This PDF file contains the front matter associated with SPIE Proceedings Volume 7899, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

Carotid atherosclerosis has been identified as a potential risk factor for cerebrovascular events, but information about its direct effect on the risk of recurrent stroke is limited due to incomplete diagnosis. The combination of vascular ultrasound, strain rate and spectroscopic photoacoustics could improve the timely diagnosis of plaque status and risk of rupturing. Current ultrasound techniques can noninvasively image the anatomy of carotid arteries. The spatio-temporal variation in displacement of different regions within the arterial wall can be derived from ultrasound radio frequency data; therefore an ultrasound based strain rate imaging modality can be used to reveal changes in arterial mechanical properties. Additionally, spectroscopic photoacoustic imaging can provide information on the optical absorption properties of arterial tissue and it can be used to identify the location of specific tissue components, such as lipid pools. An imaging technique combining ultrasound, strain rate and spectroscopic photoacoustics was tested on an excised atherosclerotic rabbit aorta. The ultrasound image illustrates inhomogeneities in arterial wall thickness, the strain rate indicates the arterial segment with reduced elasticity and the spectroscopic photoacoustic image illustrates the accumulation of lipids. The results demonstrated that ultrasound, strain rate and spectroscopic photoacoustic imaging are complementary. Thus the integration of the three imaging modalities advances the characterization of atherosclerotic plaques.

We demonstrate intravascular photoacoustic imaging of human coronary atherosclerotic plaque. We specifically imaged lipid content, a key factor in vulnerable plaques that may lead to myocardial infarction. An integrated intravascular photoacoustics (IVPA) and ultrasound (IVUS) catheter with an outer diameter of 1.25 mm was developed. The catheter comprises an angle-polished optical fiber adjacent to a 30 MHz single-element transducer. The ultrasonic transducer was optically isolated to eliminate artifacts in the PA image. We performed measurements on a cylindrical vessel phantom and isolated point targets to demonstrate its imaging performance. Axial and lateral point spread function widths were 110 μm and 550 μm, respectively, for PA and 89 μm and 420 μm for US. We imaged two fresh human coronary arteries, showing different stages of disease, ex vivo. Specific photoacoustic imaging of lipid content, is achieved by spectroscopic imaging at different wavelengths between 1180 and 1230 nm.

In this work, we show, for the first time to our knowledge, that multispectral optoacoustic tomography (MSOT) can deliver high resolution images of activatable molecular probe's distribution, sensitive to matrix metalloproteinases (MMP), deep within optically scattering human carotid specimen. It is further demonstrated that this method can be used in order to provide accurate maps of vulnerable plaque formations in atherosclerotic disease. Moreover, optoacoustic images can simultaneously show the underlining plaque morphology for accurate localization of MMP activity in three dimensions. This performance directly relates to small animal screening applications and to clinical potential as well.

A major obstacle in understanding the mechanism of ischemic stroke is the lack of a tool to noninvasively or minimally invasively monitor cerebral hemodynamics longitudinally. Here, we applied optical-resolution photoacoustic microscopy (OR-PAM) to longitudinally study ischemic stroke induced brain injury in a mouse model with transient middle cerebral artery occlusion (MCAO). OR-PAM showed that, during MCAO, the average hemoglobin oxygen saturation (sO₂) values of feeder arteries and draining veins within the stroke core region dropped ~10% and ~34%, respectively. After reperfusion, arterial sO₂ recovered back to the baseline; however, the venous sO2 increased above the baseline value by ~7%. Thereafter, venous sO₂ values were close to the arterial sO₂ values, suggesting eventual brain tissue infarction.

A photoacoustic (PA) imaging system was developed to achieve high sensitivity for the detection and characterization of vascular anomalies in the breast in the mammographic geometry. Signal detection from deep in the breast was achieved by a broadband 2D PVDF planar array that has a round shape with one side trimmed straight to improve fit near the chest wall. This array has 572 active elements and a -6dB bandwidth of 0.6-1.7 MHz. The low frequency enhances imaging depth and increases the size of vascular collections displayed without edge enhancement. The PA signals from all the elements go through low noise preamplifiers in the probe that are very close to the array elements for optimized noise control. Driven by 20 independent on-probe signal processing channels, imaging with both high sensitivity and good speed was achieved. To evaluate the imaging depth and the spatial resolution of this system,2.38mm I.D. artificial vessels embedded deeply in ex vivo breasts harvested from fresh cadavers and a 3mm I.D. tube in breast mimicking phantoms made of pork loin and fat tissues were imaged. Using near-infrared laser light with incident energy density within the ANSI safety limit, imaging depths of up to 49 mm in human breasts and 52 mm in phantoms were achieved. With a high power tunable laser working on multiple wavelengths, this system might contribute to 3D noninvasive imaging of morphological and physiological tissue features throughout the breast.

Currently, most of the cancers in the ovary are detected when they have already metastasized to other parts of the body. As a result, ovarian cancer has the highest mortality of all gynecological cancers with a 5-year survival rate of 30% or less [1]. The reason is the lack of reliable symptoms as well as the lack of efficacious screening techniques [2,3]. Thus, there is an urgent need to improve the current diagnostic techniques. We have investigated the potential role of co-registered photoacoustic and ultrasound imaging in ovarian cancer detection. In an effort to bring this technique closer to clinical application, we have developed a co-registered ultrasound and photoacoustic transvaginal probe. A fiber coupling assembly has been developed to deliver the light from around the transducer for reflection geometry imaging. Co-registered ultrasound and photoacoustic images of swine ovaries through vagina wall muscle and human ovaries using the aforementioned probe, demonstrate the potential of photoacoustic imaging to non-invasively detect ovarian cancer in vivo.

Coregistered optoacoustic (OA) and ultrasound (US) images obtained using a dual modality optoacoustic/ultrasonic breast imaging system are used together for enhanced diagnostic capabilities in comparison to each individual technology. Therefore, an operator-independent delineation of diagnostically relevant objects (in our case breast tumors) with subsequent automatic analysis of image features is required. We developed the following procedure: 1) Image filtering is implemented on a US image to minimize speckle noise and simultaneously preserve the sharpness of the boundaries of the extended objects; 2) Boundaries of the objects of interest are automatically delineated starting with an initial guess made by an operator; 3) Both US and OA images are analyzed using the detected boundaries (contrast, boundary sharpness, homogeneity of the objects and background, boundary morphology parameters are calculated). Calculated image characteristics can be used for statistically independent evaluation of structural information (US data) and vascularization (OA data) of the studied breast tissues. Operator-independent delineation of the objects of interest (e.g. tumors and blood vessels) is essential in clinical OA spectroscopy (using multiple laser wavelengths to quantify concentrations of particular tissue chromophores, such as oxy- and deoxy- hemoglobin, water, and lipids). Another potential application of the suggested image analysis algorithm could be in OA imaging system design, when system performance should be evaluated in terms of quality of the images reconstructed from the well-defined objects of interest. The discussed principles of image analysis are illustrated by using real clinical US and OA data.

Photoacoustic tomography provides both structural and functional imaging in vivo based on optical absorption contrast. A novel imaging system that incorporates a two-dimensional matrix ultrasound probe for combined photoacoustic and ultrasonic three-dimensional (3D) volumetric imaging is presented. The system consists of a tunable dye laser pumped by a Nd:YAG laser, a commercial ultrasound imaging system (Philips iU22) with a two-dimensional matrix transducer (Philips X7-2, 2500 elements, 2-7 MHz), and a multichannel data acquisition system which allows us to acquire RF channel data. Compared with alternative 3D techniques, this system is attractive because it can generate co-registered 3D photoacoustic and ultrasound images without mechanical scanning. Moreover, the lateral resolution along the azimuth and elevational directions are measured to be 0.77 ± 0.06 mm and 0.96 ± 0.06 mm, respectively, based on reconstructed photoacoustic images of phantoms containing individual human hairs. Finally, in vivo 3D photoacoustic sentinel lymph node mapping using methylene blue dye in a rat model is demonstrated.

Ultrasound imaging is being widely used in clinics to obtain diagnostic information non-invasively and in real time. A high-resolution ultrasound imaging platform, Vevo (VisualSonics, Inc.) provides in vivo, real-time images with exceptional resolution (up to 30 microns) using high-frequency transducers (up to 80 MHz). Recently, we built optoacoustic systems for probing radial artery and peripheral veins that can be used for noninvasive monitoring of total hemoglobin concentration, oxyhemoglobin saturation, and concentration of important endogenous and exogenous chromophores (such as ICG). In this work we used the high-resolution ultrasound imaging system Vevo 770 for visualization of the radial artery and peripheral veins and acquired corresponding optoacoustic signals from them using the optoacoustic systems. Analysis of the optoacoustic data with a specially developed algorithm allowed for measurement of blood oxygenation in the blood vessels as well as for continuous, real-time monitoring of arterial and venous blood oxygenation. Our results indicate that: 1) the optoacoustic technique (unlike pure optical approaches and other noninvasive techniques) is capable of accurate peripheral venous oxygenation measurement; and 2) peripheral venous oxygenation is dependent on skin temperature and local hemodynamics. Moreover, we performed for the first time (to the best of our knowledge) a comparative study of optoacoustic arterial oximetry and a standard pulse oximeter in humans and demonstrated superior performance of the optoacoustic arterial oximeter, in particular at low blood flow.

We developed a noninvasive, optoacoustic diagnostic platform for monitoring of multiple physiologic variables in inpatients and outpatients. One of the most important applications of this platform is noninvasive, rapid assessment and management of circulatory shock, a common condition in critically ill patients. At present, monitoring of circulatory shock requires measurement of central venous blood oxygenation using invasive procedures such as insertion of catheters in central veins. Hemoglobin saturation below 70% in central veins indicates circulatory shock that requires immediate treatment. We built a portable optoacoustic system for noninvasive measurement of central venous oxygenation. In this study we used the optoacoustic system and clinical ultrasound imaging systems for rapid optoacoustic probing of these veins. The optoacoustic system utilizes a custom-made, sensitive optoacoustic probe that was developed in our laboratory for monitoring of blood oxygenation in deep blood vessels. The studies were performed in human subjects with different geometry (depth, size) of the veins. The ultrasound imaging systems permitted rapid identification of specific blood vessels for optoacoustic probing. We developed a novel algorithm for continuous, realtime, and precise measurement of blood oxygenation in blood vessels. Precision of central venous oxygenation measurement obtained in the study was very high: 1%. Our results indicate that the combination of optoacoustics and ultrasound imaging systems can provide more rapid and accurate assessment and management of the circulatory shock.

We have successfully implemented a focused ultrasonic transducer for photoacoustic endoscopy. The photoacoustic endoscopic probe's ultrasound transducer determines the lateral resolution of the system. By using a focused ultrasonic transducer, we significantly improved the endoscope's spatial resolution and signal-to-noise ratio. This paper describes the technical details of the ultrasonic transducer incorporated into the photoacoustic endoscopic probe and the experimental results from which the transducer's resolution is quantified and the image improvement is validated.

Improper placement or positioning of an endotracheal tube may be lethal. Correct placement and positioning of endotracheal tubes is an essential component of life support during resuscitation from cardiac arrest or severe multiple trauma, during mechanical ventilatory support and during most surgical procedures under general anesthesia. To properly ventilate the lungs, endotracheal tubes must be inserted into the trachea rather than the esophagus, must be properly positioned in the mid-trachea and must remain properly positioned. We proposed to use optoacoustic technique for noninvasive monitoring of endotracheal tube placement and positioning. In this work we developed a compact, near infrared optoacoustic system for this application and performed in vitro tests of the system. The tests were performed in tissue phantoms (simulating overlying tissue) with an endotracheal tube. The optoacoustic measurements were noninvasively performed from the skin surface using custom-made optoacoustic probes. The placement and positioning of the endotracheal tubes were monitored with submillimeter axial and millimeter lateral resolution using the optoacoustic system. The obtained data indicate that optoacoustics can provide real-time, precise, cost-effective monitoring of placement and positioning of endotracheal tubes.

Brachytherapy is a technique commonly used in the treatment of prostate cancer that relies on the precise placement of small radioactive seeds near the tumor location. The advantage of this technique over traditional radiation therapies is that treatment can be continuous and uniform, resulting in fewer clinic visits and a shorter treatment duration. Two important phases of this treatment are needle guidance for implantation, and post-placement verification for dosimetry. Ultrasound is a common imaging modality used for these purposes, but it can be difficult to distinguish the seeds from surrounding tissues, often requiring other imaging techniques such as MRI or CT. Photoacoustic imaging may offer a viable alternative. Using a photoacoustic system based on an L7- 4 array transducer and a realtime ultrasound array system capable of parallel channel data acquisition streamed to a multi-core computer via PCI-express, we have demonstrated imaging of these seeds at an ultrasound depth of 16 mm and laser penetration depths ranging up to 50 mm in chicken tissue with multiple optical wavelengths. Ultrasound and photoacoustic images are coregistered via an interlaced pulse sequence. Two laser pulses are used to form a photoacoustic image, and at these depths, the brachytherapy seeds are detected with a signal-to-noise ratio of over 26dB. To obtain this result, 1064nm light was used with a fluence of 100mJ/cm², the ANSI limit for human skin exposure at this wavelength. This study demonstrates the potential for photoacoustic imaging as a candidate technology for brachytherapy seed placement guidance and verification.

We have implemented a hand-held photoacoustic and ultrasound probe for image-guided needle biopsy using a modified clinical ultrasound array system. Pulsed laser light was delivered via bifurcated optical fiber bundles integrated with the hand-held ultrasound probe. We photoacoustically guided needle insertion into rat sentinel lymph nodes (SLNs) following accumulation of indocyanine green (ICG). Strong photoacoustic image contrast of the needle was achieved. After intradermal injection of ICG in the left forepaw, deeply positioned SLNs (beneath 2-cm thick chicken breast) were easily indentified in vivo and in real time. Further, we confirmed ICG uptake in axillary lymph nodes with in vivo and ex vivo fluorescence imaging. These results demonstrate the clinical potential of this hand-held photoacoustic system for facile identification and needle biopsy of SLNs for cancer staging and metastasis detection in humans.

Mainstream optical microscopy technologies normally detect fluorescence or scattering, which may require undesirable labeling, but cannot directly sense optical absorption, which provides essential biological functional information. Here we reported in vivo and label-free subwavelength-resolution photoacoustic microscopy (SW-PAM) by using a waterimmersion optical objective with a 1.23 NA. Capable of detecting nonfluorescent endogenous pigments, SW-PAM provides exquisitely high optical-absorption contrast. And, as a result of background-free detection, the sensitivity of SW-PAM to optical absorption reaches 100%. SW-PAM was demonstrated with wide-field optical microscopy by imaging gold nanospheres, ex vivo cells, and in vivo vasculature and melanoma. It was shown that SW-PAM has approached the ultimate diffraction-limited optical resolution-220 nm resolution at 532 nm wavelength. Subcellular organelles, such as melanosomes, can be resolved by SW-PAM. Vasculature and early-stage melanoma were imaged with 21:1 and 34:1 contrasts, respectively, without labeling. For all these applications, SW-PAM has contrasts orders of magnitude higher than wide-field optical microscopy. Therefore, SW-PAM is expected to join the mainstream microscopy technologies.

Optical-resolution photoacoustic microscopy has demonstrated utility in imaging and characterizing microvasculature networks in vivo. This work presents a novel imaging system that integrates optical-resolution photoacoustic microscopy and fluorescence confocal microscopy for simultaneous photoacoustic and fluorescence imaging. The integrated system can acquire intrinsically registered photoacoustic and fluorescence images in a single scan. Capable of providing micrometer scale microscopic imaging of both optical absorption and fluorescence contrasts, the system demonstrates in vivo imaging of oxygen saturation and oxygen partial pressure in mouse ears.

Many diseases, normal decay and physiological functions are closely related to alterations in the metabolic rate of oxygen (MRO₂). In this study, we demonstrate that all the parameters for MRO₂ quantification can be simultaneously obtained by optical-resolution photoacoustic microscopy (OR-PAM). MRO₂ of the mouse ear under normothermia (31 °C skin temperature) and controlled systematic hyperthermia (42 °C skin temperature) was studied. As a result of hyperthermia, the MRO₂ increased by 34.1%. The tumor hypermetabolism was also demonstrated by longitudinally monitoring a melanoma growing on a mouse ear. The results show that OR-PAM, as a single noninvasive imaging modality, is well suited for quantitative MRO₂ measurement in microenvironments.

To image beyond the quasi-ballistic photon regime, photoacoustic tomography systems must rely on diffuse photons; however, there still exists an optimal illumination pattern that results in the largest number of photons reaching a target at a given depth. Many photoacoustic imaging systems incorporate weak optical focusing through oblique or dark-field illumination, but these systems are not often optimized for deep light delivery. Multiple parameters and constraints, particularly for in vivo imaging, need to be considered to determine the optimal illumination scheme for a given system: beam diameter, incident angle, pulse repetition rate, laser fluence, and target depth. Monte Carlo simulations of varied beam geometries and incident angles show the best optical illumination schemes for different imaging depths. Further an analytic model based on the diffusion theory provides a rapid method of determining the optimal beam size and incident angle for a given target depth and agrees well with the simulations. The results reveal the most efficient optical focal position to maximize the number of photons delivered to a target depth, therein maximizing the PA signal. The principles and results discussed here are not limited to the system investigated, but can be applied to other system configurations to improve the photoacoustic signal strength.

In this paper the feasibility of optical-resolution photoacoustic micro-endoscopy (OR-PAME) using image guide fibers and a unique fiber laser system is demonstrated. The image guide consists of 30,000 individual fibers in a bundle 800 μm in diameter and the diode-pumped, pulsed Ytterbium fiber laser can provide repetition rates up to 600 kHz. Phantom studies indicate 7+/-2 μm resolution. The compact, flexible nature of the image guide and the small footprint of the apparatus make it ideal for photoacoustic micro-endoscopy, as well as increasing the usability of OR-PAM for potential clinical applications.

Photoacoustic tomography detecting ultrasound signals generated from photon absorption provides optical absorption contrast in vivo for structural, functional and molecular imaging. Although photoacoustic tomography technology has grown fast in recent years, real-time photoacoustic imaging with cellular spatial resolution are still strongly demanded. We developed a photoacoustic microscopy which has video-rate imaging capability with cellular spatial resolution. The system consists of a single-element focused ultrasound transducer, a fiber-based light-delivery subsystem, a voice-coil translation stage, a motion controller, and a data acquisition subsystem. A compact cube is employed to split optical and acoustic beams. The mass of the entire scanning photoacoustic probe is less than 40 grams, which minimizes potential vibrations and inertial effects, therefore, makes it capable to scan fast. The imaging system is capable of acquiring 20 cross-sectional (B-scan) images per second over 9 mm, and up to 40 B-scan images per second over 1 mm. Focused laser beams provide a lateral resolution of five microns. Confocal deployment of optical and acoustic focuses provides higher SNR than optical scanning approach. Micron-sized carbon particles flowing in silicone tubing and in vivo blood flows were imaged in video-rate, which demonstrated the capability to image highly dynamic biological processes in vivo with cellular resolution. This real-time high-resolution photoacoustic imaging system provides a promising approach for various in vivo imaging and quantitative studies.

We have developed a novel, hybrid imaging modality, Transient Absorption Ultrasonic Microscopy (TAUM), which fuses photoacoustic microscopy with non-linear microscopy. Photoacoustic microscopy is well known for its ability to image chromophores deep (> 1 mm) in scattering media with spatial resolutions in the 10s of microns. Non-linear microscopy is well known for its exquisite spatial resolution in three dimensions. This superior spatial resolution is attributed to the fact that the collected signal has a non-linear dependence on the light intensity. This dependence confines the signal to a very small focal volume, producing optically resolved voxels. Transient absorption is a non-linear process often used to map the excited state lifetimes of molecules exhibiting low fluorescence quantum efficiency. This sensitivity to non-radiative transitions makes transient absorption an attractive process to combine with photoacoustic imaging. We have built a prototype transient absorption ultrasonic microscope, implementing off-axis photoacoustic detection to allow the use of a high-quality, high numerical aperture objective. This high-quality, commercial lens is required to provide the tight focusing needed to optimize non-linear effects. We have demonstrated the increased spatial resolution of TAUM by imaging Rhodamine 6G in a capillary tube. The capillary cross-section is fully resolved, suggesting an axial resolution of < 10 microns. A 6 MHz transducer was used in this experiment, which results in an axial resolution of ~ 400 microns when used in a traditional photoacoustic microscope. Boasting the superior penetration depth and absorption contrast offered by photoacoustic emission and complemented by spatial resolutions comparable to confocal microscopy, we believe that Transient Absorption Ultrasonic Microscopy has excellent potential for producing volumetric images with cellular/subcellular resolution in three dimensions deep inside living tissue.

Photoacoustic microscopy (PAM) offers label-free, optical absorption contrast. A high-speed, high-resolution PAM system in an inverted microscope configuration with a laser pulse repetition rate of 100,000 Hz and a stationary ultrasonic transducer was built. Four-dimensional in vivo imaging of microcirculation in mouse skin was achieved at 18 three-dimensional volumes per second with repeated two-dimensional raster scans of 100 by 50 points. The corresponding twodimensional B-scan (50 A-lines) frame rate was 1800 Hz, and the one-dimensional A-scan rate was 90,000 Hz. The lateral resolution is 0.23±0.03 μm for Au nano-wire imaging, which is 2.0 times below the diffraction limit.

In this paper, we report the experimental investigation of a novel fitting procedure which can detect and quantitatively characterize the optical contrasts of targets using diffuse optical tomography (DOT)-assisted photoacoustic tomography. The hybrid system combines a 64-channel photoacoustic system with a 9-source, 14-detector frequency-domain DOT system. A white probe was used to house the ultrasound transducer, the optical sources and detectors. The experiment was performed in the reflection mode which is more realistic to clinical applications. The fitting procedure included a complete photoacoustic forward model, which incorporated an analytical model of light transport and a model of acoustic propagation. Using the structural information from the PAT images and the background information from DOT measurements, the photoacoustic forward model was used to recover the target absorption coefficient quantitatively. Phantom absorbers, 1 cm in diameter, with absorption coefficients ranging from 0.08 to 0.28 cm^-1 were imaged at depths of up to 3.0 cm. The fitting results were at least 85% of their true values for both high and low contrast targets. Blood sample in a thin tube of radius 0.6 mm, that was simulating a blood vessel, was also imaged, and the reconstructed images and fitted absorption coefficients are presented. These results illustrate the promising application of this fitting procedure for tissue absorption coefficient characterization and consequently breast cancer diagnosis.

Quantitative measurements of the chromophores concentration in vivo present a challenge in photoacoustic imaging. The obtained signal depends on the absorbed optical density which is the product of absorption coefficient and local fluence. As a result of wavelength-dependent optical attenuation and scattering, the local fluence in biological media varies with depth and the optical wavelength. This fluence heterogeneity needs to be compensating in the order to recover the absolute absorption coefficient. In this paper we describe a new approach to recover the absolute optical absorption coefficient from measured PA signals based in combination between photoacoustic and acousto-optic signals. The present method is based on two principles, a given photon trajectory through a scattering medium can be travelled in two directions with equal probability and photons which traverse a certain volume can be labeled in that volume with the use of focused ultrasound. We give proof of the principle using Monte Carlo simulation and we demonstrate the experimental feasibility of the technique in tissue-mimicking phantom by correcting a fluence heterogeneity caused by the optical diffusion.

Optical absorption is closely associated with many physiologically important parameters, such as the concentration and oxygen saturation of hemoglobin, and it can be used to quantify the concentrations of non-fluorescent molecules. We introduce a method to quantify the absolute optical absorption based upon the acoustic spectra of photoacoustic (PA) signals. This method is self-calibrating and thus insensitive to variations in optical fluence. Factors such as the detection system bandwidth and acoustic attenuation can affect the quantification but can be canceled by measuring the acoustic spectra at two optical wavelengths. This method has been implemented on various PA systems, including optical-resolution PA microscopy, acoustic-resolution PA microscopy, and reconstruction based PA array systems. We quantified the optical absorption coefficients of phantom samples at various wavelengths. We also quantified the oxygen saturation of hemoglobin in live mice.

We use femtosecond laser pulses absorbed in a metallic transducer, namely the picosecond ultrasonics technique, for the remote optical generation and detection of GHz acoustic frequencies in single cells by pump-probe sampling. Samples are MC3T3 cells adhering on a TiAl4V alloy substrate. Both pump and probe beams are focused at the cell/transducer interface. The pump absorption yields a temperature rise in the absorbing substrate and a picosecond acoustic pulse is generated through the thermoelastic effect. The probe beam is partially reflected from the metallic interface and partially scattered by the acoustic wavefront propagating in the transparent cell. The change of reflectivity of the cell is measured as a function of the pump-probe time delay. Interferences arise from the two probe contributions causing the so-called Brillouin oscillations. Optical phase variations due to acoustic-induced changes in cell thickness are simultaneously measured. The result of a semi-analytical calculation is fitted to the experimental data. Acoustic frequencies are detected at 30 GHz in the nucleus of single osteoblast cells.

A photoacoustic (PA) imaging methodology utilizing coded optical excitation and correlation signal processing has been described. The basic principles of using relatively long coded waveforms and a matched filter signal compression to increase signal-to-noise ratio (SNR) and axial resolution are common in conventional radar and sonar systems. To emphasize these similarities, the proposed technique is called the photoacoustic sonar (or radar). We describe the implementation of the PA sonar using a near-IR intensity modulated continuous wave laser source and frequency-domain correlation processing of the acoustic response. Application of the PA sonar for imaging of biological materials with discrete chromophores was studied using tissue mimicking phantoms. The SNR gain achieved with linear chirps is analyzed and compared with conventional time-domain photoacoustics.

The interferometric measurement of laser induced thermoelastic expansion of tissue samples can be used to estimate their optical, thermal and mechanical properties. This method was used to assess the Gruneisen coefficient and optical attenuation depth for native and coagulated ex-vivo bovine liver and porcine kidney samples. The results demonstrate decreases of 54% and 60% in the optical attenuation depth in bovine liver and porcine kidney after coagulation, respectively. The Gruneisen coefficient of native porcine kidney was determined to be 58% smaller (p < 0.05) that native bovine liver. The measured Gruneisen coefficients for native and coagulated ex-vivo porcine kidney were 0.07 ± 0.03 and 0.105 ± 0.02, respectively, whereas the Gruneisen coefficients for native and coagulated liver were 0.126 ± 0.036 and 0.127 ± 0.04, respectively. Our measurements indicate significant inter sample variability due likely to inherent variations in tissue optical absorption and surface preparation.

We developed an optoacoustic biosensor intended for the detection of bloodborne microorganisms using immunoaffinity reactions of antibody-coupled gold nanorods as contrast agents specifically targeted to the antigen of interest. Optoacoustic responses generated by the samples are detected using a wide band ultrasonic transducer. The sensitivity of the technique has been assessed by determining minimally detectable optical density which corresponds to the minimum detectable concentration of the target viral surface antigens. Both ionic solutions and gold nanorods served as the contrast agent generating the optoacoustic response. The sensitivity of Nano-LISA is at least OD=10-6 which allows reliable detection of 1 pg/ml (depending on the commercial antibodies that are used). Adequate detection sensitivity, as well as lack of non-specific cross-reaction between antigens favors NanoLISA as a viable technology for biosensor development.

In this report, we present a novel 3D photoacoustic computed tomography (PACT) system for small-animal whole-body imaging. The PACT system, based on a 512-element full-ring transducer array, received photoacoustic signals primarily from a 2-mm-thick slice. The light was generated by a pulse laser, and can either illuminate from the top or be reshaped to illuminate the sample from the side, using a conical lens and an optical condenser. The PACT system was capable of acquiring an in-plane image in 1.6 s; by scanning the sample in the elevational direction, a 3D tomographic image could be constructed. We tested the system by imaging a cylindrical phantom made of human hairs immersed in a scattering medium. The reconstructed image achieved an in-plane resolution of 0.1 mm and an elevational resolution of 1 mm. After deconvolution in the elevational direction, the 3D image was found to match well with the phantom. The system was also used to image a baby mouse in situ; the spinal cord and ribs can be seen easily in the reconstructed image. Our results demonstrate that the PACT system has the potential to be used for fast small-animal whole-body tomographic imaging.

As an emerging imaging technique that combines high optical contrast and ultrasonic detection, photoacoustic tomography (PAT) has been widely used to image optically absorptive objects in both human and animal tissues. PAT overcomes the depth limitation of other high-resolution optical imaging methods, and it is also free from speckle artifacts. To our knowledge, water has never been imaged by PAT in biological tissue. Here, for the first time, we experimentally imaged water in both tissue phantoms and biological tissues using a near infrared (NIR) light source. The differences among photoacoustic images of water with different concentrations indicate that laser-based PAT can usefully detect and image water content in tissue.

Multispectral Optoacoustic Tomography (MSOT) is an emerging technique for high resolution macroscopic imaging with optical and molecular contrast. We present cardiovascular imaging results from a multi-element real-time MSOT system recently developed for studies on small animals. Anatomical features relevant to cardiovascular disease, such as the carotid arteries, the aorta and the heart, are imaged in mice. The system's fast acquisition time, in tens of microseconds, allows images free of motion artifacts from heartbeat and respiration. Additionally, we present in-vivo detection of optical imaging agents, gold nanorods, at high spatial and temporal resolution, paving the way for molecular imaging applications.

Optoacoustic tomography can visualize optical contrast in tissues while capitalizing on the advantages of ultrasound, such as high spatial resolution and fast imaging capabilities. We report herein on a novel multi-spectral optoacoustic tomography system capable of resolving dynamic contrast at video rate and showcase its performance by monitoring kidney perfusion after injection of Indocyaningreen (ICG).

Photoacoustic tomography can provide high resolution 3D images of vascular networks, making it well suited to characterising the development of tumour vasculature and its response to treatment. In this study, photoacoustic images to depths of up to 9 mm were obtained using an all optical ultrasound detection scheme. Two type of colorectal tumours (LS174T and SW1222) implanted subcutaneously in a mouse were studied. 3D photoacoustic images were obtained in vivo revealing the different vascular architectures of each tumour type and their evolution over a period of several days. The results suggest that photoacoustic imaging could play a role in providing essential pre-clinical information on tumour pathophysiology and eliciting the biological mechanisms underlying anti-angiogenic therapies and other treatments.

Acousto-optic imaging is based on ultrasound modulation of multiply scattered light in thick media. We experimentally demonstrate the possibility to perform a self-adaptive wave-front holographic detection at 790 nm, within the optical therapeutic window where absorption of biological tissues is minimized. A high-gain Te-doped Sn2P2S6 bulk crystal is used for this purpose. We image optical absorbing objects embedded within a thick scattering phantom by use of pulsed ultrasound to get a dynamic millimetric axial resolution. Our technique represents an interesting approach for breast cancer detection.

Diffuse optical techniques in tissue are insensitive to oxygenation changes inside large blood vessels, due to the high optical absorption relative to the surrounding tissue. To overcome this a hybrid technique of diffuse light modulated by focused ultrasound (US) was used to detect an acousto-optic (AO) signal from a large (1 cm diameter) blood-filled tube surrounded by a turbid medium. An injection of microbubbles, a contrast agent used in clinical diagnostic US, amplified this AO signal to an experimentally detectable level. The blood was diluted to vary its optical absorption, and a resulting change in the magnitude of the AO signal was observed. A mechanism by which microbubbles can enhance USmodulation of light is proposed by deriving a 2^nd order approximation to the Rayleigh-Plesset equation of motion for a bubble in an US field. A Monte Carlo (MC) model of a deep blood vessel geometry has also been developed: this takes into account the optical scattering from oscillating microbubbles in the blood, which is expected to vary spatially and temporally. Results of the MC model show that the US-modulated light signal is more sensitive to oxygenation changes within the blood vessel than a diffuse optical signal. Experimental results show a significant enhancement of the USmodulated optical signal when microbubbles were introduced.

In turbid media such as biological tissues, light undergoes multiple scattering. Consequently, it is not possible to focus light at depths beyond one transport mean free path in such media. To break through this limit, we proposed and experimentally demonstrated a novel technique, based on ultrasonic encoding of diffused laser light and optical time reversal, which effectively focuses light into a turbid medium. In the experimental implementation of the Time-Reversed Ultrasonically Encoded (TRUE) optical focusing, a turbid medium was illuminated by a laser beam with a long coherence length. The incident light was multiply scattered inside the medium and ultrasonically encoded within the ultrasonic focal zone. The wavefront of the ultrasonically encoded light was then time reversed by a Phase Conjugate Mirror (PCM) outside the medium. The time-reversed (or phase conjugated) optical wavefront traced back the trajectories of the ultrasonically encoded diffused light, and converged to the ultrasonic focal zone. With a commercially available photorefractive crystal as the PCM, the main approaches for increasing focusing depth are to improve the efficiencies of ultrasonic encoding and time reversal. Our recent experiments showed that light can be focused into a 5-mm thick tissue-mimicking phantom (optical thickness = 50, i.e., geometric thickness = 50 mean free paths) with a dynamically adjustable focus. The TRUE optical focusing opens a door to focusing light into turbid media or manipulating light-matter interactions.

In pulsed ultrasound modulated optical tomography (USMOT), as an ultrasound (U/S) pulse propagates, it performs as a scanning probe within the sample, and modulates the scattered light spatially distributed along the axis of propagation. By detecting and processing the modulated signal, the information along the U/S axis of the sample (1D image) is studied. The signal is modelled as a convolution of the U/S pulse and the ultrasonic and optical properties of the medium along the U/S focus. Based upon this model, a Maximum Likelihood (ML) method for image reconstruction is established. The ML data inversion technique for a pulsed USMOT signal is, for the first time to our knowledge, investigated both theoretically and practically. The ML method inverts the pulsed USMOT signal to retrieve the spatially varying properties of the sample along the U/S scanning column, and then the optical absorption property can be acquired. The results show that the ML method can serve as a useful fitting tool for a pulsed USMOT signal even in the presence of noise. The work illustrates the application of this iterative algorithm on simulated and experimental data. Experimental results using 5cm thick animal tissue phantoms (scattering coefficient μ_s is 6.5cm^-1) demonstrate that the resolution is better than 100μm using a 10MHz transducer.

We introduce a novel experimental design for non-invasive scanning optoacoustic microscopy that utilizes a parabolic surface for ultrasound focusing. We demonstrate that off-axis parabolic mirrors made of sufficiently high acoustic impedance materials work as ideal reflectors in a wide range of apertures and provide lossless conversion of a spherical acoustic wavefront into a plane wave. We further test the performance of a custom optoacoustic imaging setup which was developed and built based on these principles. The achieved resolution limit of 0.3 mm, with NA of 0.5 and the transducer bandwidth of 5 MHz, matches the resolution limit defined by diffraction. Although further improvements of current experimental setup are required to achieve resolution similar to leading microscopy systems, this proof-of-concept work demonstrates the viability of the proposed design for optoacoustic microscopy applications.

Polymer Bragg Grating Waveguides (BGW) are demonstrated as ultrasound detectors. The device is fabricated by direct electron beam lithography technique using SU-8 as the core material with grating features fabricated on the side walls of the rib waveguide. The main motivation for this design is the linear geometry of the device, which can be used in a linear array facilitating high frequency ultrasound imaging. The fabricated BGW device has a grating periodicity of 530 nm and the grating length is 500 μm. The device is tested for optical resonance spectrum. The BGW device is characterized both optically and acoustically. The BGW device is experimentally demonstrated for the detection of ultrasound waves emitted by a 25 MHz transducer. Detection sensitivity depends on optimal grating design for a steep resonance. The results demonstrate the potential use of BGW devices in highly compact array of optoacoustic detectors for high sensitivity ultrasound detection and photoacoustic imaging.

A miniature (250 μm outer diameter) photoacoustic probe for endoscopic applications has been developed. It comprises a single delivery optical fibre with a transparent Fabry Perot (FP) ultrasound sensor at its distal end. The fabrication of the sensor was achieved by depositing a thin film multilayer structure comprising a polymer spacer sandwiched between a pair of dichroic dielectric mirrors on to the tip of a single mode fiber. The probe was evaluated in terms of its acoustic bandwidth and sensitivity. Ultra high acoustic sensitivity has been achieved with a concave FP interferometer cavity design, which effectively suppresses the phase dispersion of multiple reflected beam within the cavity to achieve high finesse. The noise equivalent noise (NEP) achieved is 8 Pa over a 20 MHz bandwidth. Backward mode operation of the probe is demonstrated by detecting photoacoustic signals in a variety of phantoms designed to simulate endoscopic applications. A side-viewing probe is also demonstrated illustrating an all-optical design for intravascular imaging applications.

It is difficult to distinguish between tumor cells and surrounding cells without staining as is done in histology. We developed tyrosinase-catalyzed melanin as a reporter gene for photoacoustic tomography. Tyrosinase is the primary enzyme responsible for the production of melanin and alone is sufficient to produce melanin in non-melanogenic cells. Two cell lines were created: a stably transfected HeLa line and a transiently transfected 293 line. A phantom experiment was performed with the 293 transfected cells 48 hours post transfection and the results compared with oxygenated whole blood, B16 melanoma and 293 control cells. An in vivo experiment was performed using the transfected HeLa cells xenografted into a nude mouse ear, and then imaged. The results show strong contrast for tyrosinase-catalyzed melanin in both the 293 cells in the tube phantom as well as the in vivo result showing melanin in a nude mouse ear. Transfection increased expression in 293 cells 159 fold and image contrast compared to blood by as much as 50 fold. Due to the strong signal obtained at longer wavelengths and the decrease of blood signal at the same wavelengths, tyrosinase catalyzed melanin is a good candidate as a molecular imaging contrast agent for photoacoustic tomography.

Optical reporter genes, such as green fluorescence protein, are powerful research tools that allow visualization of gene expression. We have successfully used tyrosinase as a reporter gene for photoacoustic imaging. Tyrosinase is the key regulatory enzyme in the production of melanin which has a broad optical absorption spectrum. MCF-7 cells were stably transfected with tyrosinase under the control of an inducible promoter. For photoacoustic experiments, MCF-7 cells were resuspended at 10⁸ cells/mL and injected in 700 μm (inner diameter) plastic tubing. Photoacoustic signal of MCF-7 cells expressing tyrosinase were >20-fold greater than those of untransfected MCF-7 cells. Photoacoustic signal of tyrosinaseexpressing MCF-7 cells were approximately 2-fold lesser and greater than those of blood at 576 and 650 nm, respectively, suggesting that photoacoustic signal from blood and tyrosinase-expressing cells can be separated by dualwavelength analysis. Photoacoustic signal from tyrosinase-expressing MCF-7 cells covered by chicken tissue could even be detected at a laser penetration depth of 4 cm, suggesting that tyrosinase can be used to image gene expression in relatively deep tissues. The current data suggests that tyrosinase is a strong reporter gene for photoacoustic imaging.

Conventional photoacoustic imaging system has limited temporal resolution and hence prohibits the applications in areas such as real-time 3D imaging. In this study, an ultrafast photoacoustic imaging system with its frame rate up to 2,000Hz is demonstrated. An ultrasound transducer with plane wave excitation and a high pulse repetition rate laser are utilized to acquire the data in parallel. Additionally, the 3D data acquisition which approaches the video rate is achieved when the volume data are collected by swept scanning of a motor. The synthetic aperture focusing technique (SAFT) based on the concept of the virtual source in the elevation plane is applied to improve the imaging quality. The 3D imaging has a frame rate of 12Hz to cover a square region of 19.2mm × 19.2mm.

The feasibility of making spatially resolved measurements of blood flow using pulsed photoacoustic Doppler techniques has been explored. Doppler time shifts were quantified via cross-correlation of pairs of photoacoustic waveforms generated within a blood-simulating phantom using pairs of laser light pulses. The photoacoustic waves were detected using a focussed or planar PZT ultrasound transducer. For each flow measurement, a series of 100 waveform pairs was collected. Previous data processing methods involved rejection of poorly correlated waveform pairs; the modal velocity value and standard deviation were then extracted from the selected distribution of velocity measurements. However, the data selection criteria used in this approach is to some extent arbitrary. A new data analysis protocol, which involves averaging the 100 cross-correlation functions and thus uses all of the measured data, has been designed in order to prevent exclusion of outliers. This more rigorous approach has proved effective for quantifying the linear motion of micron-scale absorbers imprinted on an acetate sheet moving with velocities in the range 0.14 to 1.25 ms-1. Experimental parameters, such as the time separation between the laser pulses and the transducer frequency response, were evaluated in terms of their effect on the accuracy, resolution and range of measurable velocities. The technique was subsequently applied to fluid phantoms flowing at rates less than 5 mms-1 along an optically transparent tube. Preliminary results are described for three different suspensions of phenolic resin microspheres, and also for whole blood. Velocity information was obtained even under non-optimal conditions using a low frequency transducer and a low pulse repetition frequency. The distinguishing advantage of pulsed rather than continuous-wave excitation is that spatially resolved velocity measurements can be made. This offers the prospect of mapping flow within the microcirculation and thus providing insights into the perfusion of tumours and other pathologies characterised by abnormalities in flow status.

Current systems designed for deep photoacoustic (PA) imaging typically use a low repetition rate, high power pulsed laser to provide a ns-scale pulse illuminating a large tissue volume. Acoustic signals recorded on each laser firing can be used to reconstruct a complete 2-D (3-D) image of sources of heat release within that region. Using broad-beam excitation, the maximum frame rate of the imaging system is restricted by the pulse repetition rate of the laser. An alternate illumination approach is proposed based on fast scanning by a low energy (~ 1 mJ) high repetition rate (up to a few kHz) narrow laser beam (~1 mm) along the tissue surface over a region of interest. A final PA image is produced from the summation of individual PA images reconstructed at each laser beam position. This concept can take advantage of high repetition rate fiber lasers to create PA images with much higher frame rates than current systems, enabling true real-time integration of photoacoustics with ultrasound imaging. As an initial proof of concept, we compare conventional broad beam illumination to a scanned beam approach in a simple model system. Two transparent teflon tubes with diameters of 1.6 mm and 0.8 mm were filled with ink having an absorption coefficient of 5 cm^-1. These tubes were buried inside chicken breast tissue acting as an optical scattering medium. They were separated by 3 mm or 10 mm to test spatial and contrast resolution for the two scan formats. The excitation wavelength was 700 nm. The excitation source is a traditional OPO pumped by a Q-switched Nd:YAG laser with doubler. Photoacoustic images were reconstructed using signals from a small, scanned PVDF transducer acting as an acoustic array. Two different illumination schemes were compared: one was 15 mm x 10 mm in cross section and acted as the broad beam; the other was 5 mm x 2 mm in cross section (15 times smaller than the broad beam case) and was scanned over an area equivalent to broad beam illumination. Multiple images obtained during narrow beam scanning were added together to form one PA image equivalent to the single-shot broad beam one. Results of the phantom study indicate that PA images formed by narrow beam scanning excitation can be equivalent to one shot broad beam illumination in signal to noise ratio and spatial resolution. Future studies will focus on high repetition-rate laser sources and scan formats appropriate for real-time, integrated deep photoacoustic/ultrasonic imaging.

Multiple cardiovascular inflammatory biomarkers were simultaneously imaged in vivo using antibody conjugated gold nanorods (GNRs) injected into a mouse model of vascular injury stimulated by a photochemical reaction of Rose Bengal dye to green light. Mixed solutions of ICAM-1 antibody conjugated GNRs (715 nm) and E-selectin antibody conjugated GNRs (800 nm) were injected to bind to their respective inflammatory markers on the luminal surface of the inferior vena cava of a mouse. Photoacoustic intensity was measured by a commercial ultrasound probe synchronized to a pulsed laser (10-18 mJ/cm²) at 715 nm or 800 nm clearly identified the upregulation of targeted biomarkers. Histopathology on the harvested tissues confirmed inflammation. The feasibility of simultaneous photoacoustic molecular imaging of inflammation responses in cardiovascular system using a commercial ultrasound system has been demonstrated in vivo.

We previously reported the detection of bacterial antigen with immunoaffinity reactions using laser optoacoustic spectroscopy and antibody-coupled gold nanorods (Ab-NR) as a contrast agent specifically targeted to the antigen of interest. The Nano-LISA (Nanoparticle Linked Immunosorbent Assay) method has been adapted to detect three very common blood-borne viral infectious agents, i.e. human T-lymphotropic virus (HTLV), human immunodeficiency virus (HIV) and hepatitis-B (Hep-B). These agents were used in a model test panel to illustrate the performance of the Nano-LISA technique. A working laboratory prototype of a Nano-LISA microplate reader-sensor was assembled and tested against the panel containing specific antigens of each of the infectious viral agents. Optoacoustic (OA) responses generated by the samples were detected using the probe beam deflection technique, an all-optical, non-contact technique. A LabView graphical user interface was developed for control of the instrument and real-time display of the test results. The detection limit of Nano-LISA is at least 1 ng/ml of viral antigen, and can reach 10 pg/ml, depending on the binding affinity of the specific detection antibody used to synthesize the Ab-NR. The method has sufficient specificity, i.e. the detection reagents do not cross-react with noncomplementary antigens. Thus, the OA microplate reader, incorporating NanoLISA, has adequate detection sensitivity and specificity for use in clinical in vitro diagnostic testing.

Magnetic nanoparticles (MNPs) have been used extensively ex vivo for cellular and molecular separations. We recently showed that a coupled nanoparticle combining a superparamagnetic core with a thin, isolated gold shell providing strong absorption in the near infrared can be used for magnetomotive photoacoustic imaging (mmPA), a new technique in which magnetic manipulation of the particle during PA imaging greatly enhances molecular contrast specificity. This particle can also be biologically targeted for in vivo applications, where mmPA imaging provides a spatially localized readout of magnetic manipulations. As an initial test of potential in vivo molecular assays and integrated molecular therapeutics using magnetic manipulation of nanoparticles, we present experiments demonstrating PA readout of trapped magnetic particles in a flow field. An aqueous solution containing a concentration of 0.05-mg/ml 10-μM superparamagnetic iron oxide particles flowed in a 1.65-mm diameter Zeus PTFE (Teflon) sublite wall tubing at three velocities of 0.8, 1.5 and 3.0-mm/s. Opposed permanent magnets separated by 40-mm were positioned on both sides of the tube. As expected, the targeted objects can be magnetically captured and accumulated locally. By translating the magnets, a dynamic magnetic field (0.1-0.3-T) was alternately generated on the side of the tube closest to one of the magnets and created a synchronous PA motion from accumulated targeted objects. This synchronized motion can be used to differentiate the stationary background or other PA sources moving asynchronously with magnetic manipulations (e.g., moving blood) from targeted cells moving synchronously with the magnetic field. This technology can potentially provide sensitive molecular assays of cellular targets travelling in the vasculature (e.g., metastatic tumor cells).

Development of new and efficient contrast agents is of fundamental importance to improve detection sensitivity of smaller lesions. Within the family of nanomaterials, carbon nanotubes (CNT) not only have emerged as a new alternative and efficient transporter and translocater of therapeutic molecules but also as a photoacoustic molecular imaging agent owing to its strong optical absorption in the near-infrared region. Drugs, Antibodies and nucleic acids could functionalize the CNT and prepare an appropriate system for delivering the cargos to cells and organs. In this work, we present a novel photoacoustic contrast agent which is based on a unique hypoxic marker in the near infrared region, 2-nitroimidazole -bis carboxylic acid derivative of Indocyanine Green conjugated to single walled carbon nanotube (SWCNT-2nitroimidazole-ICG). The 2-nitroimidazole-ICG has an absorption peak at 755 nm and an extinction coefficient of 20,5222 M^-1cm^-1. The conjugation of this marker with SWCNT shows more than 25 times enhancement of optical absorption of carbon nanotubes in the near infrared region. This new conjugate has been optically evaluated and shows promising results for high contrast photoacoustic imaging of deeply located tumors. The conjugate specifically targets tumor hypoxia, an important indicator of tumor metabolism and tumor therapeutic response. The detection sensitivity of the new contrast agent has been evaluated in-vitro cell lines and with in-vivo tumors in mice.

Photoacoustic imaging is a dual imaging modality capable of producing images with inherent contrast on par to optical imaging techniques but with depth penetration and resolution similar to ultrasound imaging techniques. This is accomplished by a short laser pulse, capable of producing acoustic waves that contain characteristic information determined by the optical properties of the system. In this paper, a method was utilized to acquire an experimental imaging operator of a photoacoustic system with 30 transducers organized in a hemispherical array. Two imaging operators were derived from the experimental matrix in order to delineate the contribution of signals to the imaging operator that stemmed separately from photoacoustic signal and noise. After producing three individual imaging operators, singular value decomposition was performed on each. The magnitudes of the singular values produced from the decomposition were plotted against their index and the trends in the singular values were examined in order to understand the behavior of the photoacoustic system as it pertains to the quantity of photoacoustic signal and noise recorded in the useful imaging volume. As well, plots of the object space singular vectors at indices of specific interest were plotted in order to qualitatively confirm the expectation of information content in the singular vectors.

The use of a pulsed fibre laser as an excitation source for photoacoustic tomography has been investigated. Fibre lasers have the advantage of being compact, robust and efficient compared to traditional excitation sources used for photoacoustic tomography (e.g. Q-switched Nd:YAG pumped OPO or dye systems). Their high pulse repetition frequencies and adjustable pulse duration, shape and duty cycle also enables a wide range of time and frequency domain excitation methods to be investigated. A 1060nm, 20W fibre laser was used to generate acoustic waves in a tissue mimicking phantom composed of blood filled tubes immersed in a 1% solution of intralipid (μ'_s=1mm^-1) . The laser was then combined with a Fabry Perot photoacoustic imaging system to obtain 3D images of a tissue mimicking phantom and an in vivo image of the vasculature of the palm of a volunteer. This study has demonstrated that pulsed fibre lasers have potential application as an excitation source for photoacoustic imaging of superficial blood vessels.

Photoacoustic (PA) images generally suffer from high clutter levels since only one-way acoustic beam forming is used to reconstruct an image. Several methods have been presented in the ultrasound (US) literature to suppress sidelobes and reduce artifacts due to phase aberrations. Notable is a class of methods using the dual apodization with cross-correlation (DAX) method. Although a very powerful tool, DAX weighting can create artifacts in complex source environments, generally underestimating the strength of weak point scatterers and speckle regions while overestimating noise signals. This fact can work to our advantage, however, in visualizing microvessels or locating regions with a significant concentration of contrast agents using PA imaging. We examined the use of PA imaging combined with DAX processing to obtain high-contrast images of a black dye inclusion placed ex vivo into fresh bovine tissue. The tissue sample was imaged with an interleaved, real-time US/PA system including a pulse laser source operating at 20 Hz. A 5MHz linear array transducer was used both for conventional US imaging and to detect the PA signal at 720 nm wavelength. Results suggest that PA imaging with DAX combined with ultrasound imaging can produce high-contrast and high-spatial-resolution visualization of particle inclusions.

Obtaining quantified optoacoustic reconstructions is an important and longstanding challenge, mainly caused by the complex heterogeneous structure of biological tissues as well as the lack of accurate and robust reconstruction algorithms. The recently introduced model-based inversion approaches were shown to eliminate some of reconstruction artifacts associated with the commonly used back-projection schemes, while providing an excellent platform for obtaining quantified maps of optical energy deposition in experimental configurations of various complexity. In this work, we introduce a weighted model-based approach, capable of overcoming reconstruction challenges caused by perprojection variations of object's illumination and other partial illumination effects. The universal weighting procedure is equally shown to reduce reconstruction artifacts associated with other experimental imperfections, such as non-uniform transducer sensitivity fields. Significant improvements in image fidelity and quantification are showcased both numerically and experimentally on tissue phantoms.

Mesenchymal stem cells (MSCs) are versatile in many tissue engineering applications and have the potential to be used for cellular therapies because they can differentiate into many cell types. Specifically, the use of MSCs for the treatment of ischemic disease is promising because MSCs can express characteristics of vascular cells. MSCs can promote vascular growth at the site of injury after delivery using a PEGylated fibrin gel. In order to quantitatively assess in vivo delivery and differentiation of MSCs, a non-invasive and high-resolution imaging technique is required. In this study, the combined ultrasound and photoacoustic imaging was demonstrated to monitor MSCs labeled with citrate-stabilized gold nanoparticles (Au NPs). It was observed that uptake of nanoparticles did not have a significant effect on cell viability and proliferation over a two-week period. Four different cell concentrations of either the non-labeled MSCs or the Au NP labeled MSCs were embedded in the tissue mimicking gelatin phantom. The ultrasound and photoacoustic signals were acquired and quantitatively analyzed to assess sensitivity and accuracy of the developed imaging approach. Furthermore, based on the results, the feasibility of in vivo ultrasound and photoacoustic imaging of MSCs was discussed.

Delivery of contrast agents and their interaction with cells is emerging as an important tool in cancer imaging and therapy. An alternative to traditional molecular targeting schemes that induce endocytotic uptake of contrast agents in cells is presented here. Specifically, the application of high-intensity, focused ultrasound (HIFU) was used to enhance uptake of gold nanorods in pancreatic cancer cells in vitro. A significant increase was observed in gold nanorod uptake when cells were incubated with nanorods and treated with HIFU. Additionally, inclusion of liquid-filled, perfluorocarbon (PFC) microdroplets in cell samples incubated with nanorods and treated with HIFU exhibited greater uptake of gold over those samples exposed to HIFU without microdroplets. Furthermore, the level of acoustic pressure required to increase nanoparticle uptake did not significantly decrease cell viability. Therefore, improved intracellular delivery of nanoparticle contrast agents is possible using HIFU without compromising cell viability. Since nanoparticle delivery is mechanically induced, this method can apply to a broad range of cancer imaging and therapy applications.

We have investigated the potential of emerging photoacoustic imaging and nuclear imaging in monitoring of drug delivery by using a newly developed dual-modality contrast agent. After the contrast agent composed of gold nanorods (GNRs) was produced, it was radiolabeled by [¹²⁵I] with high yield and without disturbing the optical properties of the contrast agent. Photoacoustic and nuclear imaging were conducted to visualize the distribution of GNRs in articular tissues of rat tail joints in situ. Findings from the two modalities corresponded well with each other. Using the current imaging systems, GNRs down to a concentration of 10 pM in biological tissues and with a radioactive label of 5 μCi can be imaged. Moreover, by radiolabeling the GNRs, the in vivo behaviors of the contrast agent can be monitored conveniently using γ-camera, allowing validation of the findings from emerging photoacoustic technique. Enabled by the high sensitivity of nuclear imaging, whole-body and longitudinal studies of the biodistribution of GNRs contrast agent can be performed noninvasively and repeatedly in the same animal. The highly efficient method reported here provides an extensively useful tool for the guidance of design and development of new gold nanoparticles as target-specific agents for both diagnostics and therapy.

Convergence of light towards a desired location in optically diffusive and aberrative media is highly relevant to optical methods of biomedical imaging. In this study, we demonstrated the feasibility of employing photoacoustic signals originating from an optically absorptive target as feedback for shaping the incident wavefront to increase optical energy density at the absorptive target. The wavefront of a collimated laser beam was shaped by an array of two-dimensional MEMS deformable mirrors and then transmitted through optically scattering paraffin. The phase of light reflected by each mirror was varied (0-2π) iteratively to maximize the amplitude of the photoacoustic signal. The photoacoustic signal potentially provides a non-invasive and reliable feedback for manipulating spatial phase distribution of light to achieve focusing in diffusive media and may facilitate optical imaging at greater depths.

Visualizing cells in three-dimensional (3D) scaffolds has been one of the major challenges in tissue engineering. Current imaging modalities have limitations. Microscopy, including confocal microscopy, cannot penetrate deeply (> 300 μm) into the scaffolds; X-ray micro-computed tomography (micro-CT) requires staining of the structure with a toxic agent such as osmium tetroxide. Here, we demonstrate photoacoustic microscopy (PAM) of the spatial distribution and temporal proliferation of melanoma cells inside three-dimensionally porous scaffolds with thicknesses over 1 mm. Melanoma cells have a strong intrinsic contrast which is easily imaged by label-free PAM with high sensitivity. Spatial distributions of the cells in the scaffold were well-resolved in PAM images. Moreover, we chronically imaged the same cell/scaffold constructs at different time points over 2 weeks. The number of cells in the scaffold was quantitatively measured from the PAM volumetric information. The cell proliferation profile obtained from PAM correlated well with that obtained using the traditional 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We believe that PAM will become a useful imaging modality for tissue engineering applications, especially when thick scaffold constructs are involved, and that this modality can also be extended to image other cell types labeled with contrast agents.

Photoacoustic (PA) imaging has been used to image soft tissue due to its high contrast and high spatial resolution. The generation of PA signal is based on the object's absorption characteristic to the emitted electromagnetic energy. Typically, a Q-switched Nd:YAG laser providing mJ pulse energy is suitable for biomedical PA applications. However, such laser is relatively bulky and expensive. An alternative way is to use a diode laser. A diode laser can generate laser pulse at much higher pulse repetition frequency (PRF). However, the output power of the diode laser is too low for effective PA generation. One method to overcome this problem is to increase the transmission energy using coded excitation. The coded laser signals can be transmitted by a diode laser with high PRF and the signal intensity of the received signal can be enhanced using pulse compression. In this study, we proposed a chirp coded excitation algorithm for a diode laser. Compared to Golay coded excitation seen in the literature, the proposed chirp coded excitation requires only a single transmission. Chirp-coded PA signal was generated by tuning the pulse duration of individual laser pulses in time domain. Result shows that the PA signal intensity can be enhanced after matched filtering. However, high range side-lobes are still present. The compression filter is an important tool to reduce the range side-lobes, which is subject to further investigation.

We propose and experimentally demonstrate a new photoacoustic (PA) excitation and analysis method which achieves an almost complete utilization of the available time and frequency windows. The method, which enables spectral and spatial characterization of flow, is based on interleaving tens of tone-burst sequences at equally spaced frequencies. Depending on the application, the interleaved signals can be generated by a single optical source or by multiple sources, possibly at different wavelengths. Upon reception, the responses corresponding to the different tone-burst sequences are spectrally de-multiplexed. As demonstrated in the current work, this method can be used to improve the SNR of PA systems based on optical sources with limited peak power. Alternatively, if the interleaved excitation signals are at different wavelengths, the PA responses can be used for multispectral characterization of the medium.

By combining the chemical specificity of stimulated Raman excitation with the deep penetration of photoacoustic imaging, molecular imaging can be achieved without external markers deep into the biological tissue.

Various optical structures have been investigated for high-frequency optoacoustic generation via thermoelastic effect, including metal films, mixture of polydimethylsiloxane (PDMS) and carbon black, two-dimensional (2-D) gold nanostructure with PDMS film, etc. However, they suffer from either low light absorption efficiency which affects the amplitude of generated ultrasound, or thick films that attenuate the amplitude and restrict its spectra bandwidth. Here we propose a novel one-dimensional photonic crystal-metallic (PCM) structure, which can be designed to absorb 100% optical energy of specific wavelengths in a total-internal-reflection geometry. The unique configuration enables us to choose suitable polymer films on top of the metallic structure, which can act as an ideal ultrasound transmitter to generate broad-band ultrasound with high conversion efficiency. Experimental results show that the PCM structure generated several times stronger ultrasound pressure than our previously demonstrated 2-D gold nanostructures [Appl. Phys. Lett. 89, 093901 (2006)]. Moreover, the generated ultrasound exhibited almost the same frequency spectrum as the input laser pulse (duration width 6 ns). This shows that the PCM structure has great potential to generate broad-band ultrasound signal. It is also important to mention that the simple PCM structure with the polymer film forms a Fabry-Pérot resonator and can play a role of an ultrasound receiver, which provides a convenient method to construct a broad-band and all-optical ultrasound transducer.

Photoacoustic (PA) Imaging can estimate the spatial distribution of oxygen saturation (sO₂) and total hemoglobin concentration (HbT) in blood, and be co-registered with B-Mode ultrasound images of the surrounding anatomy. This study will focus on the development of a PA imaging mode on a commercially available array based micro-ultrasound (μUS) system that is capable of creating such images. The system will then be validated in vivo against a complementary technique for measuring partial pressure of oxygen in blood (pO₂). The pO₂ estimates are converted to sO₂ values based on a standard dissociation curve found in the literature. Finally, the system will be used for assessing oxygenation in a murine model of ischemia, both during injury and recovery.

Photoacoustic (PA) tomography is a rapidly developing imaging modality which can provide high contrast and spatial-resolution images of light absorption distribution in tissue. However, the quantitative reconstruction of absorption distribution is still a challenge. In this study, we propose an adaptive and quantitative reconstruction algorithm for reducing amplification of noises and artifacts in deep position due to light fluence compensation. In this method, the quantitative processing is integrated into the iterative reconstruction, and absorption coefficient distribution is iteratively updated. At each iteration step, the residual is calculated from detected PA signals and the signals calculated from a forward model by using the initial pressure which is calculated from the production of voxel value and the light fluence. By minimizing the residual, the reconstructed values are converged to the true absorption coefficient distributions. Since this method uses a global optimized compensation, better CNR can be obtained. The results of simulation and phantom experiment indicate that the proposed method provide better CNR at deep region. We expect that the capability of increasing imaging depth will broaden clinical applications.

We explore the feasibility of using photoacoustic imaging based on a focused transducer to characterizing acute myocardial ischemia at different stage. In this study, we blocked rat left anterior coronary descending artery (LAD) to induce the acute myocardial ischemia. The results show that the intensity and areas of photoacoustic images of myocardial decrease with the LAD time increasing, which suggests that photoacoustic imaging has a potential for diagnosis of acute myocardial ischemia.

Three-dimensional scaffolds provide physical support and an adjustable microenvironment to facilitate vascularization of ischemic tissues; however, in vivo imaging of scaffold functioning is still challenging. Micro-CT, the current frequentlyused imaging modality for scaffold characterization, provides poor contrast for wet scaffold, which limits its in vivo applications. In this paper, dual modes of photoacoustic microscopy (PAM), using acoustic resolution PAM (AR-PAM) and optical resolution PAM (OR-PAM), were employed for imaging scaffolds in blood as well as in chicken breast tissues. By choosing different wavelengths, 570 nm and 638 nm, we spectroscopically differentiated the photoacoustic signals generated from blood and from carbon nanotube incorporated scaffolds. The ex vivo experiments demonstrated a lateral resolution of 45 μm and a maximum penetration of ~2 mm for AR-PAM, and a lateral resolution of 3 μm and a maximum penetration of ~660 μm for OR-PAM. OR-PAM further quantified the average pore size of scaffolds to be 100-200 μm in diameter. Our results suggest that PAM is a promising tool for in vivo monitoring of scaffold-induced angiogenesis as well as the degradability of scaffolds themselves.

In this study, we characterized a newly developed imaging system, "dual illumination mode photoacoustic tomography (PAT) system". The PAT system can simultaneously or separately illuminate biological tissues from a forward and backward direction toward an array transducer. The shape of the custom-made transducer is rectangular, which allows direct illumination of tissue surfaces in front of the array transducer through a holding plate from the backward direction. The transducer frequency was designed at 1 MHz by considering the trade-off relationship between ultrasound attenuation and image resolution. A Ti:Sa laser optically pumped with a Q-switched Nd:YAG laser, having a tunable wavelength of 700 to 900 nm, was chosen for deep light penetration in tissues. The laser light was sufficiently expanded and homogenized to keep the level of laser-pulse fluence on the sample surface under the ANSI safety limit. System performance was tested with phantoms. The results of our study showed that the system visualized all the absorbers embedded in a 50-mm-thick tissue-mimicking phantom with a lateral resolution of 2~3 mm.

In this study, we propose an advanced model-based reconstruction algorithm for three-dimensional photoacoustic imaging. The algorithm is based on accurate forward photoacoustic models and an optimization algorithm which minimizes the square of the error between the measured acoustic signals and the signals predicted by the forward models. The forward photoacoustic models incorporate system-configuration and detector-dependent factors such as frequency response and finite size effect. A conjugate gradient-based optimization algorithm is used for reconstructing images. In addition, we make use of the symmetry and locality of the photoacoustic waves in the computations of the forward photoacoustic models in order to reduce the memory requirements and computation time in three-dimensional image reconstruction. The results show that the proposed algorithm provides high-resolution and high-quality photoacoustic images.

Ultrasonic detectors are commonly calibrated by finding their response to incident plane waves. However, in optoacoustics, the response to broadband point sources is required. To induce such sources using the optoacoustic effect, the illuminated object's dimensions must be smaller than the resolution achievable by the optoacoustic system. The main difficulty in such measurements is that the magnitude of the field emitted by such sources is proportional to their dimensions, and thus may be weak compared to parasitic sources in the setup. In this work we experimentally demonstrate two methods for calibrating acoustic detectors. In both methods, acoustic sources are optoacoustically induced in large optically absorbing slabs. Despite the large dimensions of the illuminated objects, the geometry used yields wide-band acoustic fields, which are perceived by the detectors as originating from point sources.

Photoacoustic systems can produce high-resolution, high-contrast images of vascular structures. To reconstruct images at very high-resolution, signals must be collected from many transducer locations, which can be time consuming due to limitations in transducer array technology. A method is presented to quickly discriminate between normal and abnormal tissue based on the structural morphology of vasculature. To demonstrate that the approach may be useful for cancer detection, a special simulator that produces photoacoustic signals from 3D models of vascular tissue is developed. Results show that it is possible to differentiate tissue classes even when it is not possible to resolve individual blood vessels. Performance of the algorithm remains strong as the number of transducer locations decreases and in the presence of noise.

In industrialized countries, cardiovascular diseases remain the main cause of mortality. The detection of atherosclerosis and its associated plaque using imaging techniques allows studying the efficacy of new drugs in vivo. Intravascular ultrasound (IVUS) imaging has been demonstrated to be a powerful tool to uncover structural information of atherosclerotic plaques. Recently, intravascular photoacoustic (IVPA) has been combined with IVUS imaging to add functional and/or molecular information. The IVPA/IVUS combination has been demonstrated in phantoms and ex vivo tissues to provide relevant information about the composition of the plaque, as well as its vulnerability. In this work, we extend previous work by developing a combined IVPA/IVUS system using a rotating ultrasound transducer in a catheter to which an optical fiber is attached. In addition, a third modality was included through fluorescence detection in the same fiber at a distinct wavelength from PA, opening the door to complementary information using fluorescence activatable probes. Cylindrical silicon phantoms with inclusions containing fluorophores or ink were used to validate the system. Bleaching of the fluorophore by the pulsed laser used for photoacoustic was quantified. IVUS images were obtained continuously and used to co-register photoacoustic and fluorescence signals.

We compare imaging systems using continuous-wave (CW) lasers with a chirped intensity modulation frequency to pulsed lasers. We show that the resolution is the same in both cases. We also compare the signal-to-noise ratio (SNR) of the two systems assuming the fluence is set by the American National Standards Institute (ANSI) limits. Although the SNR depends on several parameters, we find that it is about 20 dB to 30 dB larger for pulsed lasers for reasonable values of the parameters. However, CW diode lasers have the advantage of being compact and relatively inexpensive, which may outweigh the slightly lower SNR in many applications.

Optical systems based on near infrared spectroscopy probe biological tissue oxygenation of a relatively large region. The acousto-optic (AO) method can tag photons by focused ultrasound in a region of interest within the tissue for potential localized oxygenation sensing. This study aimed to compare the regional sensitivity between the optical and AO sensing techniques. The regional sensitivity was defined as the amount of change observed in the measured signal in response to a small localized change in the optical absorption. In both reflection and transmission configurations, optical systems based on a single source and detector have been shown to be more sensitive to the absorption variation in the superficial region. The results demonstrate that the AO sensitivity region depends on the location of the ultrasound focal region as well as the distribution of the optical fluence. In the transmission mode, the optimal AO sensitivity region has been found to be in the ultrasound focal region. In the reflection mode, however, the optimal sensitivity region of AO does not always coincide with the location of the ultrasound focal region. Instead, the AO measurement is also sensitive to the local absorption change in regions between the ultrasound focal region and the optical probes. In general, the AO method can probe deeper into the phantom as compared to the optical measurements.

We present a fast inversion algorithm for quantitative two- and three-dimensional optoacoustic tomography. The algorithm is based on an accurate and efficient forward model, which eliminates the need for regularization in the inversion and can achieve real-time performance. The reconstruction speed and other algorithmic performances are demonstrated using numerical simulation studies and experimentally on tissue-mimicking optically heterogeneous phantoms and small animals. In the experimental examples, the model-based reconstructions manifested correctly the effect of light attenuation through the objects and did not suffer from the artifacts which usually afflict the commonly used filtered backprojection algorithms, such as negative absorption values.

One of the major challenges of optoacoustic imaging is that it involves relatively weak acoustic signals, which need to be detected with high signal-to-noise ratio (SNR). Because the SNR is generally proportional to the area of the detector's face, large detectors are commonly used. Although the use of such detectors improves the SNR, it may lead to significant signal distortion resulting in artifacts in the reconstructed optoacoustic image. In this work we developed a method for simulating the spatially dependent frequency response of acoustic detectors with arbitrary surface shapes. The frequency response is incorporated into a forward model for optoacoustic propagation. Our method can be used for designing detectors with desired properties and reducing reconstruction artifacts caused by the response of finite-size detectors.

We designed and fabricated a probe for endoscopic photoacoustic imaging with a structure that includes an optical fiber to deliver laser pulses and polymer microring resonators on transparent fused silica substrate. To calibrate the performance of the probe, a 6 μm carbon fiber embedded in an agarose gel phantom was imaged. At a depth of 2.7 mm from the probe edge, radial resolution up to 21 μm and transverse resolution of 750 μm were obtained. Transverse resolution can be improved using fibers with small core sizes. Experimental results show that the devised probe has potential for high-resolution photoacoustic endoscopy.

Recently, photoacoustic imaging has been intensively studied for blood vessel imaging, and shown its capability of revealing vascular features suggestive of malignancy of breast cancer. In this study, we explore the feasibility of visualization of micro-calcifications using photoacoustic imaging. Breast micro-calcification is also known as one of the most important indicators for early breast cancer detection. The non-ionizing radiation and speckle free nature of photoacoustic imaging overcomes the drawbacks of current diagnostic tools - X-ray mammography and ultrasound imaging, respectively. We employed a 10-MHz photoacoustic imaging system to verify our idea. A sliced chicken breast phantom with granulated calcium hydroxyapatite (HA) - major chemical composition of the breast calcification associated with malignant breast cancers - embedded was imaged. With the near infared (NIR) laser excitation, it is shown that the distribution of ~500 μm HAs can be clearly imaged. In addition, photoacoustic signals from HAs rivals those of blood given an optimal NIR wavelength. In summary, photoacoustic imaging shows its promise for breast micro-calcification detection. Moreover, fusion of the photoacoustic and ultrasound images can reveal the location and distribution of micro-calcifications within anatomical landmarks of the breast tissue, which is clinically useful for biopsy and diagnosis of breast cancer staging.

Photo-acoustic imaging (PAI) is a hybrid imaging modality, which can offer a high contrast tomographic image with ultrasound like resolution in depth of centimeters. Additionally, it has been studied well as functional imaging modality using characteristics that can distinguish by absorption spectra. Our purpose is to investigate the image quality and potential of photo-acoustic (PA) image as a preliminary study toward the medical diagnosis applications. For this purpose, firstly we focused on the difference of image quality between photo-acoustic image and ultrasound image using array transducers system. Secondly we examined the effect of illumination method on photo-acoustic image quality. We compared both photo-acoustic image and ultrasound image of a phantom using original PAI experimental apparatus system with 192ch PZT array probe. Resolution of PAI and ultrasound image could be revealed, based on the optimized reconstruction method for each using each element data. We demonstrated higher resolution and higher contrast of photo-acoustic image than ultrasound image. To examine the effect on photo-acoustic image quality, we analyzed depth-dependent signal attenuation in scattering media under various illumination methods such as the expansion of illumination area. Our analysis with experiments and Monte Carlo simulation are performed to show the necessity for illumination optimization depend on the application and probe size.

In this study, we report on using a 50-MHz functional photoacoustic microscopy (PAM) to transcranially image the cross-section and hemoglobin oxygenation (SO₂) changes of single mouse cortical vessels in response to left forepaw electrical stimulation. Three difference levels of the cortical vessels (i.e., with different-sized diameters of 350, 100 and 55 μm) on activated regions were marked to measure their functional cross-section and SO₂ changes as a function of time. Electrical stimulation of the mouse left forelimb was applied to evoke functional changes in vascular dynamics of the mouse somatosensory cortex. The applied current pulses were with a pulse frequency of 3 Hz, pulse duration of 0.2 ms, and pulse amplitude of 2 mA. The cerebrovascular cross-section changes, which indicate changes in cerebral blood volume (CBV), were probed by images acquired at 570 nm, a hemoglobin isosbestic point, while SO₂ changes were monitored by the derivatives of 560-nm images normalized to 570-nm ones. The results show that vessel diameter and SO₂ were significantly dilated and increased when compared with those of the controlled ones. In summary, the PAM shows its promise as a new imaging modality for transcranially functional quantification of single vessel diameter (i.e., CBV) and SO₂ changes without any contrast agents applied during stimulation.

The reconstruction algorithms commonly used in photoacoustic tomography do not account for the effects of acoustic attenuation on the measured time-domain signals. For experimental measurements made in biological tissue, acoustic attenuation causes the high frequency components of the generated ultrasound signals to be significantly reduced. When this signal loss is neglected, it manifests as a depth dependent magnitude error and blurring of features within the reconstructed photoacoustic image. Here, the approach described by Treeby et al. [Inverse Problems 26(11), p. 115003, 2010] is applied to the reconstruction of high-resolution threedimensional photoacoustic images of vascular networks around the abdomen of a pregnant female mouse. The reconstruction is based on the idea of time reversal in which a numerical model of the acoustic forward problem is run backwards in time. Compensation of acoustic attenuation in the inverse problem is achieved by using a forward model that accurately accounts for the frequency dependent attenuation experimentally observed in biological tissue. The regularisation of the inverse problem is discussed, and the methodology demonstrated through the reconstruction of several images. Clear improvements in image magnitude and resolution are seen when attenuation compensation is included.

Photoacoustics has been widely studied as a combined imaging modality of both optical and acoustical methods. The merits of the photoacoustic imaging are realizing the full potentials of pulsed laser-tissue interaction. As the photoacoustic waves can be induced at chromophores by pulsed lased irradiation through a thermoelastic process, it covers a wide range of frequency. In order to take advantages of the wide range frequency characteristics, we employed not PZT, but piezoelectronic copolymer film, P(VDF/TrFE) film, with various thickness ranging from 20 to 100 μm as photoacoustic transducers. Because blood vessels play a key role in homeostasis, we experimentally demonstrated blood vessels phantom using second harmonic generation of Q-switched Nd:YAG laser and Ti:sapphire nanosecond laser pulses through optical fiber transmission. The detected photoacoustic waveforms showed distinctive time-of-flight signals. The photoacoustic signals were sensitive to temperature, absorption coefficients of chromophores, and diameters of the phantom vessels. Hemoglobin oxygen saturation could be easily derived from the multi wavelength photoacoustic signals using differential optical absorption characteristics. These results proved the functional quantitative photoacoustic imaging using the signal characteristics. A multivariate photoacoustic imaging approach must be promising to convenient diagnosis.

A modified quantitative inversion algorithm is presented that minimizes the effects of internal acoustic reflections or scattering in tomographic optoacoustic images. The inversion procedure in our model-based algorithm consists in solving a linear system of equations in which each individual equation corresponds to a given position of the acoustic transducer and to a given time instant. Thus, the modification that we propose in this work consists in weighting each equation of the linear system with the probability that the measured wave is not distorted by reflection or scattering phenomena. We show that the probability that a reflected or scattered wave is detected at a given position and at a given instant is approximately proportional to the size of the area in which the original wave could have been generated, which is dependent on the position of the transducer and on the time instant, so that such probability can be used to weight each equation of the linear system. Thereby, the contribution of the waves that propagate directly to the transducer to the reconstructed images is emphasized. We experimentally test the proposed inversion algorithm with tissue-mimicking agar phantoms in which air-gaps are included to cause reflections of the acoustic waves. The tomographic reconstructions obtained with the modification proposed herein show a clear reduction of the artefacts due to these acoustic phenomena with respect to the reconstructions yielded with the original algorithm. This performance is directly related to in-vivo small animal imaging applications involving imaging in the presence of bones, lungs, and other highly mismatched organs.

Ultrasonic attenuation in biomaterials limits the quality and resolution of ultrasonic imaging. This work presents a simple and reliable method to investigate acoustic attenuation of biological tissue samples and liquids in order to improve reconstruction algorithms for photoacoustic imaging. For this purpose broadband high-frequency single transmission measurements were performed. The spectra of the acquired signals were compared to reference measurements in distilled water. Unfocused broadband piezoelectric transducers were used as ultrasound source and detector. Moreover, laser generated ultrasound, which provides more intensity and signals with higher bandwidth, was used to measure acoustic attenuation. Only few studies concerned with attenuation of fat tissue performed broadband high frequency measurements and to our knowledge none of those used the simple and reliable single transmission approach with unfocused ultrasound. Our results for acoustic attenuation in olive oil show good agreement with literature. Many studies indicate linear frequency increase of attenuation of fat tissue. However, we observed significant non-linear frequency behaviour of porcine subcutaneous fat tissue at room temperature with a power-law exponent of around 1.45.

Our goal is to develop a photo-acoustic imaging (PAI) system which offers functional image of living tissues and organs with high resolution. In order to obtain high resolution image, we implemented the Fourier transform reconstruction algorithm which determines an optical absorption distribution from photo-acoustic (PA) signals. However, resolutions of reconstructed images were restricted by the sensor directionality, finite scan width and frequency band width. There was an essential requirement to optimize the sensor specification. In this study, we demonstrated relationship between image resolution and sensor specification by simulation and experiment. In our experimental system, PA signals were acquired by line scanning of our fabricated P(VDF/TrFE) film sensor. As results of simulations and experiments, lateral resolutions of PA images were restricted by the directionality of sensor. Furthermore, by limiting scan width and frequency band width, lateral resolution is decreased at deep region. The optimum sensor specification depends on the imaging region due to some trade-offs, for example, a sensor with wider directionality has less sensitivity, wider scan in same step increases acquisition time. Therefore, the results could indicate the possibility of optimizing sensor directionality, scan width and frequency band width for various depths and volumes of imaging region.

We developed a second-generation optical-resolution photoacoustic microscope (OR-PAM) with a novel acoustic detection scheme, which improved upon the sensitivity of our first-generation system by 18 dB. Moreover, translating the imaging head instead of the living object improved the scanning speed by a factor of 5, widening the field of view within the same acquisition time. The anatomy and hemoglobin oxygen saturation of an entire mouse ear was imaged in vivo.

The question of whether particle size affects modulation efficiency, defined as the ratio of ultrasound-modulated fluorescence (UMF) signal to DC (direct current) signal, of the fluorescence emission from four different sized fluorescent particles was investigated experimentally. The four particles are streptavidin-conjugated Alexa Fluo 647 (~5 nm in diameter) and three carboxylate-modified fluorescent microspheres (FM) with different diameters of 0.02, 0.2, and 1.0 μm. Modulation efficiency was evaluated as a function of the fluorophore size and fluorophore concentration. The modulation efficiency was improved about two times when the size of the fluorescent particles is increased from 5 nm to 1 μm. This result implies that using large fluorescence particles can slightly improve the modulation efficiency but the improvement is limited.

A combined ultrasonic and photoacoustic imaging system is presented that is capable of deep tissue imaging. The system consists of a modified clinical ultrasound array system and tunable dye laser pumped by a Nd:YAG laser. The system is designed for noninvasive detection of sentinel lymph nodes and guidance of needle biopsies for axillary lymph node staging in breast cancer patients. Using a fraction of the American National Standards Institute (ANSI) safety limit, photoacoustic imaging of methylene blue achieved penetration depths of greater than 5 cm in chicken breast tissue. Photoacoustic imaging sensitivity was measured by varying the concentration of methylene blue dye placed at a depth of 3 cm within surrounding chicken breast tissue. Signal-to-noise ratio, noise equivalent sensitivity, and axial spatial resolution were quantified versus depth based on in vivo and chicken breast tissue experiments. The system has been demonstrated in vivo for detecting sentinel lymph nodes in rats following intradermal injection of methylene blue. These results highlight the clinical potential of photoacoustic image-guided identification and needle biopsy of sentinel lymph nodes for axillary staging in breast cancer patients.

This work investigates a forward model associated with intra-lumenal detection of acoustic signal originated from transient thermal-expansion of the tissue. The work is specific to intra-lumenal thermo-acoustic tomography (TAT) which detects the contrast of tissue dielectric properties with ultrasonic resolution, but it is also extendable to intralumenal photo-acoustic tomography (PAT) which detects the contrast of light absorption properties of tissue with ultrasound resolution. Exact closed-form frequency-domain or time-domain forward model of thermally-induced acoustic signal have been studied rigorously for planar geometry and two other geometries, including cylindrical and spherical geometries both of which is specific to external-imaging, i.e. breast or brain imaging using an externally-deployed applicator. This work extends the existing studies to the specific geometry of internal or intra-lumenal imaging, i.e., prostate imaging by an endo-rectally deployed applicator. In this intra-lumenal imaging geometry, both the source that excites the transient thermal-expansion of the tissue and the acoustic transducer that acquires the thermally-induced acoustic signal are assumed enclosed by the tissue and on the surface of a long cylindrical applicator. The Green's function of the frequency-domain thermo-acoustic equation in spherical coordinates is expanded to cylindrical coordinates associated with intra-lumenal geometry. Inverse Fourier transform is then applied to obtain a time-domain solution of the thermo-acoustic pressure wave for intra-lumenal geometry. Further employment of the boundary condition to the "convex" applicator-tissue interface would render an exact forward solution toward accurate reconstruction for intra-lumenal thermally-induced acoustic imaging.

We developed a tri-modal system combining photoacoustic (PA) tomography, thermoacoustic (TA) tomography, and ultrasound (US) imaging. Acquired images of an excised dog prostate were compared to histology results. All three modalities can image distinct features. Features like the urethra were shown in both TA and US images, but TA gave a higher contrast-to-noise ratio. Fibrous tissue was more clearly imaged by TA, while the duct structure was better shown in PA images. These experimental results demonstrate the potential advantages of our tri-modal imaging system.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology providing visualization of superficial structures in vivo with optical-absorption contrast. High resolution is possible as the lateral spatial resolution is determined by the optical spot size rather than acoustic detection. The imaging speed is dictated by both the beam scanning speed and the laser pulse repetition rate. We are developing a realtime OR-PAM system that uses a high repetition rate pulsed laser and high speed XY mirror galvanometers. We have demonstrated OR-PAM imaging by employing a diode-pumped pulsed Ytterbium fiber laser with a pulse repetition rate ranging from 20 kHz - 600 kHz, second harmonic generation at a wavelength of 532 nm and average output power up to 13 W. In our study, we utilized 0.13μJ ~1-ns pulses. A photoacoustic probe consisting of a 45-degree glass prism in an optical index-matching fluid is used to transmit the focused output of the laser to the sample and also to reflect exiting photoacoustic signals to an ultrasound transducer. Phantom studies with a ~7.5-μm carbon fiber demonstrate the ability to image with ~7-μm optical lateral spatial resolution. Combined with a fast-scanning mirror oscillating at 800 (B-scan) lines per second, we demonstrate a system capable of C-scan imaging at 4 frames per second. These near-realtime frame-rates should permit clinical applications.

Metal needles are commonly used for drug delivery or biopsy collection in clinical settings. Needle deflection and deformation can occur when inserting needles into soft, non-homogeneous tissues which can affect the location accuracy of insertion. Therefore, the ability to visualize both anatomical surrounding structures and the advancing needle is required. Ultrasound is commonly used for image-guidance of needles; however, specular reflections from the metal surface can deflect the acoustic beam away from the transducer when the needle is even slightly angled from the US transducer thereby rendering the needle invisible in the image. Photoacoustic imaging has been proposed for guidance of metal needles and other metal objects in-vivo. The high optical absorption coefficient of stainless steel can provide high photoacoustic imaging contrast. The photoacoustic signal is produced omni-directionally from the metal surface allowing for greater detection of needles at increasing injection angles compared to ultrasound imaging. In the current work, needles were inserted into excised tissue and imaged using an ultrasound array transducer and a pulsed 800 nm laser. The results showed that at a shallow 10° insertion angle, the photoacoustic ratio of needle signal to background was four-times higher compared to ultrasound. Furthermore, the surrounding tissue composition was observed to have an effect on photoacoustic signal enhancement which correlated with the change of the Grüneisen coefficient of the surrounding tissue environment, suggesting that the photoacoustic signal amplitude could be used to ascertain surrounding tissue composition. Photoacoustic imaging provides sufficient depth penetration for this application and offers excellent image contrast.

Herein we suggest and experimentally validate a novel thermoacoustic imaging method that relies on near-field exposure of the object to ultrashort impulses of safe radiofrequency energies. The physical rationale behind the Near-field Radiofrequency Tomography (NRT) is the well known ability of biological tissues to absorb a very significant portion of energy when closely coupled to radiofrequency and microwave sources. Compared to existing thermoacoustic imaging implementations, NRT offers a significantly simpler and cost-effective technology that uses high energy impulses instead of expensive and inefficient carrier-frequency amplification methods, making it possible to achieve significantly better imaging resolution without compromising thermoacoustic signal strength.

Functional photoacoustic microscopy is a valuable tool in quantifying hemoglobin oxygenation within single vessels. In several functional studies with this tool, quantitative sO2 measurements were taken both in vitro and in vivo. Although in vitro measurements of sO₂ showed high agreement with expected values from premade samples, in practice, in vivo measurements were less accurate. The reported values of 70%-100% sO₂ in the arteries present large deviations from the expected range of 95-100%. Several factors, such as fluence wavelength dependence, optical wavelength range, and transducer central frequency have been suggested and investigated in order to understand these discrepancies. Despite additional knowledge of systematic errors arising from such factors, measuring the absolute value of sO₂ in vivo remains a challenge. All previous studies assumed linear dependence of the photoacoustic signal on absorption and used the linear least squares model. However, several factors, such as wavelength calibration errors, photodiode-wavelength dependence, and intensity dependent non-linearity, all of which may have a significant effect on the final calculation, have not been investigated. Here we evaluate both in vitro and in vivo the effects on sO₂ measurements of photodiode wavelength dependence, laser wavelength accuracy, and intensity dependent absorption of oxygenated and deoxygenated hemoglobin. We show that these factors may contribute significantly to the deviations in sO₂ calculations in vivo.

Multispectral optoacoustic (photoacoustic) tomography (MSOT) exploits the high resolutions provided by ultrasound imaging technology in combination with the more biologically relevant optical absorption contrast. Traces of molecules with different spectral absorption profiles, such as blood (oxy- and de-oxygenated) and biomarkers can be recovered using multiple wavelengths excitation and a set of methods described in this work. Three unmixing methods are examined for their performance in decomposing images into components in order to locate fluorescent contrast agents in deep tissue in mice. Following earlier works we find Independent Component Analysis (ICA), which relies on the strong criterion of statistical independence of components, as the most promising approach, being able to clearly identify concentrations that other approaches fail to see. The results are verified by cryosectioning and fluorescence imaging.

Total Internal Reflection Photoacoustic Spectroscopy (TIRPAS) is a method that exploits the evanescent field of a nanosecond duration laser pulse reflecting off a glass/water interface to generate photoacoustic responses. These photoacoustic events are generated in light absorbing analytes suspended in the fluid medium in contact with the glass that are within the penetration depth of the evanescent wave. This method has been employed in previous studies by Hinoue et al. Hinoue et al. used an optically chopped HeNe laser at 632.8 nm to detect Brilliant Blue FCF dye at different angles of incidence. In recent years, the advent of high power nanosecond pulsed tunable lasers has allowed for the re-visitation of the TIRPAS idea under stress confinement and orders of magnitude larger peak energy conditions. Compared to conventional detection methods, this approach has the potential to detect much smaller quantities of disease indicators, such as circulating tumor cells and hemazoin crystals in malaria, than other optical methods. The detection limit of the TIRPAS system was quantified using chlorazol black solution with an absorption coefficient of 55 cm^-1 at 532 nm. Interaction with the evanescent field was verified by varying the angle of incidence of the probe laser beam that generated the photoacoustic waves, thereby changing the penetration depth of the evanescent field as well as the photoacoustic spectroscopy effect from angled excitation.

Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are inadequately sensitive. Patients must wait until secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and flow through the blood or lymph system can provide data for diagnosing and monitoring cancer. Our group utilizes the photoacoustic effect to detect metastatic melanoma cells, which contain the pigmented granule melanin. As a rapid laser pulse irradiates melanoma, the melanin undergoes thermo-elastic expansion and ultimately creates a photoacoustic wave. Thus, melanoma patient's blood samples can be enriched, leaving the melanoma in a white blood cell (WBC) suspension. Irradiated melanoma cells produce photoacoustic waves, which are detected with a piezoelectric transducer, while the optically transparent WBCs create no signals. Here we report an isolation scheme utilizing two-phase flow to separate detected melanoma from the suspension. By introducing two immiscible fluids through a t-junction into one flow path, the analytes are compartmentalized. Therefore, the slug in which the melanoma cell is located can be identified and extracted from the system. Two-phase immiscible flow is a label free technique, and could be used for other types of pathological analytes.

In the reconstruction process of photo acoustic experiments, it was observed that adding a passive element to the experimental setup, improves the quality of the reconstruction of the object. This contribution analyzes this effect in some detail. We consider a cylindrical configuration. We start from an artificial and theoretically constructed optical absorption distribution that radiates sound waves when interrogated by the optical pulse. We analyze in the experimental setup the addition of the passive element to this example. The reported investigation is a part of a larger study on the existence, uniqueness and stability of photo acoustic inverse source reconstructions.

An acoustic and photoacoustic characterization of micron-sized perfluorocarbon (PFC) droplets is presented. PFC droplets are currently being investigated as acoustic and photoacoustic contrast agents and as cancer therapy agents. Pulse echo measurements at 375 MHz were used to determine the diameter, ranging from 3.2 to 6.5 μm, and the sound velocity, ranging from 311 to 406 m/s of nine droplets. An average sound velocity of 379 ± 18 m/s was calculated for droplets larger than the ultrasound beam width of 4.0 μm. Optical droplet vaporization, where vaporization of a single droplet occurred upon laser irradiation of sufficient intensity, was verified using pulse echo acoustic methods. The ultrasonic backscatter amplitude, acoustic impedance and attenuation increased after vaporization, consistent with a phase change from a liquid to gas core. Photoacoustic measurements were used to compare the spectra of three droplets ranging in diameter from 3.0 to 6.2 μm to a theoretical model. Good agreement in the spectral features was observed over the bandwidth of the 375 MHz transducer.

One of the leading causes of medical malpractice claims in emergency medicine is the misdiagnosis of the presence of foreign bodies. Radiolucent foreign bodies are especially difficult to differentiate from surrounding soft tissue, gas, and bone using existing clinical imaging modalities. Because many radiolucent foreign bodies have sufficient contrast for imaging in the optical domain, we are exploring the use of laser-induced optoacoustic imaging for the detection of foreign bodies, especially in orbital and craniofacial injuries, in which the foreign bodies are likely to lie within the penetration depth of visible and near infrared wavelengths. In order to evaluate the performance of optoacoustic imaging for clinical detection and characterization, common foreign bodies have been scanned over a range of visible and near infrared wavelengths to obtain the spectroscopic properties of the materials commonly associated with these foreign bodies. The foreign bodies are also being embedded in realistic ex vivo tissue phantoms to evaluate the changes that may occur in the spectroscopic absorption of the materials due to the interaction with tissue absorbers. Ultimately, we anticipate that spectroscopic characterization will help identify specific wavelengths to be used for imaging foreign bodies that will provide useful diagnostic data about the material properties of the object, thereby enabling the characterization, as well as the location, of the objects. This information will aid the clinician in choosing the optimal treatment course for the patient.

Accurate lymph node analysis is essential to determine the prognosis and treatment of patients suffering from melanoma. The initial results of a tomographic photoacoustic modality to detect melanoma metastases in resected lymph nodes are presented based on phantom models and a human lymph node. The results show melanoma metastases detection is feasible and the setup is capable of distinguishing absorbing structures down to 1 mm. In addition, the use of longer laser wavelengths could result in an image containing a higher contrast ratio. Future research shall be focused on using the melanin characteristics to improve contrast and detection possibilities.

In this study, we applied an integrated photoacoustic imaging (PAI) and high intensity focused ultrasound (HIFU) system to noninvasively monitor the thermal damage due to HIFU ablation in vivo. A single-element, spherically focused ultrasonic transducer, with a central frequency of 5MHz, was used to generate a HIFU area in soft tissue. Photoacoustic signals were detected by the same ultrasonic transducer before and after HIFU treatments using different wavelengths. The feasibility of combined contrast imaging and treatment of solid tumor in vivo by the integrated PAI and HIFU system was also studied. Gold nanorods were used to enhance PAI during the imaging of a CT26 tumor, which was subcutaneously inoculated on the hip of a BALB/c mouse. Subsequently, the CT26 tumor was ablated by HIFU with the guidance of photoacoustic images. Our results suggested that the tumor was clearly visible on photoacoustic images after the injection of gold nanorods and was ablated by HIFU. In conclusion, PAI may potentially be used for monitoring HIFU thermal lesions with possible diagnosis and treatment of solid tumors.

Photoacoustic imaging offers a new and complementary contrast mechanism to the traditional structural contrast of ultrasound. While the combination of these two modes has been demonstrated in the past with single-element transducers, array transducers offer clear advantages in both modes by eliminating mechanical scanning and allowing image formation from a single excitation. Given the abundance of commercially available ultrasound systems, it is desirable to use them as much as possible. However, these systems often only allow access to beamformed RF data. We discuss the applicability of ultrasound beamformers for photoacoustic imaging, and find that with only software-defined control over the speed of sound, walking aperture reconstruction is optimally performed using a speed correction factor of 1.414. When sector-scanning is used, a different strategy is required. We also demonstrate a new photoacoustic-ultrasound imaging system based on a Verasonics ultrasound array system. The system streams raw channel data to a 6-core PC at up to 1.4GB/s via PCI-Express, allowing interlaced ultrasound and photoacoustic data to be acquired and reconstructed at realtime rates. Using an L7-4 linear array transducer, we demonstrate the performance of this system and discuss potential applications. The system should provide new opportunities for clinical and pre-clinical imaging.

The specificity of both molecular and functional photoacoustic (PA) images depends on the accuracy of the photoacoustic absorption spectroscopy. Because the PA signal is a product of both the optical absorption coefficient and the local light fluence, quantitative PA measurements of absorption require an accurate estimate of the optical fluence. Lightmodeling aided by diffuse optical tomography (DOT) methods can be used to provide the required fluence map and to reduce errors in traditional PA spectroscopic analysis. As a proof-ofconcept, we designed a phantom to demonstrate artifacts commonly found in photoacoustic tomography (PAT) and how fluence-related artifacts in PAT images can lead to misrepresentations of tissue properties. Specifically, we show that without accounting for fluence-related inhomogeneities in our phantom, errors in estimates of the absorption coefficient from a PAT image were as much as 33%. To correct for this problem, DOT was used to reconstruct spatial distributions of the absorption coefficients of the phantom, and along with the surface fluence distribution from the PAT system, we calculated the fluence everywhere in the phantom. This fluence map was used to correct PAT images of the phantom, reducing the error in the estimated absorption coefficient from the PAT image to less than 5%. Thus, we demonstrate experimentally that combining DOT with PAT can significantly reduce fluence-related errors in PAT images, as well as produce quantitatively accurate, highresolution images of the optical absorption coefficient.

Photoacoustic tomography is an imaging technique used for a wide range of biomedical applications. In this imaging modality, an object is illuminated with a pulsed optical field that induces an acoustic pressure wave related. The resulting pressure wave is directly related to the heating of the object (optical absorption). Knowledge of the resultant pressure wave away from the source allows for reconstruction of the absorbed optical energy density within the object. Most analytic reconstruction algorithms for photoacoustic tomography are based on the assumption of a homogeneous background for the acoustic field. The effects of changes in the density and speed of sound of various types of biological tissue are not presently accounted for in reconstruction algorithms. In this work, photoacoustic tomography is considered when the primary acoustic source is embedded in a planar layered medium whose speed of sound and densities are known, but vary from layer to layer. An exact propagation model, valid for acoustic wave propagation in dispersive and absorptive layered media, is presented. This model accounts for multiple reflections of acoustic waves between the layers. An inversion model is presented for acoustic data acquired on a plane parallel to the layered medium. The acquired data are shown to be simple linear combinations of plane waves generated at the source. Numerical simulations will illustrate the method in a number of situations relevant to biomedical imaging.

Spectrum analysis of the radio-frequency signals from photoacoustic imaging is performed to generate parameters for tissue characterization that are calibrated for the response of the imaging system. Calibrated photoacoustic spectra were obtained by dividing the photoacoustic spectra from tissue by the spectrum of a point absorber. The resulting quasi-linear spectra were fit by linear regression, and midband fit, slope, and intercept were computed from the best-fit line. These spectral parameters were compared between tumors resulting from a prostate adenocarcinoma model and adjacent normal tissue in a murine model. The mean midband fit and intercept showed significant differences between regions of tumor and adjacent normal tissue. These initial results suggest that such frequency-domain analysis can provide a quantitative method for tissue characterization using photoacoustic imaging.

Researchers have been using single element transducers for photoacoustic microscopy (PAM), but such systems have limited depth of field due to a single focus. The aim for this project was to develop a high-frequency annular array transducer for improved depth-of-field PAM. We have designed a concave 40 MHz ultrasound transducer which has 8 annular array elements with equal area. The outer ring is 12 mm in diameter, the geometric focus is 12 mm, and the space between each annulus is 100 μm. The array was fabricated by lithographically patterning metalized polyimide film to define back electrodes and signal leads. 9-micron-PVDF film was then press-fit into the array pattern with epoxy as a backing material and a single drop of epoxy as a bonding layer. The array exhibits high sensitivity to high-frequency photoacoustic signals. Dynamic focusing of amplified and digitized signals permits extended depth-of-field imaging compared to the single-element transducer case. Dark-field light-delivery and 3-axis motorized scanning permits 3-D photoacoustic microscopy. Imaging performance in phantoms is discussed.

Co-registering ultrasound (US) and photoacoustic (PA) imaging is a logical extension to conventional ultrasound because both modalities provide complementary information of tumor morphology, tumor vasculature and hypoxia for cancer detection and characterization. In addition, both modalities are capable of providing real-time images for clinical applications. In this paper, a Field Programmable Gate Array (FPGA) and Digital Signal Processor (DSP) module-based real-time US/PA imaging system is presented. The system provides real-time US/PA data acquisition and image display for up to 5 fps^* using the currently implemented DSP board. It can be upgraded to 15 fps, which is the maximum pulse repetition rate of the used laser, by implementing an advanced DSP module. Additionally, the photoacoustic RF data for each frame is saved for further off-line processing. The system frontend consists of eight 16-channel modules made of commercial and customized circuits. Each 16-channel module consists of two commercial 8-channel receiving circuitry boards and one FPGA board from Analog Devices. Each receiving board contains an IC† that combines. 8-channel low-noise amplifiers, variable-gain amplifiers, anti-aliasing filters, and ADC's^‡ in a single chip with sampling frequency of 40MHz. The FPGA board captures the LVDSξ Double Data Rate (DDR) digital output of the receiving board and performs data conditioning and subbeamforming. A customized 16-channel transmission circuitry is connected to the two receiving boards for US pulseecho (PE) mode data acquisition. A DSP module uses External Memory Interface (EMIF) to interface with the eight 16-channel modules through a customized adaptor board. The DSP transfers either sub-beamformed data (US pulse-echo mode or PAI imaging mode) or raw data from FPGA boards to its DDR-2 memory through the EMIF link, then it performs additional processing, after that, it transfer the data to the PC^** for further image processing. The PC code performs image processing including demodulation, beam envelope detection and scan conversion. Additionally, the PC code pre-calculates the delay coefficients used for transmission focusing and receiving dynamic focusing for different types of transducers to speed up the imaging process. To further speed up the imaging process, a multi-threads technique is implemented in order to allow formation of previous image frame data and acquisition of the next one simultaneously. The system is also capable of doing semi-real-time automated SO₂ imaging at 10 seconds per frame by changing the wavelength knob of the laser automatically using a stepper motor controlled by the system. Initial in vivo experiments were performed on animal tumors to map out its vasculature and hypoxia level, which were superimposed on co-registered US images. The real-time system allows capturing co-registered US/PA images free of motion artifacts and also provides dynamitic information when contrast agents are used.

Clinical photoacoustic (PA) imaging relies on illuminating objects at depth. To do this, it is important to optimise the illumination geometry with respect to the sensitivity pattern of the acoustic receiver, taking optical scattering into account. The three-dimensional point spread function (3D PSF) measured at various depths as a function of the optimisation variables, is being explored to determine its usefulness for this purpose. The 3D PSF of a reflection mode photoacoustic scanner was measured by acquiring a series of PA images of the tip of a 0.25mm radius graphite rod placed at a depth of 2 cm, by translating the photoacoustic linear array transducer and illumination optics in the elevational direction. This was done for a series of angles and separations of the fibre optic illuminators, for a background medium of 1% intralipid, which simulates, to first order, the optical scattering that would be experienced in tissue. The background noise was found to be influenced by the illumination geometry, and may have been associated with PA clutter generated by absorption in the background medium. The angle of illumination and distance separating fibre optic illuminators were found to be weakly optimum at around 76 degrees and 15.5mm respectively, where the PSF amplitude passed through a weak maximum. As expected, the shape of the 3D PSF was found to be independent of illumination geometry. However, the combination of using the tip of a graphite rod as a point object, and plotting the 3D PSF as a means of locating the peak signal, appears to be a successful method of studying the effect of illumination variables on signal strength. Ultimately when complete, this optimisation should enable the clarity images at the depth of interest to be maximised.

Optoacoustic Tomography (OAT) is an emerging hybrid imaging technique with great potential for a wide range of biomedical imaging applications. Assuming point-like transducers, analytic algorithms are available for image reconstruction, but they are applicable only when the measured data are densely sampled on an aperture that encloses the object. In many cases of practical interest, however, measurements may be limited in number and are acquired on an incomplete aperture. Total variation (TV) minimization has been proved to be a powerful tool for limited-data reconstruction. However, most previous studies of limited-data OAT were based on an approximate imaging model that assumed point-like transducers, which limits the improvements on the reconstructed OAT image quality. In this work, we develop and investigate an iterative reconstruction algorithm incorporating ultrasonic transducer properties applicable for limited-data OAT. The algorithm is based on the minimization of the image TV subject to a data consistency condition, and is conceptually and mathematically distinct from classic iterative reconstruction algorithms. Preliminary computer-simulation studies are conducted to investigate the proposed algorithm. These studies reveal that the constrained, total variation minimization algorithm can yield accurate reconstructions in many limited-data applications where classic algorithms do not perform well.

A microbubble-based imaging/therapeutic agent is introduced. Specifically, gold nanoparticles (AuNRs) are encapsulated in microbubbles (MBs) for both ultrasound (US) imaging and laser-induced thermotherapy (LIT). In addition, this agent, AuNR-MB, takes albumin microbubble as a carrier and includes the AuNRs that maintain the original absorption peak at around 760nm. AuNR-MBs in different sizes are synthesized. Imaging is first performed to evaluate its feasibility. The enhanced PA and US signals in polyacrylamide gel for in vitro study are measured. The PA spectroscopy is then performed and the results generally agree with the measured optical absorption although its peak is slightly broadened and shifted possibly due to mixing. In phantoms, the contrast is 1.531, 2.447, 2.085, 1.994, 0.768, and 0.573 at wavelength of 720, 760, 800, 860, 900, and 940 nm respectively. Finally, the application of the new agent to LIT is presented. A continuous wave laser at 800 nm is used to heat the samples with the power at 1W. The photoacoustic (PA) intensity in the region of interest (ROI) is increased by an average of 5.2dB. The increased signal level implies that the temperature in the ROI can be increased to 44.3°C in aqueous filled setup. Furthermore, the dual-modality agent has the potential to be used in HIFU therapy, drug delivery and loading of DNA for gene transfer.

Photoacoustic imaging (PAI) was employed to detect small animal brain activation after the administration of cocaine hydrochloride. Sprague Dawley rats were injected with different concentrations (2.5, 3.0, and 5.0 mg per kg body) of cocaine hydrochloride in saline solution through tail veins. The brain functional response to the injection was monitored by photoacoustic tomography (PAT) system with horizontal scanning of cerebral cortex of rat brain. Photoacoustic microscopy (PAM) was also used for coronal view images. The modified PAT system used multiple ultrasonic detectors to reduce the scanning time and maintain a good signal-to-noise ratio (SNR). The measured photoacoustic signal changes confirmed that cocaine hydrochloride injection excited high blood volume in brain. This result shows PAI can be used to monitor drug abuse-induced brain activation.

Photoacoustic imaging is a promising technique combining high ultrasonic resolution and high optical contrast. However, quantification has proved rather challenging. In this paper, we present a non-iterative reconstruction strategy with multiple-optical-sources for reconstruction of absorption, scattering perturbations as well as the spatially varying Grüeneisen parameter from a known turbid background. We term this method the multiple-illumination photoacoustic tomography (MI-PAT). While numerical challenges still exist, we demonstrated that the linearized MI-PAT framework we propose has orders of magnitude improved condition number compared with Continuous-Wave Diffuse Optical Tomography (CW-DOT).

Light-absorbing films have been utilized as optoacoustic transmitters for all optical ultrasound transducers. For these thin-film transmitters, however, optoacoustic conversion efficiencies (OCEs) and the output pressures have not been evaluated over a broadband and high frequency range. Here, we characterized such optoacoustic performance with high precision by using optical microring ultrasound detectors (OMUDs) under a plane wave configuration. We obtained ultrasound pulses minimizing diffraction-induced signal distortion, while maintaining broadband spectral information up to 100 MHz owing to the detector wideband response. In order to find an efficient thermal transfer medium for optoacoustic generation, we compared the OCEs and the output pressures for various polymers. Finally, a 2-D gold nanostructure with the polymer layer was characterized over the broadband frequency range.

Recently, we have developed a combined photoacoustic and high-frequency Doppler ultrasound system with a single element transducer to estimate the metabolic rate of oxygen consumption in small animal models. However, the long scanning time due to mechanical motion may be a limitation of our swept-scan system. In this work, the single element transducer was replaced by a clinical array transducer which may provide more accurate flow velocity estimations, higher frame rates, improved penetration depth, and improved depth-of-field due to dynamic focusing capabilities. We used an array system from Verasonics Inc. which enables flexible pulse-sequence programming and parallel channel data acquisition, along with a pulsed laser and optical parametric oscillator. For flow estimation, we implemented a flash- Doppler sequence which transmits ensembles of plane-wave excitations. Echo signals are beamformed and subjected to wall-filtering and Kasai flow estimation algorithms. High frame rates over a wide region can be achieved. Combined interlaced photoacoustic and Doppler imaging on flow phantoms has been performed on this system. We demonstrate the ability to image animal blood to depths of 1.5-cm with high signal-to-noise with both modalities. The light penetration is 2-cm. We discuss the performance of Doppler flow estimation and photoacoustic oxygen saturation estimation and their role in future work of estimating oxygen consumption.

The metabolic rate of oxygen consumption (MRO₂) quantifies tissue metabolism, which is important for diagnosis of many diseases. For a single vessel model, the MRO₂ can be estimated in terms of the mean flow velocity, vessel crosssectional area, total concentration of hemoglobin (CHB), and the difference between the oxygen saturation (sO₂) of blood flowing into and out of the tissue region. In this work, we would like to show the feasibility to estimate MRO₂ with our combined photoacoustic and high-frequency ultrasound imaging system. This system uses a swept-scan 25-MHz ultrasound transducer with confocal dark-field laser illumination optics. A pulse-sequencer enables ultrasonic and laser pulses to be interlaced so that photoacoustic and Doppler ultrasound images are co-registered. Since the mean flow velocity can be measured by color Doppler ultrasound, the vessel cross-sectional area can be measured by power Doppler or photoacoustic imaging, and multi-wavelength photoacoustic methods can be used to estimate sO₂ and C_HB, all of these parameters necessary for MRO₂ estimation can be provided by our system. Experiments have been performed on flow phantoms to generate co-registered color Doppler and photoacoustic images. To verify the sO₂ estimation, two ink samples (red and blue) were mixed in various concentration ratios to mimic different levels of sO₂, and the result shows a good match between the calculated concentration ratios and actual values.

Spectroscopic photoacoustic microscopy (PAM) requires a pulsed nanosecond laser with tunable wavelength, but such lasers are expensive and have poor wavelength switching speed. We are developing a rapidly tunable system based on a high repetition rate supercontinuum source. A supercontinuum is produced by propagating 0.6 ns duration pulses from an 7.5 kHz Q-switched Nd:YAG microchip laser through 7 meters of photonic crystal fiber (PCF). Wavelength selection is achieved with a rapidly tunable prism-based monochromator, where an actuator-controlled mask selects the desired wavelength band. Ten different wavelength bands (570 to 930 nm) are acquired in less than 1 second for each image pixel. Each wavelength has a bandwidth of 40 nm. The PAM system employs optical focusing of the excitation beam and detection with a 25 MHz spherically focused f/3 transducer. Multiwavelength imaging is tested on phantoms with different color inks. The inks were correctly identified by processing the multiwavelength images with a linear discriminant analysis. A major advantage of our tunable source is the high repetition rate and rapid access to widely separated wavelengths. These promising results suggest the potential of our wavelength agile source for spectroscopic photoacoustic microscopy.

Scalability is a key feature of photoacoustic microscopy (PAM). Reports have shown that PAM systems can be designed to possess sub-micron resolution at shallow depths or penetrate centimeters deep at the expense of resolution while the number of resolved pixels in the depth direction remains high. This capability to readily tune the imaging parameters while maintaining the same inherent contrast could be extremely useful for a variety of biomedical applications. Human skin, with its layered vascular structure whose dimensions scale with depth, provides an ideal imaging target to illustrate this advantage. Here, we present results from in vivo human skin imaging experiments using two different PAM systems, an approach which enables better characterization of the cutaneous microvasculature throughout the imaging depth. Specifically, we show images from several distinct areas of skin: the palm and the forearm. For each region, the same area was imaged with both an optical-resolution PAM (OR-PAM) and an acoustic-resolution PAM (AR-PAM), and the subsequent images were combined into composite images. The OR-PAM provides less than 5 μm lateral resolution, capable of imaging the smallest capillary vessels, while the AR-PAM enables imaging at depths of several millimeters. Several structures are identifiable in the ORPAM images which cannot be differentiated in AR-PAM images, namely thin epidermal and stratum corneum layers, undulations in the dermal papillae, and capillary loops. However, the AR-PAM provides images of larger vessels, deeper than the OR-PAM can penetrate. These results demonstrate how PAM's scalability can be utilized to more fully characterize cutaneous vasculature, potentially impacting the assessment of numerous cardiovascular related and cutaneous diseases.

Gold nanorods (GNRs) have been demonstrated as a scattering imaging agent or therapeutic agent. Because of their narrow window, biocompatibility, and uniform small size for blood circulation, GNRs are well suited to serve as imaging contrast agents. Especially, strong phothothermal (PT) effect is attractive for diagnostic (e.g. sentinel lymph node biopsy) or therapeutic (e.g. PT therapy) purposes. In this paper, we demonstrate GNRs as multipurpose agents with PT-optical coherence tomography (OCT) for imaging sentinel lymph node. The results show that GNRs are promising for imaging contrast enhancement for visualizing the detailed functions of SLN.

We used a three-dimensional optical tomogaphy system that was previously developed to create high contrast maps of optical absorbance of mice tissues. In this study, animals were scanned before and after injection of gold nanorods (GNRs) at different time periods. As-synthesized GNRs were purified from hexadecyltrimethylammonium bromide (CTAB) and coated with polyethylene glycol (PEG) to obtain GNR-PEG complexes suitable for in vivo applications. Intravenous administration of the purified GNR-PEG complexes to mice resulted in an enhanced contrast of normal tissues and blood vessels as compared to ordinary nude mice. In parallel with optoacoustic imaging we investigated the accumulation of GNRs in liver using invasive analytical techniques. Maximum levels of GNRs in liver macrophages were observed after 48-72 hours post-injection, followed by slow clearance trend after 8 days. Optoacoustic imaging revealed redistribution of GNR in mouse organ and tissues: in the initial hours, accumulation of GNRs is seen predominantly in the periphery of the mouse, while a gradual increase of GNR levels in liver, spleen and kidneys is seen in 1 and 24 hours.

Photoacoustic imaging and optical coherence tomography are two emerging imaging modalities which provide complementary optical absorption and scattering contrasts for biological tissues. While photoacoustic imaging provides tissue vasculature information, optical coherence tomography offers micron-scale morphological imaging with penetration depths of 1~3 mms. Pulse-echo ultrasound is readily available from photoacoustic system and it provides tissue structure information at deeper depths with resolutions scalable with the transducer frequency. We present a prototype endoscope that consists of a ball lensed optical coherence tomography probe, a right-angled multimode fiber for delivering the laser beam for photoacoustic imaging, and a high frequency ultrasound transducer of 35 MHz center frequency. The overall diameter is 5mms. Porcine ovaries were imaged ex vivo to demonstrate the capability of this new combined endoscopy. The microvascular and high resolution structural images at subsurface and deeper tissue range demonstrate the synergy of the combined endoscopy over each modality alone.

One of the most frequently performed blood tests, measurement of total hemoglobin concentration, requires invasive blood sampling. We developed an optoacoustic technique for noninvasive monitoring of total hemoglobin concentration and other blood variables by probing the radial artery or other blood vessels. Recently, we designed and built a focused, wide-band, polymer-based optoacoustic transducer for blood vessel probing with high, submillimeter lateral resolution and incorporated it into a highly portable, laser diode-based optoacoustic system. The focused optoacoustic transducer combines a fiber-optic delivery system and a wide-band piezosensor. First, we experimentally measured transducer parameters (lateral resolution, sensitivity, focal length). To test the transducer capabilities in measurement of total hemoglobin concentration and other blood parameters from blood vessels, we prepared a tissue phantom simulating strongly-scattering tissues with blood vessels of different diameters, spacing, and depths. Optoacoustic signals were acquired from blood at different hemoglobin concentration and oxygenation during transducer scanning over the phantom. In vivo experiments were performed from radial arteries and peripheral veins of different size, depth, and spacing. Submillimeter lateral resolution was obtained in the in vitro and in vivo experiments. The high resolution combined with the wide-band detection of the optoacoustic waves can be used for monitoring of blood variables in blood vessels with high accuracy, sensitivity, and specificity.