanoma changes in the absorption spectrum have been observed compared to the spectrum of nonmalignant nevi. So far, optoacoustic imaging has been applied to human skin mostly in single-wavelength mode, providing anatomical information but no functional information. In this work, we excited the tissue by a tunable laser source in the spectral range from 413-680 nm with a repetition rate of 50 Hz. The laser was operated in wavelengthsweep mode emitting consecutive pulses at various wavelengths that allowed for automatic co-registration of the multispectral datasets. The multispectral raster-scan optoacoustic mesoscopy (MSOM) system provides a lateral resolution of <60 μm independent of wavelength. Based on the known absorption spectra of melanin, oxyhemoglobin, and deoxyhemoglobin, three-dimensional absorption maps of all three absorbers were calculated from the multispectral dataset.

Fabry-Perot (FP) polymer film sensors offer high acoustic sensitivity, small element sizes, broadband frequency response and optical transmission to enable high resolution, backward mode photoacoustic (PA) imaging. Typical approaches to sensor fabrication involve the deposition of stacks of alternating dielectric materials to form interferometer mirrors, which are separated by a polymer spacer. If hygroscopic soft dielectric materials are used, a protective polymer layer is typically required. In this study, methods for the deposition of water-resistant, hard dielectric materials onto polymers were explored to improve the robustness and performance of the sensors. This involved the optimisation of the fabrication process, the optical and acoustic characterisation of the sensors, and a comparison of the frequency response with the output of an acoustic forward model. The mirrors, which were separated by a 20 μm Parylene spacer, consisted of eight double layers of Ta₂O₅ and SiO₂ deposited onto polymer substrates using temperature-optimised electron vapour deposition. The free spectral range of the interferometer was 32 nm, its finesse F_R = 91, and its visibility V = 0.72. The noise-equivalent pressure was 0.3 kPa (20 MHz bandwidth). The measured frequency response was found to be more resonant at 25 MHz compared to sensors with soft dielectric mirrors, which was also in good agreement with the output of a forward model of the sensor. The sensors were used in a PA scanner to acquire 3-D images in tissue phantoms.

Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

The increasing use of mouse models for human brain disease studies, coupled with the fact that existing high-resolution functional imaging modalities cannot be easily applied to mice, presents an emerging need for a new functional imaging modality. Utilizing both mechanical and optical scanning in the photoacoustic microscopy, we can image spontaneous cerebral hemodynamic fluctuations and their associated functional connections in the mouse brain. The images is going to be acquired noninvasively with a fast frame rate, a large field of view, and a high spatial resolution. We developed an optical resolution photoacoustic microscopy (OR-PAM) with diode laser. Laser light was raster scanned due to XY-stage movement. Images from ultra-high OR-PAM can then be used to study brain disorders such as stroke, Alzheimer’s, schizophrenia, multiple sclerosis, autism, and epilepsy.

We describe the ongoing development and performance of a high-pulse-energy wavelength-cycling laser system for three-dimensional optoacoustic tomography of the breast. Joule-level energies are desired for achieving the required penetration depths while maintaining safe fluence levels. Wavelength cycling provides a pulse sequence which repeatedly alternates between two wavelengths (approximately 756 and 797 nm) that provide differential imaging. This improves co-registration of captured differential images and quantification of blood oxygen saturation. New design features have been developed for and incorporated into a clinical prototype laser system, to improve efficacy and ease of use in the clinic. We describe the benefits of these features for operation with a clinical pilot optoacoustic / ultrasound dual-modality three-dimensional imaging system.