This PDF file contains the front matter associated with SPIE Proceedings Volume 7564, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

In photoacoustic (PA) imaging of microvascular networks, the transverse component of the blood flow that is perpendicular to the acoustic probing beam is usually dominant. We propose a new method to measure the transverse flow, based on the Doppler bandwidth broadening. The bandwidth broadening is inversely proportional to the transit time spent by the absorbers passing through the focus. Because the photoacoustic signal in one A-scan has a wide band, multiple successive A-scans are used to estimate the relatively small signal variance. Then the bandwidth broadening can be calculated from the standard derivation of the Doppler spectrum. By exploiting the pulse excitation and bidirectional raster motor scanning, threedimensional structural and flow information can be obtained simultaneously. From a flow of a suspension of carbon particles (diameter: 6 μm), transverse flow speeds from 0 to 2.5 mm/s were measured using optical-resolution photoacoustic microscopy. The bandwidth broadening at each speed was in good agreement with the theoretical prediction. The blood flow in a mouse brain was also imaged.

Using realtime ultrasound array photoacoustic microscopy (UA-PAM), we demonstrated the feasibility of noninvasive in vivo imaging of human pulsatile dynamics, as well as 3-D dynamic imaging of sentinel lymph nodes (SLNs) in a murine model. The system, capable of realtime B-scan imaging at 50 Hz and high-speed 3-D imaging, was validated by imaging the subcutaneous microvasculature in rats and humans. After the validation, a human superficial palmar was imaged, and its pulsatile dynamics monitored, with 20-ms B-scan imaging temporal resolution. In addition, noninvasive photoacoustic sentinel lymph node (SLN) mapping with high spatial resolution has the potential to reduce the false negative rate and eliminate the use of radioactive tracers. Upon intra-dermal injection of Evans blue, the system maps SLNs accurately in mice and rats. Furthermore, the ~6 s 3-D imaging temporal resolution offers the capability to quantitatively and noninvasively monitor the dye dynamics in SLNs in vivo through sequential 3-D imaging. The demonstrated capability suggests that high-speed 3-D photoacoustic imaging should facilitate the understanding of the dynamics of various dyes in SLNs, and potentially help identify SLNs with high accuracy. With the results shown in this study, we believe that UA-PAM can potentially enable many new possibilities for studying functional and physiological dynamics in both preclinical and clinical imaging settings.

Photoacoustic imaging has emerged as a promising technique for visualizing optically absorbing structures with ultrasonic spatial resolution. Since it relies on optical absorption of tissues, photoacoustic imaging is particularly sensitive to vascular structures even at the micro-scale. Power Doppler ultrasound can be used to detect moving blood irrespective of Doppler angles. However, the sensitivity may be inadequate to detect very small vessels with slow flow velocities. In this work, we merge these two synergistic modalities and compare power Doppler ultrasound images with high-contrast photoacoustic images. We would like to understand the advantages and disadvantages of each technique for assessing microvascular density, an important indicator of disease status. A combined photoacoustic and highfrequency ultrasound system has been developed. The 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. Experiments have been performed on flow phantoms to test the capability of our system and signal processing methods. Work in progress includes in vivo color flow mapping. This combined system will be used to perform blood oxygen saturation and flow estimations, which will provide us with the parameters to estimate the local rate of metabolic oxygen consumption, an important indicator for many diseases.

We demonstrate the feasibility of optical angiography on live mice using a new photoacoustic computed tomography (PCT) scanner. The scanner uses a sparse array of discrete ultrasound detectors geometrically arranged to capture 128 simultaneous radial "projections" through a 25-mm-diameter volume of interest. Denser sets of interleaved radial projections are acquired by rotating the sparse array continuously about its vertical axis during data acquisition. The device has been designed specifically for imaging laboratory mice, which remain stationary during data collection. Angiographic data are acquired at a rate of 1280 radial projections per second following a bolus injection of 2 mg/mL of indocyanine green (ICG).

A major obstacle in studying angiogenesis is the inability to noninvasively image neovascular development in an individual animal. We applied optical-resolution photoacoustic microscopy (OR-PAM) to determine the kinetics of hypoxia-inducible factor-1 (HIF-1)-mediated angiogenesis in a transgenic mouse model. During continuous 30-day activation of HIF-1α, we used OR-PAM to monitor alterations in microvasculature in transgenic mice compared to nontransgenic mice. OR-PAM has demonstrated the potential to precisely monitor antiangiogenic therapy of human cancers, allowing for rapid determinations of therapeutic efficacy or resistance.

Advances in the brain functional imaging greatly facilitated the understanding of neurovascular coupling. For monitoring of the microvascular response to the brain electrical stimulation in vivo we used optical-resolution photoacoustic microscopy (OR-PAM) through the cranial openings as well as transcranially. Both types of the vascular response, vasoconstriction and vasodilatation, were clearly observed with good spatial and temporal resolution. Obtained results confirm one of the primary points of the neurovascular coupling theory that blood vessels could present vasoconstriction or vasodilatation in response to electrical stimulation, depending on the balance between inhibition and excitation of the different parts of the elements of the neurovascular coupling system.

The vulnerability of atherosclerotic plaques that are formed in the arterial walls due to atherosclerosis depends on both their distribution and composition. The distribution of the plaques can be imaged using an intravascular ultrasound (IVUS) imaging which is a clinically approved minimally-invasive method. The recently introduced intravascular photoacoustic (IVPA) imaging may be used to obtain the necessary information about the composition of the plaques. Previous studies using excised rabbit arteries have demonstrated that the combined IVUS/IVPA imaging may simultaneously provide the morphology and functional information of plaques. However, for in-vivo IVUS/IVPA imaging, an integrated IVUS/IVPA imaging catheter capable both of delivering light into a vessel lumen with consequent detection of photoacoustic transients and of probing the arterial walls in pulse-echo mode is required. In the current study, an advanced prototype of the integrated IVUS/IVPA imaging catheter based on a 40-MHz single-element ultrasound transducer and a 600-μm-core single optical fiber is introduced. Unlike previously reported prototypes, the current integrated IVUS/IVPA imaging catheter is capable of cross-sectional imaging of vessel walls via mechanical rotation of the catheter. The performance of the integrated IVUS/IVPA catheter was evaluated in tissue-mimicking phantoms with and without the presence of blood in a lumen. The results of our study suggest that the approach used to develop integrated IVUS/IVPA imaging catheter can be successfully translated to the clinical environment for in-vivo combined IVUS/IVPA imaging.

The combination of intravascular ultrasound and intravascular photoacoustic imaging has been proposed. In this study, we propose a scan head design that is sufficiently small to fit in the tip of the catheter. In addition, the design is also suitable for ultra high frame rate imaging. The scan head consists of a single element, ring-shaped transducer for sideward ultrasound transmission. The transducer has a diameter of 3mm. On acoustic detection, we propose the use of a polymer microring array. For demonstration purposes, a single micro-ring is used with mechanical scanning in this study. For optical illumination, a multimode fiber with a cone-shaped mirror is used. Note that only a single ultrasound/laser pulse is required to acquire an ultrasound/photoacoustic image frame. Phantom imaging results are demonstrated.

Using contrast agents with desired targeting moiety and optical absorption, intravascular photoacoustic imaging may be used to identify various biomarkers expressed during the progression of atherosclerotic lesions. In this paper, we present intravascular photoacoustic imaging of macrophages in the atherosclerotic lesions using bio-conjugated gold nanoparticles as the contrast agent. Atherosclerotic lesions were created in the aorta of a New Zealand white rabbit subjected to a high cholesterol diet and balloon injury. The rabbit was injected with 20 nm spherical gold nanoparticles conjugated with antibodies. The macrophages with internalized gold nanoparticles were imaged by intravascular photoacoustic imaging in the near infrared range; this was possible because of plasmon resonance coupling between closely spaced gold nanoparticles internalized by macrophages. The multi-wavelength intravascular photoacoustic images of the diseased aorta were analyzed to identify the presence and location of macrophages labeled with gold nanoparticles. Spectroscopic intravascular photoacoustic image showing the distribution of gold nanoparticles was further confirmed by the gold-specific silver staining of the tissue crosssection. The results of our study suggest that molecular intravascular photoacoustic imaging can be used to image macrophages in atherosclerosis.

Intravascular photoacoustic (IVPA) imaging that aims to detect atherosclerotic plaques with differential composition is studied computationally and experimentally. IVPA images are usually reconstructed by simply aligning photoacoustic signals with scan conversion, which results in images with severe blurring and increases the difficulty in signal detection. The scanning aperture in IVPA, in contrast to other photoacoustic tomography applications, is enclosed within the imaged object. Consequently, quantitative image reconstruction becomes infeasible, as the data sufficiency condition for stable image reconstruction is not satisfied in such a limited-view scanning. However, useful information regarding certain plaque boundaries can still be reconstructed, which can facilitate plaque detection. In this study, strategies for limited-view reconstruction will be investigated for the IVPA scanning geometry. Computer simulations are carried out to validate the developed method.

Recently it has been shown that multiwavelength photoacoustic imaging has the potential to discriminate between normal and atheromatous areas of arterial tissue when operating in the 740-1300nm wavelength range. At this wavelength range the absorption spectrum of lipids and normal arterial tissue are significantly different allowing discrimination between one another. Also, this wavelength range has the advantage of being relatively weakly absorbed by blood. This obviates the need for a saline flush if implemented using an intravascular imaging probe. In this study we investigate the possibility of identifying regions of high lipid concentration from 2D multiwavelength photoacoustic images of vascular tissue by exploiting the unique spectral features of lipids. Recognising regions of high lipid concentration would be useful to identify plaques which are likely to rupture (vulnerable plaques). To investigate this, samples of post mortem human aortas were imaged at a range of near-infrared (NIR) wavelengths and compared to histology. Photoacoustic images were also obtained when illuminating the sample through blood. This study demonstrated that lipid rich atheromatous plaques can clearly be identified using multiwavelength photoacoustic imaging.

In this study, we further developed our photoacoustic endoscopic system to produce three-dimensional images of internal organs by performing pullback C-scans. Employing the side-scanning photoacoustic endoscopic probe discussed in the Optical Society of America's journal Optics Letters, we could acquire successive B-scan images by pulling back the probe with a motorized linear stage. We demonstrate the endoscopic system's volumetric imaging ability through imaging of a metal wire phantom and an in situ rat rectum.

We developed a unique reflection-mode photoacoustic technique sensitive to optical scattering in turbid media. We focused a small laser spot on to the surface of a turbid medium and captured the photoacoustic signal by a focused ultrasound transducer. The amplitude of the photoacoustic signal for different surface illumination spot locations is an effective estimate of the Green's function of light transport in turbid media. Our results for different concentrations of Intralipid indicate that this method is capable of distinguishing small changes in the reduced scattering coefficient. In this work, we present experimental measurements for an Intralipid phantom with reduced scattering coefficients of 3, 4, and 5 cm^-1, and show that Monte Carlo simulations of light transport accurately reproduce experimental curves. This means that we can estimate transport-regime optical properties of the media given a suitable fitting algorithm.

Purpose: The purpose of this study is to develop a new 3-D iterative Monte Carlo algorithm to recover the heterogeneous distribution of molecular absorbers with a solid tumor. Introduction: Spectroscopic imaging (PCT-S) has the potential to identify a molecular species and quantify its concentration with high spatial fidelity. To accomplish this task, tissue attenuation losses during photon propagation in heterogeneous 3D objects is necessary. An iterative recovery algorithm has been developed to extract 3D heterogeneous parametric maps of absorption coefficients implementing a MC algorithm based on a single source photoacoustic scanner and to determine the influence of the reduced scattering coefficient on the uncertainty of recovered absorption coefficient. Material and Methods: This algorithm is tested for spheres and ellipsoids embedded in simulated mouse torso with optical absorption values ranging from 0.01-0.5/cm, for the same objects where the optical scattering is unknown (μ_s'=7-13/cm), and for a heterogeneous distribution of absorbers. Results: Systemic and statistical errors in ma with a priori knowledge of μ_s' and g are <2% (sphere) and <4% (ellipsoid) for all ma and without a priori knowledge of ms' is <3% and <6%. For heterogenenous distributions of ma, errors are <4% and <5.5% for each object with a prior knowledge of ms' and g, and to 7 and 14% when μ_s' varied from 7-13/cm. Conclusions: A Monte Carlo code has been successfully developed and used to correct for photon propagation effects in simulated objects consistent with tumors.

Photoacoustic reconstruction for linear scanning geometry includes the delay-and-sum method, the spectral-domain method and the time-domain based method. In practice, the data collection using the planar detection geometry is not full-view, causing the details of the reconstructed object to be blurred and distorted. In addition to the exact formulation, we adopt a heuristic reconstruction method. In this paper, we demonstrate photoacoustic reconstruction for linear scanning geometry by formulating the image reconstruction into an optimization problem, and solve the problem with the particle swarm optimization (PSO) method. In this method, first we guess the initial optical energy distribution. According to photoacoustic model, described by the Helmholtz equation, the generated photoacoustic wave can be collected with planar detection geometry. The spherical Radon transform is adopted for the simulation of arbitrarily guessed optical energy distribution. Next we compare the collected signals generated from the guessed optical energy distribution with the measured signals by the sum of squared differences. By minimizing the error sum among various guesses, the initial optical energy distribution is obtained. In this regard, no limited-view is encountered. To guess the initial distribution efficiently such that the sum of the squared differences is minimized is an optimization problem with the dimension of unknowns being the size of the initial optical energy distribution. PSO is a derivative-free and population-based stochastic method that has been used to solve various optimization problems due to its simplicity and efficiency. High computational costs aroused from a large number of particles required can be alleviated with the use of the graphic processing units (GPUs). The proposed reconstruction method based on the PSO algorithm along with the spherical Radon transform is implemented on a NVIDIA Telsa C1060 GPU.

Most image reconstruction algorithms for biomedical photoacoustic tomography make the assumption that the optically-generated ultrasonic waves are recorded by pressure detectors with an omni-directional response. In other words, the detectors are assumed to sample the pressure field exactly at a point. In practice this is rarely the case as real detectors have a finite size and often respond not purely to pressure changes but to some combination of acoustic pressure and pressure gradient (or other derivatives). This can make them less sensitive to pressure waves at some angles. The effect of this sensor directionality on photoacoustic tomography was considered here for the case of time-reversal image reconstruction. The ultrasound simulation toolbox k-Wave was used to perform the study.

The data acquisition speed in photoacoustic computed tomography (PACT) is limited by the laser repetition rate and the number of parallel ultrasound detecting channels. Reconstructing PACT image with a less number of measurements can effectively accelerate the data acquisition and reduce the system cost. Recently emerged Compressed Sensing (CS) theory enables us to reconstruct a compressible image with a small number of projections. This paper adopts the CS theory for reconstruction in PACT. The idea is implemented as a non-linear conjugate gradient descent algorithm and tested with phantom and in vivo experiments.

Acousto-optic (AO) signals can be very weak and the aim of this work was to investigate their amplification using microbubbles. The acoustic pressure radiated by the microbubbles produces refractive index changes in the surrounding medium, and this is proposed as an additional mechanism which modulates the phase of photons. The analytical form of this additional modulation is derived based on the Rayleigh-Plesset equation, which describes microbubble oscillations, in the case where the microbubble oscillations are linear under low applied ultrasound pressure. We show that microbubbles can increase the modulation depth of the AO signal using Monte Carlo simulations. The increase in modulation depth is dependent on the applied ultrasound frequency and the resonance frequency of the microbubbles.

Since optoacoustic tomography is considered a high-resolution modality, determination of the absolute detection limit, as it relates to the sensitivity of biomarker detection is not straightforward. This is due to the fact that experimental determination of the sensitivity as a function of target size remains difficult since no established technique exists so far to reproducibly create very small targets containing well-defined concentrations of markers. We combine theoretical analysis with imaging results for large amounts of the marker and place the measured value on the appropriate parameter-dependent signal intensity curve. A performance estimate of the particular experimental system and the expected signal-to-noise-ratio for smaller amounts of markers can then be made.

Photoacoustic imaging is a novel imaging method for medical and biological applications, combining the advantages of Diffuse Optical Imaging (high contrast) and Ultrasonic Imaging (high spatial resolution). A short laser pulse hits the sample. The absorbed energy causes a thermoelastic expansion and thereby launches a broadband ultrasonic wave (photoacoustic signal). The distribution of absorbed energy density is reconstructed from measurements of the photoacoustic signals around the sample. For collecting photoacoustic signals either point like or extended, integrating detectors can be used. The latter integrate the pressure at least in one dimension, e.g. along a line. Thereby, the three dimensional imaging problem is reduced to a two dimensional problem. For a tomography device consisting of a scanning line detector and a rotating sample, fiber-based detectors made of polymer have been recently introduced. Fiber-based detectors are easy to use and possess a constant, high spatial resolution over their entire active length. Polymer fibers provide a better impedance matching and a better handling compared with glass fibers which were our first approach. First measurement results using polymer fiber detectors and some approaches for improving the performance are presented.

A non-metallic interferometric silica optical fiber ultrasonic wideband sensor is presented for optoacustic imaging applications. We characterized the ultrasonic sensitivity of the optical fiber sensor within the frequency range of 1 to 5 MHz. We compared detection of real world optoacoustic signals, generated from an optically absorbing object embedded in a tissue mimicking phantom, between our sensor and an array of piezoelectric transducers. Reconstructed two dimensional acoustic images of the phantom are presented and compared with images obtained with the Laser Optoacoustic Imaging System, LOIS-64B, demonstrating the feasibility of our fiber optic sensor as a wideband ultrasonic sensor.

Photoacoustic Imaging (also known as thermoacoustic or optoacoustic imaging) is a novel imaging method which combines the advantages of Diffuse Optical Imaging (high contrast) and Ultrasonic Imaging (high spatial resolution). A short laser pulse excites the sample. The absorbed energy causes a thermoelastic expansion and thereby launches a broadband ultrasonic wave (photoacoustic signal). This way one can measure the optical contrast of a sample with ultrasonic resolution. For collecting photoacoustic signals our group introduced so called integrating detectors a few years ago. Such integrating detectors integrate the pressure in one or two dimensions -a line or a plane detector, respectively. Thereby the three dimensional imaging problem is reduced to a two or a one dimensional problem for the projections and a two or three dimensional inverse radon transform as a second step to get the three dimensional initial pressure distribution. The integrating detectors are mainly optical detectors and thus can provide a high bandwidth up to several 100 MHz. Using these detectors the resolution is often limited by the acoustic attenuation in the sample itself, because attenuation increases with higher frequencies. Stoke's equation describes the attenuation of photoacoustic generated waves in liquids very well, which results in an increase of the acoustic attenuation with the square of the frequency. For fat tissue an absorption coefficient which is approximately linear proportional to frequency is reported. Presented measurements give an exponential power law dependency with an exponent between 1.31 and 1.36 in fat tissue near the skin of a pig. These equations describing frequency dependent acoustic attenuation have been solved in the past by decomposing the pressure wave into plane waves damped in space, described by the complex part of the wave number equal to the attenuation coefficient. One main result of this paper is that for Photoacoustic Tomography another description seems to be very useful: like for a standing wave in a resonator the wave number is real but the frequency is complex. The complex part of the frequency is the damping in time. Both descriptions are equivalent, but with the complex frequency description acoustic attenuation can be included in all "k-space" methods well known in Photoacoustic Tomography just by introducing a factor describing the exponential decay in time.

Optoacoustic Tomography (OAT) is a hybrid imaging modality that combines the advantages of both optical imaging and ultrasound imaging techniques. Most existing reconstruction algorithms for OAT assume pointlike transducers, which may result in conspicous image blurring and distortions in certain applications. In this work, a new imaging model that incorporates the transducer response is employed for image reconstruction. Computer-simulation studies demonstrate that the new reconstruction method can effectively compensate for image resolution degradation associated with the transducer response.

To speed up the data acquisition in photoacoustic tomography full field detection can be used to avoid the time consuming scanning around the object. The full field detection is realized using a phase contrast method like commonly used in optical microscopy. An expanded light beam considerably larger than the object size illuminates the sample placed in the middle of the propagating light beam. Images obtained with a CCD-camera at a certain time show a projection of the instantaneous pressure field (phase object) in a given direction. The reconstruction method is related to imaging with integrating line detectors, but has to be matched to the specific information in the recorded images, which is now purely spatially resolved as opposed to spatiotemporally for a single scanning detector. The reconstruction of the projection images of the initial pressure distribution is done by back propagating the observed wave pattern in Radon space. Numerical simulations and experiments are performed to show the overall adaptability of this technique in photoacoustic tomography.

For real-time optoacoustic imaging of the human body, a linear array transducer and reflection mode optical irradiation is usually preferred. Such a setup, however, results in significant image background, which prevents imaging structures at the ultimate depth limited only by the signal noise level. Therefore we previously proposed a method for image background reduction, based on displacement-compensated averaging (DCA) of image series obtained when the tissue sample under investigation is gradually deformed. Optoacoustic signals and background signals are differently affected by the deformation and can thus be distinguished. The proposed method has now been applied to imaging artificial tumors embedded inside breast phantoms. Optoacoustic images are acquired alternately with pulse-echo images using a combined optoacoustic/ echo-ultrasound device. Tissue deformation is accessed via speckle tracking in pulse echo images, and optoacoustic images are compensated for the local tissue displacement. In that way optoacoustic sources are highly correlated between subsequent images, while background is decorrelated and can therefore be reduced by averaging. We show that breast image contrast is strongly improved and detectability of embedded tumors significantly increased, using the DCA method.

We report on a new method for extraction of quantified optical absorption maps of scattering and absorbing media using sparse representation, a relatively recent and fast emerging technique in the field of signal processing. The tomographic reconstruction is facilitated by assuming slow spatial variations of illuminating optical field along with relatively sharp changes in optical absorption coefficient. As opposed to previous approaches that utilize photon transport equation in order to correct images for inhomogeneous light distribution within the imaged object, the method herein provides an estimate for photon fluence directly from the recorded optoacoustic signals. In this way a robust quantitative performance is achieved without prior knowledge of illumination geometry and optical properties of the object.

We have developed an integrated photoacoustic imaging (PAI) and high intensity focused ultrasound (HIFU) system for solid tumor treatment. A single-element, spherically focused ultrasonic transducer, with a central frequency of 5MHz, was used to generate a HIFU field in soft tissue. The same ultrasonic transducer was also used as a detector during photoacoustic imaging before and after HIFU treatments. During each experiment, targeted soft tissue was first imaged by PAI. The resulted image was used for the planning of subsequent HIFU treatment. After HIFU treatment, the sample was imaged again by PAI to evaluate the treatment result. Good contrast was obtained between photoacoustic images before and after HIFU treatment. It is concluded that PA imaging technology may potentially be combined with HIFU treatment for imaging-guided therapy.

A novel combined photoacoustic tomography (PAT), optical resolution photoacoustic microscopy (ORPAM) and optical coherence tomography (OCT) instrument has been developed for imaging biological tissues. The system is based on the use of a Fabry-Perot (FP) polymer film ultrasound sensor. This is designed to be transparent to wavelengths between 590nm and 1200nm so that photoacoustic excitation laser pulses in this spectral range can be transmitted through the sensor into the underlying tissue to allow backward mode operation. The dual PAT-ORPAM capability of the system was demonstrated by imaging a tissue phantom composed of 7μm diameter carbon fibres immersed in an optically scattering liquid. The lateral and vertical spatial resolutions in ORPAM mode are approximately 7μm and 10μm respectively for sub-mm depths. In PAT mode, the lateral spatial resolution is less than 50μm for depths up to 5mm and the vertical resolution is approximately 10μm. The transparent nature of the FP polymer film ultrasound sensor offers a convenient platform for combining other optical imaging modalities with PAT and ORPAM. To illustrate this, a frequency-domain OCT system operating at 1060nm was integrated into the system and combined PAT/OCT images of the skin of a mouse were obtained in vivo.

A device for three-dimensional (3D) photoacoustic tomography with resolution in the range of tens of micrometers is presented. It is based on a focused laser beam as detector, which propagates near the imaging object in a water bath and is part of a Mach-Zehnder interferometer. By scanning the beam relative to the object, data for 3D image reconstruction are acquired. Using a phantom consisting of human hairs, the resolution of the setup is demonstrated. Using a flea and an excised mouse heart as samples, the imaging capabilities of the device for complex biological objects are shown.

We show that it is possible to obtain high optical contrast photoacoustic images of tissue with 0.55 μm transverse resolution. To achieve high sensitivity, we used a high NA (0.85), 125 MHz spherically focused ultrasonic transducer in a confocal arrangement with a high resolution optical objective (NA=0.6). Laser pulses of a few nJ in pulse energy with durations of 1.5 ns at a 20 KHz pulse repetition rate were used to generate photoacoustic waves. Although the penetration depth is limited to hundreds of microns by both optical scattering and ultrasonic absorption, the developed technique can compete with optical microscopy, for example, in quantitative spectral measurements, in microcirculation research, or in nanoparticle detection.

Optical-resolution photoacoustic microscopy is a novel imaging technology for visualizing optically-absorbing superficial structures in vivo with lateral spatial resolution determined by optical focusing rather than acoustic detection. Since scanning of the illumination spot is required, the imaging speed is limited by the scanning speed and the laser pulse repetition rate. Unfortunately, lasers with high-repetition rate and suitable pulse durations and energies are difficult to find. We are developing compact laser sources for this application. Passively Q-switched fiber and microchip lasers with pulse repetition rates up to 300 kHz are demonstrated. Using a diode-pumped microchip laser fiber-coupled to a large mode-area Yb-doped fiber amplifier we obtained 60μJ 1-ns pulses at the frequency-doubled 532-nm wavelength. The pulse-repetition rate was determined by the power of the microchip laser pump source at 808nm and may exceed 10 kHz. Additionally, a passively Q-switched fiber laser utilizing a Yb-doped double-cladding fiber and an external saturable absorber has shown to produce 250ns pulses at repetition rates of 100-300 KHz. A photoacoustic probe enabling flexible scanning of the focused output of these lasers consisted of a 45-degree glass prism in an optical index-matching fluid. Photoacoustic signals exiting the sample are deflected by the prism to an ultrasound transducer. Phantom studies with a 7.5-micron carbon fiber demonstrate the ability to image with optical rather than acoustic resolution. We believe that the high pulse-repetition rates and the potentially compact and fiber-coupled nature of these lasers will prove important for clinical imaging applications where realtime imaging performance is essential.

Purpose: Ultrasound and optical coherence tomography (OCT) are widely used techniques for diagnostic imaging of the eye. OCT provides excellent resolution, but limited penetration. Ultrasound provides better penetration, but an order-of-magnitude poorer resolution than OCT. Photoacoustic imaging is relatively insensitive to scattering, and so offers a potential means to image deeper than OCT. Furthermore, photoacoustic imaging detects optical absorption, a parameter that is independent of that detected by conventional ultrasound or OCT. Our aim was to develop a photoacoustic system suitable for imaging the eye. Methods: We developed a prototype system utilizing a focused 20 MHz ultrasound probe with a central aperture through which optics were introduced. The prototype system produced 1-μJ, 5-nsec pulses at 532 or 1064 nm with a 20-μm spot size at a 500 Hz repetition rate. The photoacoustic probe was mounted onto computer-controlled linear stages and pulse-echo ultrasound and photoacoustic images obtained on ex vivo pig eyes and in vivo mouse eyes. Results: Lateral resolution was significantly improved by use of a laser spot size much smaller than the acoustic beamwidth. Photoacoustic signals were obtained primarily from melanin in ex vivo tissues and from melanin and hemoglobin in vivo. Image fusion allowed superposition of photoacoustic signals upon the anatomic features detected by conventional ultrasound. Conclusion: Photoacoustic imaging detects the presence of clinically relevant pigments, such as melanin and oxyand deoxy-hemoglobin, and, potentially, from other pathologic pigments occurring in disease conditions (tumors, nevii, macular degeneration). Fine-resolution photoacoustic data provides information not detected in current ophthalmic imaging modalities.

We report results of two in vivo functional human imaging experiments using photoacoustic microscopy. In Experiment 1, the hemodynamic response to an ischemic event was measured. The palm of a volunteer was imaged and a single cross-section was monitored while periodic arterial occlusions were administered using a blood pressure cuff wrapped around the upper arm and inflated to ~280 mmHg. Significant relative decreases in oxygen saturation (sO₂) and total hemoglobin (HbT) were observed during periods of ischemia. Upon release of the occlusion, significant relative increases in sO2 and HbT due to post-occlusive reactive hyperemia were recorded. Experiment 2 explored the vascular response to a local, external thermal stimulus. Thermal hyperemia is a common physiological phenomenon and thermoregulation function in which blood flow to the skin is increased to more efficiently exchange heat with the ambient environment. The forearm of a volunteer was imaged and a single cross-section was monitored while the imaged surface was exposed to an elevated temperature of ~46°C. Due to thermal hyperemia, relative increases in sO₂ and HbT were measured as the temperature of the surface was raised. These results may contribute as clinically relevant measures of vascular functioning for detection and assessment of vascular related diseases.

Photoacoustic microscopy (PAM) is a high-contrast, high-resolution imaging modality used primarily for imaging hemoglobin and melanin. Important applications include mapping of the microvasculature and melanoma tumor margins. We have developed a novel photoacoustic microscope design, which substantially simplifies construction by enabling the use of unmodified commercial optics and ultrasonic transducers. Moreover, the simple design may be readily incorporated into a standard light microscope, thus providing a familiar imaging platform for clinical researchers. A proof-of-concept Off-Axis PAM system with a lateral resolution of 26 μm and a modest axial resolution of 410 μm has been assembled and characterized using tissue samples. We have derived the appropriate equations to describe the relevant design parameters and verified the equations via measurements made on our prototype Off-Axis PAM system. A consequence of the simple design is a reduction in axial resolution compared to coaxial designs. The reduction is inversely proportional to the cosine of the angle between excitation and detection and equal to 15% and 41% for angles of 30º and 45º, respectively. While resolution is negatively affected by off-axis detection, the ability to measure weak signals at depth is enhanced. Off-axis detection has an inherent dark-field quality; chromophores excited outside the numerical aperture of the ultrasonic detector will not be detected. The physical geometry of Off-Axis PAM enables the placement of the ultrasonic transducer at the minimum distance from the sample with no obstructions between the sample and transducer. This may prove to be an additional advantage of Off-Axis PAM over designs that incorporate long working distance ultrasonic transducers and/or require the propagation of the acoustic wave through the laser excitation optics to achieve co-axial detection.

Photoacoustic imaging exploits contrast mechanisms that depend on optical and thermomechanical properties of optical absorbers. The photoacoustic signal bandwidth is dictated by the absorber size and the laser pulse width. In this work we demonstrate that photoacoustic signals can be detected from micron and sub-micron particles. We anticipate applications to include cellular imaging with nanometer sized contrast agents such as gold nanoshells, nanorods, and nanocages. An existing acoustic microscopy system was used (the SASAM 1000, kibero GmbH). This platform is developed on an Olympus IX81 optical microscope with a rotating column that has an optical condenser for transmission optical microscopy and an acoustic module for the acoustic microscopy. The adapted optoacoustic module consists of a Qswitched Nd:YAG solid-state-laser (Teem Photonics, France) generating sub-nanosecond pulses. Scans were acquired of microparticles (1 μm black Toner particles) and cells. The confocal arrangement allowed high signal to noise ratio photoacoustic signals (>30 dB) to be detected at approximately 400 MHz. The particles of various sizes produced signals of different frequency content. In imaging mode, the full width half maximum (FWHM) was measured to be 3.6 μm for the 400 MHz transducer which is in general agreement theory for a 0.3 NA objective (4.3μm). Moreover, images are generated from single melanoma cells, generated by the endogenous contrast from the intracellular melanin.

The characteristic absorption spectrum of hemoglobin depends upon the amount of oxygen the hemoglobin carries. This property of the hemoglobin enables one to image blood oxygen saturation distribution in biological tissues by using spectroscopic photoacoustic tomography. In photoacoustic imaging, the amplitude of photoacoustic signal induced by optical absorption is proportional to light energy deposition which is the product of the optical absorption coefficient and local light fluence at the imaging target. Since the attenuation of light in biological tissues are wavelength dependent, the spectrum of local light fluence at a target tissue beneath the sample surface is different from the spectrum of the incident light fluence above the surface. An unknown spectrum of the light fluence in the sample prevents us from obtaining quantitative functional images such as oxygen saturation and hemoglobin concentration in the sample by photoacoustic tomography. We developed a new technique of using an optical contrast agent with known optical absorption spectrum to obtain the accurate spectrum of light fluence at a subsurface target tissue such as a blood vessel beneath the sample surface. The technique has been validated by obtaining an accurate absorption spectrum of a micro-flow vessel buried in strong optical scattering media including diluted whole milk and chicken breast tissue. In this work, we further explored the capability of this technique through the experiments on tissue mimicking phantoms and living animals. By using this technique we were able to obtain accurate blood oxygen saturation in vessels buried at different depths in an optical scattering medium. Also, the oxygenation levels in main arteries in rat tails have been quantified more accurately in a noninvasive manner.

Nanotechnology and the various properties of gold nanoparticles (AuNPs) are quickly changing the field of cancer detection and treatment. Photoacoustic detection methods show an increase in sensitivity using gold nanoparticle antibody conjugation, which selectively targets melanoma cancer cells. Instead of targeting melanoma tumors, we tag single cells, analogous to circulating metastatic melanoma cells. Using an in vitro, stationary cell system and planar samples, we demonstrate an average of 24% improved optical detectability of melanoma cells tagged with AuNPs over unprocessed melanoma cells. Tagged cells showed a raised plateau of absorbance from 470nm to 550nm. Untagged cells showed a general decline in absorption as wavelength increased. The results of our study have the potential to not only better develop photoacoustic detection of melanoma, but also extend the viability and use of photoacoustics into detection of otherwise unpigmented cancers.

Photoacoustic tomography is a non-invasive imaging technique based on the detection of broadband acoustic emissions generated by the absorption of light in tissue. This technique utilises the high contrast of optical imaging with high resolution from ultrasound imaging. However, the ability to detect these emissions above the noise level ultimately limits the depth to which imaging can be performed. Introduction of light-absorbing gold nanoparticles can improve the signal-to-noise ratio in tissue, through greater optical absorption and targeting specific cell populations, thereby enhancing contrast and the ability to delineate tissue types. For sufficiently high laser fluence incident on a nanoparticle, a transient vapour cavity is formed and undergoes inertial collapse, generating a broadband emission and possibly additional contrast. However, the laser fluence required to achieve this typically exceeds the maximum permissible exposure (MPE) for tissue. Through the combination of ultrasonic and optical pulses, the light and sound thresholds required to repeatedly generate inertial cavitation were reduced to 11.1 mJ/cm² and 1.5 MPa respectively. Experiments employed a transparent acrylamide gel possessing a small (<600 μm) spherical region doped with 80 nm diameter gold nanoparticles and simultaneously exposed to pulsed laser light (532 nm) and pulsed ultrasound (1.1 MHz). The amplitude of broadband emissions induced by both light and sound exceeded that produced by light alone by almost two orders of magnitude, thereby facilitating imaging a deeper depth within tissue. 2D images of doped regions generated from conventional photoacoustic and ultrasound-enhanced emissions are presented and compared.

Ultrasonic (US) imaging is the most common real-time modality, providing multiple dimensional changes in morphology for clinical practice. Photoacoustic (PA) imaging has demonstrated great promise as a new functional and molecular imaging tool. However, absorption in background tissue also generates a PA signal and limits the specific contrast of molecular contrast agents. To increase the linear range of these agents, the background must be suppressed. Magnetic nanoparticles provide a new possibility to increase contrast by magnetomotive manipulation during imaging. A multi-functional imaging system integrating US and PA imaging with magnetic manipulation can take advantage of each single modality by providing anatomical images and molecular function with greater contrast. However, one key issue for multi-functional imaging is how to spatially combine and temporarily synchronize US and PA imaging with magnetomotive instrumentation. In this study, we built a simple system to integrate US and PA imaging with magnetomotive capability. We evaluated this system by measuring the motion of a phantom including magnetic nanoparticles (MNPs) using US when these particles were subjected to a time-varying magnetic field.

The unique electrical properties of Single-Walled Carbon Nanotubes make them good candidates for thermoacoustic contrast agents. Theoretical considerations suggest that nanotubes are capable of greatly increasing a material's absorption of electromagnetic radiation. We describe these properties and discuss our measurements of aqueous nanotube solutions and nanotube-infused tissue mimicking phantoms. We discuss results and the difficulties currently associated with making these measurements on nanotubes.

Sentinel lymph node biopsy (SLNB) has become the standard method for axillary staging in breast cancer patients, relying on invasive identification of sentinel lymph nodes (SLNs) following injection of blue dye and radioactive tracers. While SLNB achieves a low false negative rate (5-10%), it is an invasive procedure requiring ionizing radiation. As an alternative to SLNB, ultrasound-guided fine needle aspiration biopsy has been tested clinically. However, ultrasound alone is unable to accurately identify which lymph nodes are sentinel. Therefore, a non-ionizing and noninvasive detection method for accurate SLN mapping is needed. In this study, we successfully imaged methylene blue dye accumulation in vivo in rat axillary lymph nodes using a Phillips iU22 ultrasound imaging system adapted for photoacoustic imaging with an Nd:YAG pumped, tunable dye laser. Photoacoustic images of rat SLNs clearly identify methylene blue dye accumulation within minutes following intradermal dye injection and co-registered photoacoustic/ultrasound images illustrate lymph node position relative to surrounding anatomy. To investigate clinical translation, the imaging depth was extended up to 2.5 cm by adding chicken breast tissue on top of the rat skin surface. These results raise confidence that photoacoustic imaging can be used clinically for accurate, noninvasive SLN mapping.

Purpose: The objective is to develop a multivariate in vivo hemodynamic model of tissue oxygenation (MiHMO₂) based on 3D photoacoustic spectroscopy. Introduction: Low oxygen levels, or hypoxia, deprives cancer cells of oxygen and confers resistance to irradiation, some chemotherapeutic drugs, and oxygen-dependent therapies (phototherapy) leading to treatment failure and poor disease-free and overall survival. For example, clinical studies of patients with breast carcinomas, cervical cancer, and head and neck carcinomas (HNC) are more likely to suffer local reoccurrence and metastasis if their tumors are hypoxic. A novel method to non invasively measure tumor hypoxia, identify its type, and monitor its heterogeneity is devised by measuring tumor hemodynamics, MiHMO₂. Material and Methods: Simulations are performed to compare tumor pO₂ levels and hypoxia based on physiology - perfusion, fractional plasma volume, fractional cellular volume - and its hemoglobin status - oxygen saturation and hemoglobin concentration - based on in vivo measurements of breast, prostate, and ovarian tumors. Simulations of MiHMO₂ are performed to assess the influence of scanner resolutions and different mathematic models of oxygen delivery. Results: Sensitivity of pO₂ and hypoxic fraction to photoacoustic scanner resolution and dependencies on model complexity will be presented using hemodynamic parameters for different tumors. Conclusions: Photoacoustic CT spectroscopy provides a unique ability to monitor hemodynamic and cellular physiology in tissue, which can be used to longitudinally monitor tumor oxygenation and its response to anti-angiogenic therapies.

The preferential absorption of near infrared light by blood makes photoacoustic imaging well suited to visualising vascular structures in soft tissue. In addition, the spectroscopic specificity of tissue chromophores can be exploited by acquiring images at multiple excitation wavelengths. This allows the quantification of endogenous chromophores, such as oxy- and deoxyhaemoglobin, and hence blood oxygenation, and the detection of exogenous chromophores, such as functionalised contrast agents. More importantly, this approach has the potential to visualise the spatial distribution of low concentrations of functionalised contrast agents against the strong background absorption of the endogenous chromophores. This has a large number of applications in the life sciences. One example is the structural and functional phenotyping of transgenic mice for the study of the genetic origins of vascular malformations, such as heart defects. In this study, photoacoustic images of mouse embryos have been acquired to study the development of the vasculature following specific genetic knockouts.

Prostate cancer is currently the most common cancer among Canadian men. Due to an increase in public awareness and screening, prostate cancer is being detected at earlier stages and in much younger men. This is raising the need for better treatment monitoring approaches. Optoacoustic imaging is a new technique that involves exposing tissues to pulsed light and detecting the acoustic waves generated by the tissue. Optoacoustic images of a tumour bearing mouse and an agematched control were acquired for a 775 nm illumination using a reverse-mode imaging system. A murine model of prostate cancer, TRAMP (transgenetic adenocarcinoma of mouse prostate), was investigated. The results show an increase in optoacoustic signal generated by the tumour compared to that generated by the surrounding tissues with a contrast ratio of 3.5. The dimensions of the tumour in the optoacoustic image agreed with the true tumour dimensions to within 0.5 mm. In this study we show that there are detectable changes in optoacoustic signal strength that arise from the presence of a tumour in the prostate, which demonstrates the potential of optoacoustic imaging for the monitoring of prostate cancer therapy.

Diagnosis of edema, abnormal accumulation of water in tissue, is important for managing various traumatic injuries and diseases. However, there is no established method for real-time, noninvasive monitoring of edema. In severe extensive burn injuries, edema develops both topically and systemically due to the increased permeability of blood vessels. In this study, we examined photoacoustic (PA) monitoring of edema formed in rat burn models. Deep dermal burn with a 20% total body surface area was made in the dorsal skin of rats. Burn and its adjacent nonburn tissues were irradiated with 6-ns light pulses at 1430 nm, which is one of the absorption peak wavelengths of water in the near infrared. The PA signal amplitude increased until 12 - 24 hr postburn, and thereafter it gradually decreased to its initial level; the latter phase (after 24 hr postburn) coincided with a diuretic phase in the rats. There was a significant correlation between the PA signal amplitudes and water contents in the tissue measured by wet/dry weight method. These findings demonstrate the validity of PA measurement for real-time, noninvasive monitoring of edema.

Photoacoustic flowmetry has been used to detect single circulating melanoma cells in vitro. Circulating melanoma cells are those cells that travel in the blood and lymph systems to create secondary tumors and are the hallmark of metastasis. This technique involves taking blood samples from patients, separating the white blood and melanoma cells from whole blood and irradiating them with a pulsed laser in a flowmetry set up. Rapid, visible wavelength laser pulses on the order of 5 ns can induce photoacoustic waves in melanoma cells due to their melanin content, while surrounding white blood cells remain acoustically passive. We have developed a system that identifies rare melanoma cells and captures them in 50 microliter volumes using suction applied near the photoacoustic detection chamber. The 50 microliter sample is then diluted and the experiment is repeated using the new sample until only a melanoma cell remains. We have tested this system on dyed microspheres ranging in size from 300 to 500 microns. Capture of circulating melanoma cells may provide the opportunity to study metastatic cells for basic understanding of the spread of cancer and to optimize patient specific therapies.

The composition and concentration of exhaled volatile gases reflects the physical ability of a patient. Therefore, a breath analysis allows to recognize an infectious disease in an organ or even to identify a tumor. One of the most prominent breath tests is the ¹³C-urea-breath test, applied to ascertain the presence of the bacterium helicobacter pylori in the stomach wall as an indication of a gastric ulcer. In this contribution we present a new optical analyzer that employs a compact and simple set-up based on photoacoustic spectroscopy. It consists of two identical photoacoustic cells containing two breath samples, one taken before and one after capturing an isotope-marked substrate, where the most common isotope ¹²C is replaced to a large extent by ¹³C. The analyzer measures simultaneously the relative CO₂ isotopologue concentrations in both samples by exciting the molecules on specially selected absorption lines with a semiconductor laser operating at a wavelength of 2.744 μm. For a reliable diagnosis changes of the ¹³CO₂ concentration of 1% in the exhaled breath have to be detected at a concentration level of this isotope in the breath of about 500 ppm.

Malaria is a blood borne infection affecting hundreds of millions of people worldwide². The parasites reproduce within the blood cells, eventually causing their death and lysis. This process releases the parasites into the blood, continuing the cycle of infection. Usually, malaria is diagnosed only after a patient presents symptoms, including high fever, nausea, and, in advanced cases, coma and death. While invading the bloodstream of a host, malaria parasites convert hemoglobin into an insoluble crystal, known as hemozoin. These crystals, approximately several hundred nanometers in size, are contained within red blood cells and white blood cells that ingest free hemozoin in the blood. Thus, infected red blood cells and white blood cells contain a unique optical absorber that can be detected in blood samples using static photoacoustic detection methods. We separated the white blood cells from malaria infected blood and tested it in a photoacoustic set up using a tunable laser system consisting of an optical parametric oscillator pumped by an Nd:YAG laser with pulse duration of 5 ns. Our threshold of detection was 10 infected white blood cells per microliter, which is more sensitive than current diagnosis methods using microscopic analysis of blood.

We developed a multiparameter, noninvasive, optoacoustic diagnostic platform that will accurately and continuously measure total hemoglobin concentration, venous oxyhemoglobin saturation (both cerebral and internal jugular), and other important physiological parameters in large populations of patients. We built optoacoustic systems for monitoring of these parameters and performed clinical tests of the systems including highly-portable laser diode-based systems. We will report results of the clinical studies performed by our group to demonstrate the capabilities of the optoacoustic platform for noninvasive monitoring of multiple physiological parameters. Our data indicate that the accuracy of the optoacoustic measurements is approaching that of "gold standard" invasive techniques.

We developed an optoacoustic technique for noninvasive, accurate, and continuous monitoring of total hemoglobin concentration and venous oxyhemoglobin saturation by probing specific blood vessels. In this work we report the development and tests of novel, focused optoacoustic transducers that provide blood vessel probing with sub-millimeter lateral resolution. The focused transducers were incorporated in our highly portable, laser diode-based optoacoustic monitoring system for pre-clinical and clinical tests. Our studies demonstrated that: 1) the focused transducer response is linearly dependent on blood total hemoglobin concentration with a high correlation coefficient; and 2) the sub-millimeter lateral resolution provided higher specificity of blood vessel probing, in particular, for smaller blood vessels such as the radial artery (diameter 2-3 mm).

Purpose: The purpose of this study is to monitor in vivo the IR dose dependent response of NF-κB and tumor hemodynamics as a function of time. Material and Methods: An MDA-231 breast cancer cell line was stably transfected with a firefly luciferase gene within the NF-kappaB promoter. Tumors on the right flank irradiated with a single fractionated dose of 5Gy or 10Gy. Over two weeks, photoacoustic spectroscopy (PCT-S), bioluminescence imaging (BLI), and dynamic contrast enhanced CT (DCE-CT) was used to monitor hemoglobin status, NF-kappaB expression, and physiology, respectively. Results: From the BLI, an increase in NF-kappaB expression was observed in both the right (irradiation) and left (nonirradiated) tumors, which peaked at 8-12 hours, returned to basal levels after 24 hours, and increased a second time from 3 to 7 days. This data identifies both a radiation-induced bystander effect and a bimodal longitudinal response associated with NF-κB-controlled luciferase promoter. The physiological results from DCE-CT measured an increase in perfusion (26%) two days after radiation and both a decrease in perfusion and an increase in fp by week 1 (10Gy cohort). PCT-S measured increased levels of oxygen saturation two days post IR, which did not change after 1 week. Initially, NF-κB would modify hemodynamics to increase oxygen delivery after IR insult. The secondary response appears to modulate tumor angiogenesis. Conclusions: A bimodal response to radiation was detected with NF-kappaB-controlled luciferase reporter with a concomitant hemodynamic response associated with tumor hypoxia. Experiments are being performed to increase statistics.

We have investigated the limitations of our laser ultrasonic plane wave δ-source. In theory, the device is capable of producing an acoustic impulse with a bandwidth exceeding 30 MHz. However, a bandwidth of 12 MHz is measured with a calibrated wideband hydrophone. A test setup was designed and built. It permits the investigation of experimental parameters that alter the generated acoustic impulse: laser pulse duration, laser spatial profile, and absorber opacity. Laser energy spatial profile is the main contributor to the narrowing of the frequency band. Our findings are presented, along with further justifications for a device with very large effective area.

To assess the malignancy and progression of a tumour, parameters such as the size and number density of the microvessels are expected to be important. The optical absorption due to the blood that fills the microvessels can be visualised by optoacoustic imaging (OA). We have previously reported that increasing the inhomogeneity of absorption within a large absorbing volume produces evidence of reduced acoustic coherence which results in improved contrast and boundary detectability. Here we propose to take advantage of the expectation that the detailed nature of the inhomogeneity should influence the frequency spectrum of the OA signal. The overall aim of this work is to determine whether an analysis of the frequency spectrum of the emitted optoacoustic signal can be used to determine the scale of this absorption inhomogeneity, in particular parameters such as the characteristic size and separation of the absorbers (microvessels). In the preliminary study reported here, various gelatine-intralipid phantoms containing cylindrical wallless tubes filled with an ink solution were measured in water with a linear array ultrasound detector, using pulsedillumination that had been adjusted for an optimal distribution of light fluence with depth. Simulations of the experiments were also conducted, using a time domain acoustic propagation method. The results confirm that optoacoustic signals bear information on the sizes and distribution of the absorbers in their frequency spectra. It is shown that a simple way to determine the diameter of a single cylindrical absorber is to estimate the quefrency of the peak in the cepstrum of the measured signal. Further work is proposed to extend this to the statistical estimation of mean diameter and mean separation for an ensemble of similar absorbers and to absorbers with a diameter that is smaller than the axial resolution of the acoustic receiver.

Dynamics of the thermoelastic expansion of native and coagulated ex-vivo bovine liver tissues after their irradiation by short laser pulses were studied. The differences in optical and thermo-mechanical properties of the native and coagulated samples such as their Gruneisen coefficient and optical attenuation depth were quantitatively determined. It was found that for coagulated ex-vivo bovine liver samples, the optical attenuation depth decrease by an average of 47%. Also significant differences were observed in the dynamics of thermoelastic expansion of the tissue surface. These differences can be potentially linked to differences in thermo-mechanical properties between native and coagulated samples. The changes in these properties may be

Purpose: Our purpose is to develop and test a molecular probe that can detect the expression of neutropilin-1 receptor (NPR-1) in vivo using fluorescence imaging and photoacoustic spectroscopy. Introduction: NPR-1 is expressed on endothelial cells and some breast cancer cells, and binds to vascular endothelial growth factor VEGF165, a growth factor associated with pathological tumor angiogenesis. This receptor is coexpressed with VEGFR2 and shown to enhance the binding of VEGF165; therefore, it has the potential to be used as a marker of angiogenic activity and targeted for therapy. Material and Methods: A peptide specific to NPR-1 receptor was synthesized and conjugated to a NIR fluorochrome (IRDye800CW) and was intravenously injected into mice with breast tumors (MCF7^VEGF). Probe kinetics was monitored in vivo via near infrared fluorescence (NIRF) within an optical imager for up to 72 hours within the tumor and compared to other organs (liver, muscle) for binding specificity. A multivariate fitting algorithm was used to spectrally deconvolve the IRDye800CW from endogenous hemoglobin signature (hemoglobin concentration and oxygen saturation). Results: Dynamics of the NIR fluorescence signal within the first hour after injection indicates specific binding compared to muscle, with an average tumor-to-muscle ration of 2.00 (+/- 0.27). Spectral analysis clearly indentified the presence of the NPR-1 probe. Based on calibration data, the average tumor concentration from both NIRF and PCT-S was measured to be ~200-300nM. Conclusion: These preliminary results show the capability of PCT to image an exogenous probe in vivo in addition to its hemoglobin state.

Several papers recently have addressed the issue of estimating chromophore concentration in PAT using multiple wavelengths. The question is how does one choose the multiple wavelengths for imaging in PAT that would give the most accurate results. Previous work was based on the knowledge of the wavelength dependence of the extinction coefficient of chromophores but did not directly address this question. One would assume that the wavelength that maximizes the extinction coefficient of the chromophorewould be the most suitable. However, this may not always be the case, especially if the extinction peak of the chromophore is fairly broad. In this paper, we derive an expression for the variance of the measured signal based on the Cramer-Rao lower bound (CRLB). This lower bound on variance can be evaluated numerically for different wavelengths using the variation of the extinction coefficients and scattering coefficients with wavelength. The wavelength that gives the smallest variance will be optimal for multi-wavelength PAT to estimate the chromophore concentration. The expression for CRLB has been derived analytically for estimating the concentration of a oxy or deoxyhemoglobin contained in a background tissue-like solution using the knowledge of the illumination function in a specific geometry using a photoacoustic microscope. This approach could also be extended to the estimation of concentrations of multiple chromophores and for other geometries.

Image-guided molecular photothermal therapy using targeted gold nanoparticles acting as photoabsorbers can be used to noninvasively treat various medical conditions including cancer. Among different types of gold nanoparticles, gold nanorods are an attractive candidate for both photothermal therapy and photoacoustic imaging due to their high and tunable optical absorption cross-section. However, nanorods are not thermodynamically stable; under laser exposure, the nanorods can easily transform to spheres, thus changing their desired optical properties. In this study, gold-silica coreshell nanorods were prepared by coating silica directly onto the surface of PEGylated gold nanorods using a modified Stöber method. The nanorods were exposed to 800 nm wavelength, 7 ns pulses of light at a 10 Hz pulse repetition rate. For different fluences ranging from 0 to 8 mJ/cm², the optical extinction spectrum was measured before and after the exposure to investigate their photothermal stability. Finally, the effectiveness of gold-silica core-shell nanoparticles as a photoacoustic contrast agent and photothermal nanoabsorber was tested using inclusion-embedded phantoms and a combined ultrasound and photoacoustic imaging system. The results of our study suggest that gold-silica core-shell nanorods are excellent candidates for image-guided molecular photothermal therapy.

In this work, we have developed a selective-plane illumination multispectral optoacoustic tomography (MSOT) technique for high-resolution whole-body visualization of intact optically diffusive organisms whose sizes may vary from sub-millimeter up to a centimeter range and beyond. By combining multi-wavelength illumination, the method is shown capable of resolving tissue-specific expression of fluorescent proteins and other molecular biomarkers located deep in living optically diffuse tissues.

Gold nanoparticles have been used as contrast agent in photoacoustic imaging to increase the detection sensitivity. For example, gold nanorods (AuNRs) have been used in time-intensity based flow estimation and used as nanoprobes to target cancer cells for early diagnosis and effective treatment. In this study, we aimed at the design and synthesis of a new type of gold nanoparticles with enhanced photoacoustic response. The key hypothesis is to create a nanostructure that allows anisotropic heat release. Specifically, such a structure results in higher heat flux transmitting outwards from the ends of the particle and therefore a greater temperature gradient can be created. To achieve this, a layer of SiO₂ was coated along the longer axis of the gold nanorods, leaving both ends uncovered. These new particles are labeled as AuNR@nu-SiO₂ with non-uniform ("nu") coating of silica. Experiments were performed to demonstrate the enhanced photoacoustic response from AuNR@nu-SiO₂. The optical illumination was delivered by a Ti: Sapphire laser pumped by an Nd:YAG laser. A home-made photoacoustic transducer with a center frequency of 20 MHz detected the resulted acoustic signal. First, new types of particles coated with and without SiO₂ were compared to bare AuNR in order to show the ability of the new nanostructure to enhance photoacoustic response. Second, the shape stability of the new particles was evaluated by measuring the photoacoustic responses versus time after high power laser irradiation. Third, the effect of thickness of SiO₂ of AuNR@nu-SiO₂ ranges from 1 nm to 15 nm was also evaluated. Results show that the mean intensity in photoacoustic image increase about 5 dB for AuNR@nu-SiO₂ compared to bare AuNR. Also, it reveals that the normalized intensity for AuNR drops to below 0.6 while it is mostly larger than 0.7 in the case of AuNR@nu-SiO₂ under pulse laser irradiation. In other words, the new type of nanoparticles is less susceptible to shape transformation. Moreover, it is indicated that the photoaocustic response increases slightly with the thickness of SiO₂ and approach to an maximum response at 9 nm thickness. In short, these new particles can be used to achieve the same level of photoacoustic response with a fewer amount of particles, which means that there is less toxicity.

We have designed a protease-sensitive imaging probe for optoacoustic imaging whose absorption spectrum changes upon cleavage by a protease of interest. The probe comprises an active site, a derivative of chlorophyll or natural photosynthetic bacteriochlorophyll that absorbs in the near infrared, conjugated to a peptide backbone specific to the protease being imaged. The uncleaved molecules tend to aggregate in dimers and trimers causing a change in the absorption spectrum relative to that of the monomer. Upon cleavage, the probe molecules de-aggregate giving rise to a spectrum characteristic of monomers. We show using photospectrometry that the two forms of the probe have markedly different absorption spectra, which could allow for in vivo optoacoustic identification using a multiwavelength imaging strategy. Optoacoustic measurements using a narrow-band dye laser find spectral peaks in the two forms of the probe at the expected location. The optoacoustic signal from the uncleaved probe is found to be considerably weaker than that of the cleaved probe, perhaps due to poor optical-acoustic coupling in the aggregated molecules. However, ultimately, it is detection of the cleaved probe that is of the greatest import since it reports on the protease activity of interest. PUBLISHERS NOTE 9/1/2010: The figure numbers are corrected. If you downloaded the incorrect version of Paper 75641T and no longer have access to download the correct paper, please contact CustomerService@SPIEDigitalLibrary.org for assistance.

Gold nanoparticles have received much attention due to their potential diagnostic and therapeutic applications. Gold nanoparticles are attractive in many biomedical applications because of their biocompatibility, easily modifiable surfaces for targeting, lack of heavy metal toxicity, wide range of sizes (35-100 nm), tunable plasmonic resonance peak, encapsulated site-specific drug delivery, and strong optical absorption in the near-infrared regime. Specifically, due to their strong optical absorption, gold nanoparticles have been used as a contrast agent for molecular photoacoustic (PA) imaging of tumor. The plasmonic resonance peak of the gold nanocages (AuNCs) was tuned to the near-infrared region, and the ratio of the absorption cross-section to the extinction cross-section was approximately ~70%, as measured by PA sensing. We used PEGylated gold nanocages (PEG-AuNCs) as a passive targeting contrast agent on melanomas. After 6-h intravenous injection of PEG-AuNCs, PA amplitude was increased by ~14 %. These results strongly suggest PA imaging paired with AuNCs is a promising diagnostic tool for early cancer detection.

We present results from a clinical case study on imaging breast cancer using a real-time interleaved two laser optoacoustic imaging system co-registered with ultrasound. The present version of Laser Optoacoustic Ultrasonic Imaging System (LOUIS) utilizes a commercial linear ultrasonic transducer array, which has been modified to include two parallel rectangular optical bundles, to operate in both ultrasonic (US) and optoacoustic (OA) modes. In OA mode, the images from two optical wavelengths (755 nm and 1064 nm) that provide opposite contrasts for optical absorption of oxygenated vs deoxygenated blood can be displayed simultaneously at a maximum rate of 20 Hz. The real-time aspect of the system permits probe manipulations that can assist in the detection of the lesion. The results show the ability of LOUIS to co-register regions of high absorption seen in OA images with US images collected at the same location with the dual modality probe. The dual wavelength results demonstrate that LOUIS can potentially provide breast cancer diagnostics based on different intensities of OA images of the lesion obtained at 755 nm and 1064 nm. We also present new data processing based on deconvolution of the LOUIS impulse response that helps recover original optoacoustic pressure profiles. Finally, we demonstrate the image analysis tool that provides automatic detection of the tumor boundary and quantitative metrics of the optoacoustic image quality. Using a blood vessel phantom submerged in a tissue-like milky background solution we show that the image contrast is minimally affected by the phantom distance from the LOUIS probe until about 60-65 mm. We suggest using the image contrast for quantitative assessment of an OA image of a breast lesion, as a part of the breast cancer diagnostics procedure.

The combination of ultrasonic and photoacoustic imaging modalities has yet to be realized in the high-frequency regime (>20MHz) where spatial resolution may permit visualization of the microvasculature. In this work, we characterize the in-vivo performance of a custom ultrasound-photoacoustic B-scanning imaging system. This system utilizes a combined ultrasound/photoacoustic probe attached to a voice-coil capable of approximately 1cm lateral translation at a rate of up to 15Hz. The probe is comprised of a 25MHz ultrasound transducer, configured confocally with a conical mirror-based dark-field laser delivery system. The fast-scanning mode permits realtime ultrasound imaging. The imaging speed of the photoacoustic mode is limited by the repetition rate of the 532nm laser (up to 20Hz). Signals from the transducer are amplified by a 39dB preamp with an additional time-gain compensation stage of up to 24dB. Control of the system is through a digital input-output PCI card, which acts as a pulse-sequencer and permits software control of time-gain compensation. This setup permits interlaced pulse sequences for excellent registration of ultrasonic and photoacoustic data, as well as separate timegain compensation curves for photoacoustic and ultrasound modalities. We have managed to achieve a lateral resolution of 155 μm and an axial resolution of 40 μm. The system is used to visualize the finger and palm of a hand to almost 1cm ultrasound depths and multiple millimeter-scale photoacoustic depths. Photoacoustic images are overlaid on the ultrasound images for simultaneous visualization of the microvasculature and surrounding tissue.

Real-time temperature monitoring with high spatial resolution (~1 mm) and high temperature sensitivity (1 °C or better) is needed for the safe deposition of heat energy in surrounding healthy tissue and efficient destruction of tumor and abnormal cells during thermotherapy. A temperature sensing technique using thermoacoustic and photoacoustic measurements combined with a clinical Philips ultrasound imaging system (iU22) has been explored in this study. Using a tissue phantom, this noninvasive method has been demonstrated to have high temporal resolution and temperature sensitivity. Because both photoacoustic and thermoacoustic signal amplitudes depend on the temperature of the source object, the signal amplitudes can be used to monitor the temperature. The signal is proportional to the dimensionless Grueneisen parameter of the object, which in turn varies with the temperature of the object. A temperature sensitivity of 0.5 °C was obtained at a temporal resolution as short as 3.6 s with 50 signal averages.

We previously demonstrated that multimodal microscopy combining photoacoustic microscopy and optical coherence tomography can provide comprehensive insight into biological tissue at μm-level resolution by exploiting both optical absorption and scattering contrasts. Recently, we have developed a second-generation integrated photoacoustic and optical-coherence microscope, which can potentially be adapted for clinical applications. In this new system, we can perform photoacoustic and optical-coherence imaging simultaneously at a speed of 5,000 A-lines per second with real-time on-screen display. Also, both modalities now work in reflection mode instead of transmission mode, allowing easy access to various anatomical locations of interest. Imaging of skin and eye has been demonstrated in living small animals.

We have constructed and tested a prototype test bed that allows us to form 3D photoacoustic CT images using near-infrared (NIR) irradiation (700 - 900 nm), 3D thermoacoustic CT images using microwave irradiation (434 MHz), and 3D ultrasound images from a commercial ultrasound scanner. The device utilizes a vertically oriented, curved array to capture the photoacoustic and thermoacoustic data. In addition, an 8-MHz linear array fixed in a horizontal position provides the ultrasound data. The photoacoustic and thermoacoustic data sets are co-registered exactly because they use the same detector. The ultrasound data set requires only simple corrections to co-register its images. The photoacoustic, thermoacoustic, and ultrasound images of mouse anatomy reveal complementary anatomic information as they exploit different contrast mechanisms. The thermoacoustic images differentiate between muscle, fat and bone. The photoacoustic images reveal the hemoglobin distribution, which is localized predominantly in the vascular space. The ultrasound images provide detailed information about the bony structures. Superposition of all three images onto a co-registered hybrid image shows the potential of a trimodal photoacoustic-thermoacoustic-ultrasound small-animal imaging system.

The photoacoustic signal of an optical absorber in a turbid medium is proportional to the local laser fluence, the optical absorption coefficient and the Gruneisen parameter. The local fluence at a subsurface absorber is determined by the initial incident fluence and optical properties of the media. Knowledge of laser fluence at subcutaneous tissue locations will improve our ability to estimate local chromophore concentrations and will lead to more quantitative estimates of blood oxygen saturation with photoacoustics. By integrating an oblique incidence reflectance (OIR) system in a photoacoustic imaging system, we are able to estimate optical properties of the turbid medium. To do this, we use a unique photoacoustic probe consisting of a 45-degree optical prism in an optical index-matching fluid. An oblique CWlaser beam interrogates the tissue surface at the same location as a pulsed laser, used for photoacoustic interrogation. Photoacoustic signals collected from the tissue are deflected by the prism to a focused 10 MHz ultrasound transducer. Diffuse light from the CW-laser is collected by a CCD camera and analyzed to estimate the bulk absorption and scattering coefficients. We fixed a tube filled with known concentrations of an absorbing dye below the probe in an Intralipid bath. We obtained the OIR and photoacoustic measurements for different Intralipid concentrations (providing a μs' between 1 and 10 cm^-1). The OIR measurements were used to estimate the bulk optical parameters. Using these values, models of light transport were then used to calculate the local laser fluence to normalize the photoacoustic measurements. The corrected photoacoustic signals show direct proportionality to the tube dye concentrations irrespective of bulk turbid medium properties.

In this work we modified light illumination of the laser optoacoustic (OA) imaging system to improve the 3D visualization of human forearm vasculature. The computer modeling demonstrated that the new illumination design that features laser beams converging on the surface of the skin in the imaging plane of the probe provides superior OA images in comparison to the images generated by the illumination with parallel laser beams. We also developed the procedure for vein/artery differentiation based on OA imaging with 690 nm and 1080 nm laser wavelengths. The procedure includes statistical analysis of the intensities of OA images of the neighboring blood vessels. Analysis of the OA images generated by computer simulation of a human forearm illuminated at 690 nm and 1080 nm resulted in successful differentiation of veins and arteries. In vivo scanning of a human forearm provided high contrast 3D OA image of a forearm skin and a superficial blood vessel. The blood vessel image contrast was further enhanced after it was automatically traced using the developed software. The software also allowed evaluation of the effective blood vessel diameter at each step of the scan. We propose that the developed 3D OA imaging system can be used during preoperative mapping of forearm vessels that is essential for hemodialysis treatment.

In this report we demonstrate improved three-dimensional optoacoustic tomography in test samples. High quality tomographic data and images were obtained from phantom of mice being 2.5 cm in diameter. Capillaries filled with cupric sulfate, ferrous sulfate and nickel sulfate solutions, and immersed in a scattering medium were used for these tests. The brightness of reconstructed phantom images was found to match accurately the absorption profiles of test solutions. Hence, optoacoustic imaging can be applied in preclinical research to perform in vivo absorptivity measurements to deduce functional information on blood oxygen levels or concentration of contrast agents.

Building photoacoustic imaging systems by using stand-alone ultrasound (US) units makes it convenient to take advantage of the state-of-the-art ultrasonic technologies. However, the sometimes limited receiving sensitivity and the comparatively narrow bandwidth of commercial US probes with elements driving long cables may not be sufficient for high quality photoacoustic imaging. In this work, a high-speed photoacoustic tomography (PAT) system has been developed using a commercial US unit and a custom built 128-element PVDF transducer array. Since the US unit supports simultaneous signal acquisition from 64 parallel receive channels, PAT data for synthetic image formation from a 64 or 128 element array aperture can be acquired after a single or dual laser firing, respectively. The PVDF array provides satisfactory receiving sensitivity and uniquely broad detection bandwidth, which enables good image quality for tomographic photoacoustic imaging. A specially designed 128-channel preamplifier board that connects the preamps directly to the PVDF elements not only enables impedance matching but also further elevates the signal-to-noise ratio in detecting weak photoacoustic signals. To examine the performance of this imaging system, experiments on phantoms were conducted and the results were compared with those acquired with commercial US probes.

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. This approach was found to be effective for quantifying the linear motion of micron-scale absorbers imprinted on an acetate sheet moving with velocities in the range 0.15 to 1.50 ms^-1. The effect of the acoustic spot diameter and the time separation between the laser pulses on measurement resolution and the maximum measurable velocity is discussed. 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.

For the first time, the hemodynamics within the entire cerebral cortex of a mouse were studied by using photoacoustic tomography (PAT) non-invasively and in real time. The PAT system, based on a 512-element full-ring array with cylindrical focusing, received the PA signal primarily from a slice of about 2 mm thickness. This system can provide not only high resolution brain vasculature images but also hemodynamic functional images. We recorded the wash-in process of a photoacoustic contrast agent in a mouse brain in real time. Our results demonstrated that PAT is a powerful imaging modality to study real-time small animal neurofunctional activities that cause changes in hemodynamics.

In this work the spatial resolution provided by the array transducers in 2D optoacoustic (OA) imaging is considered numerically. The resolution is determined from the point spread function (PSF) of an array, that is a 2D OA image of a point source of the spherical acoustic wave. Numerical simulations consisted of the following steps. First, the OA signals excited by the point source and detected by each element of the array calculated using the Rayleigh integral. Radial backprojection algorithm was then employed to obtain the image of the point source in the imaging plane. The influence of the geometrical parameters of array elements, number of the detectors and a single detector bandwidth on the PSF was studied in detail. It was shown that the spatial resolution provided by the array transducer in the imaging plane can be unambiguously determined from the detector frequency band, array aperture angle and the width of a single array detector, and does not depend on the number of the detectors.

An essential problem dealing with three-dimensional optoacoustic imaging is the long data acquisition times associated with recording signals from multiple spatial projections, where signal averaging for each projection is applied to obtain satisfying signal-to-noise-ratio. This approach complicates acquisition and makes imaging challenging for most applications, especially for in vivo imaging and multispectral imaging. Instead we employ a herein introduced continuous data acquisition methodology that greatly shortens recording times over multiple projection angles and acquires high quality tomographic data without averaging. By this means a two dimensional image acquisition having 270 angular projections only takes about 9 seconds, while a full multispectral three dimensional image can normally take about 15 minutes to acquire with a single ultrasonic detector. The system performance is verified on tissue-mimicking phantoms containing known concentrations of fluorescent molecular agent as well as small animals.

Ultrasound-modulated optical tomography (UOT) combines the spatial resolution of ultrasonic waves and the spectroscopic properties of light to detect optically absorbing and/or scattering objects in highly scattering media. In this work, a double-pass confocal Fabry-Perot interferometer is used as a bandpass filter to selectively detect the ultrasoundtagged photons. The limited etendue of the confocal Fabry-Perot interferometer is compensated by using a singlefrequency laser emitting high-peak-power optical pulses. Compared to photoacoustic tomography, UOT is not only sensitive to optical absorption but also to scattering properties. In this paper, we consider the detection of absorbing and scattering objects embedded in thick (30 to 60 mm) tissue-mimicking phantoms and biological tissues. The experimental evaluation of the spatial resolution of the technique is compared to that expected from the ultrasonic beam intensity profile. Preliminary results indicate that the edge spread function is influenced by the level of absorption of the embedded object.

High intensity focused ultrasound (HIFU) is a powerful noninvasive tool for targeted tissue ablation. Monitoring of the treatment process and efficacy in real time, however, remains challenging. The tissue necrosis during the HIFU exposure leads to changes in optical absorption and scattering coefficients. In this paper, we explore the use of acousto-optic imaging (AOI), a hybrid technique that combines ultrasound with diffuse light to obtain deep-tissue optical contrast at ultrasound resolution, to sense the changes in optical contrast at depth in tissue associated with the onset formation and development of the necrosed tissue region. In this technique, the tissue to be treated is illuminated with near-infrared light and a continuous, amplitude-modulated focused ultrasound beam is used to induce thermal tissue necrosis as well as the acousto-optic (AO) interaction. The AOI signal is detected via a photorefractive crystal (PRC)-based interferometer, and then fed into a lock-in amplifier tuned to the ultrasound modulation frequency. As a thermal lesion forms in the ultrasound focal zone, which is also the zone of AO interaction, the AOI signal diminishes in amplitude owing to enhanced optical attenuation. It is further shown that the reduction of the AOI signal is correlated with the volume of ensuing lesion. Therefore, the evolution of AOI signal as a function of time provides a means for continuous monitoring of HIFU treatment process as well as exposure guidance.

Thermoacoustic image contrast is dependent on the dielectric and thermoacoustic properties of the tissue being imaged, its spatial distribution, and the polarization of the incident microwave radiation. We have designed and constructed a thermoacoustic computed tomography (TCT) test platform to study these effects in phantoms and biologic tissue (e.g., beefsteak and mice). Our results indicate that muscle and fat are easily differentiated, but the relative thermoacoustic absorption is strongly dependent upon the polarization angle of the microwave radiation and the morphology of fat and muscle tissues.

Conventional photoacoustic imaging systems excite a photoacoustic wave by illuminating an area on the order of square centimeters with millijoule laser pulses. Spatial resolution is then determined by the ultrasound transducer and is typically on the order of 100 μm. We report on a system that focuses femtosecond, nanojoule pulses to a spot with a diameter of ~ 1 μm to perform laser-scanning photoacoustics with micrometer resolution. Near-infrared femtosecond laser pulses with a pulse energy of 2.4 nanojoules excite a train of photoacoustic waves at the repetition rate of the pulsed laser (80 MHz). These photoacoustic waves are detected by an unfocused single-element ultrasound transducer tuned to 80 MHz. A radiofrequency lock-in amplifier recovers the amplitude of the frequency component of the photoacoustic signal at the pulse repetition frequency. This amplitude is an indicator of the absorption coefficient of the sample at the laser focus and at the laser wavelength. Initial experiments using a graphite rod as absorber reproducibly yield signals in the 0.2 - 2 microvolt range with a signal-to-noise ratio of 18 dB, recovered from 10 mV of broadband noise. The photoacoustic imaging system is integrated in a commercial laser-scanning two-photon fluorescence microscope, enabling simultaneous three-dimensional fluorescence- and photoacoustic imaging. One major application will be to image both morphology and oxygen saturation of microvasculature in the cerebral cortex of anesthetized rodents in vivo in the context of tumor angiogenesis. In this paper we describe the physics of femtosecond photoacoustics and demonstrate initial results.

Photothermal heterodyne imaging has demonstrated a high sensitivity of seeing single metallic nanoparticles of diameter down to 5 nm. However, rare attention has been paid to the phase of the photothermal heterodyne signal relative to that of the modulated pump beam. We show that the phase of the photothermal heterodyne signal from semiconducting nanomaterials such as silicon and germanium nanowires is around 0 degree, while that from metallic nanomaterials such as silver and gold nanoparticles (NPs) is around 180 degrees. Using this property we have been able to distinguish gold seeds from germanium nanowire (GeNW) body in a label-free and contact-free manner. A theoretical model based on light scattering by a fluctuating dielectric material was used to explain the origin of different phases in the photothermal heterodyne signal.

Using high speed photography at sub-microsecond temporal resolution, we observed laser-induced cavitation within a thin film of liquid. In accordance with the literature, focusing a pulse of a gaussian-like intensity distribution into the liquid, instigated a disk-shaped cavity. The propagation of an acoustic transient, generated during the formation and expansion of the cavity, is evidenced by secondary cavitation stimulated in the surrounding liquid. Introducing a laguerre-gauss holographic diffractive optic element into the beam path, re-distributes the optical energy into the socalled 'doughnut mode', with an axial intensity minimum. Focusing this modulated pulse into the liquid induced a ringshaped cavity with a notably different dynamic to that of the disk cavity, due primarily to the encapsulated droplet, which is present from cavity initiation through expansion and subsequent deflation. In this paper we present initial observations on the novel dynamics of the ring-shaped cavity and discuss several of the distinctive features. Particularly the secondary cavitation induced in the surrounding liquid, and the implicated multiplexing of the acoustic transient generated during the ring-cavity expansion, is of interest.

A photoacoustic correlation spectroscopy (PACS) technique was proposed for the first time. This technique is inspired by its optical counterpart-the fluorescence correlation spectroscopy (FCS), which is widely used in the characterization of the dynamics of fluorescent species. The fluorescence intensity is measured in FCS while the acoustic signals are detected in PACS. To proof of concept, we demonstrated the flow measurement of light-absorbing beads probed by a pulsed laser. A PACS system with temporal resolution of 0.8 sec was built. Polymer microring resonators were used to detect the photoacoustic signals, which were then signal processed and used to obtain the autocorrelation curves. Flow speeds ranging from 249 to 15.1 μm/s with corresponding flow time from 4.42 to 72.5 sec were measured. The capability of low-speed flow measurement can potentially be used for detecting blood flow in relatively deep capillaries in biological tissues. Moreover, similar to FCS, PACS may have many potential applications in studying the dynamics of photoacoustic beads.

Complete and continuous imaging of microvascular networks is crucial for a wide variety of biomedical applications. Photoacoustic tomography can provide high resolution microvascular imaging using hemoglobin within red blood cells (RBC) as an endogenous contrast agent. However, intermittent RBC flow in capillaries results in discontinuous and fragmentary capillary images. To overcome this problem, we used Evans Blue (EB) dye as a contrast agent for in vivo photoacoustic imaging. EB has strong optical absorption at 610 nm and distributes uniformly in the blood stream by chemically binding to albumin. By intravenous injection of EB (6%, 200 μL), complete and continuous microvascular networks-especially capillaries-of the ears of nude mice were imaged. The diffusion of EB (3%, 100 μL) leaving the blood stream was monitored for 2 hours. At lower administration dose of EB (3%, 50 μL), the clearance of the EB-albumin complex was imaged for 10 days and quantitatively investigated using a two-compartment model.

For the first time, high frequency ultrasound imaging, multiphoton tomography, and dermoscopy were combined in a clinical study. Different dermatoses such as benign and malign skin cancers, connective tissue diseases, inflammatory skin diseases and autoimmune bullous skin diseases have been investigated with (i) state-of-the-art and highly sophisticated ultrasound systems for dermatology, (ii) the femtosecond-laser multiphoton tomograph DermaInspectTM and (iii) dermoscopes. Dermoscopy provides two-dimensional color imaging of the skin surface with a magnification up to 70x. Ultrasound images are generated from reflections of the emitted ultrasound signal, based on inhomogeneities of the tissue. These echoes are converted to electrical signals. Depending on the ultrasound frequency the penetration depth varies from about 1 mm to 16 mm in dermatological application. The 100-MHz-ultrasound system provided an axial resolution down to 16 μm and a lateral resolution down to 32 μm. In contrast to the wide-field ultrasound images, multiphoton tomography provided horizontal optical sections of 0.36×0.36 mm² down to 200 μm tissue depth with submicron resolution. The autofluorescence of mitochondrial coenzymes, melanin, and elastin as well as the secondharmonic- generation signal of the collagen network were imaged. The combination of ultrasound and multiphoton tomography provides a novel opportunity for diagnostics of skin disorders.

We present a photo-acoustic concave transmitter to generate and subsequently focus high frequency ultrasound. Owing to a short time-duration of pulse laser beam, high frequency acoustic waves and tight focusing can be easily achieved. The transmitter consists of a light-absorbing film coated on a concave spherical structure. For detection, we used an optical microring ultrasound detector capable of covering a broadband and high frequency spectrum of photo-acoustic source. A spot width of ~44 μm was obtained at the focal plane. As the finite size and the specific shape of the microring cause a geometrical effect on the detection process, especially for high frequency components, we performed a 2-D spatial signal processing to remove this effect and extract a pure pressure distribution. The aperture for acoustic focusing could be optically controlled by changing the size of pulse laser beam.

We propose an acoustic 4f imaging system by using a pair of acoustic lens and an optical microring ultrasound detector (OMUD). The system was designed to have a long range imaging, and the signal strength was enhanced by a factor of ~13 by using an acryl-based acoustic lens at an imaging distance of close to 10 cm. The acoustic signal had a broadband and high frequency spectrum for a given focal distance owing to the unique characteristic of the OMUD. The imaging was obtained without using any reconstruction algorithms. Several performances of the designed system have been investigated by using photo-acoustic microspheres (301 μm in diameter) which are excited by pulsed laser beam. The resolution of images were compared, which consist of full frequency spectrum and harmonic frequency components. With high frequency (10 MHz and 15 MHz), the images showed consistently better resolutions (440 μm and 370 μm) for the microsphere. Frequency analysis of a time-domain signal waveform showed that the signal spectrum of the current system extends up to 20 MHz.

Multiphoton excitation-induced photoacoustic microscopy (MEPAM) can be used to investigate the interior of dense objects precisely and directly because the multiphoton excitation occurs only at the focal point. This method makes it possible to avoid the strong signal from the surface of dense objects. However, in the case of tissue imaging, one-photon photoacoustic signals affect the image constructed from MEPAM signals, owing to the smaller cross section of multiphoton absorption compared with that of one-photon absorption. Thus, in order to apply MEPAM for precise investigation in living tissues, it is important to enhance (or extract) only the photoacoustic signals induced by multiphoton excitation. In this study, we examined the use of frequency-selective detection (frequency filtering) in multiphotonphotoacoustic imaging by evaluating the depth discrimination and penetration. Because MEPAM signals are generated in a very small region, they include higher frequency components compared with one-photon photoacoustic signals. We measured the images at the cross sections of blood-vessel phantoms visualized by MEPAM using the high-frequency components. We found that the images visualized using only high-frequency components showed better contrast compared with those visualized using all frequency components. We conclude that the combination of frequency filtering and MEPAM demonstrates great potential for precise observation of cross sections of blood vessels in living tissues.

The complementary information provided by the ultrasound and photoacoustic imaging modalities has sparked their use in combination in recent years. We introduce a dual contrast agent capable of providing image contrast enhancement for both modalities simultaneously. The dual contrast agent is a small liquid perfluorocarbon droplet encased with bovine serum albumin and loaded with plasmonic nanoparticles. The plasmonic metal nanoparticles themselves act as a photoacoustic contrast agent. Furthermore, the perfluorocarbon droplet creates acoustic impedance mismatch between itself and the surrounding tissue, allowing it to act as an ultrasound contrast agent. Experiments demonstrating the performance of this dual contrast agent in ultrasound and photoacoustic imaging are presented. Differences in contrast mechanisms of the dual agent are highlighted, and finally, the applications for the agent are discussed.

Assessments of vascularity are important when assessing inflammation changes in tendon injuries since Achilles tendinitis is often accompanied with neovascularization or hypervascularity. In this study, we have investigated the feasibility of photoacoustic imaging in noninvasive monitoring of morphological and vascular changes in Achilles tendon injuries. Collagenase-induced Achilles tendinitis model of mice was adopted here. During collagenase-induced tendinitis, a 25-MHz photoacoustic microscopy (PAM) was used to image micro-vascular changes in Achilles tendons longitudinally up to 23 days. The positions of vessels imaged by PAM were identified by co-registration of PAM Bmode images with 25-MHz ultrasound (USM) ones. Morphological changes in Achilles tendons due to inflammation and edema were revealed by the PAM and USM images. Proliferation of new blood vessels within the tendons was also observed. Observed micro-vascular changes during tendinitis were similar to the findings in the literatures. This study demonstrates that photoacoustic imaging, owning required sensitivity and penetration, has the potential for high sensitive diagnosis and assessment of treatment performance in tendinopathy.

Segmentation in Magnetic resonance imaging (MRI) images is a widely studied problem, and techniques (supervised and unsupervised) are discussed in the literature. The basic approaches to image segmentation are based upon: (a) boundary representation, (b) regional characteristics and (c) a combination of boundary and region-based features. In this paper, we report classification of brain tissue based objects employing one of combination of boundary and region-based features as wavelet modified generic Fourier descriptor (WGFD) technique. This technique have been applied to a database consisting of 3 different class's tissues, each class consist of 50 shapes. The Euclidean distance has been calculated as a similarity measure parameter for tissue shape classification. The classification results have been carried out and it is inferred that WGFD performs for brain tissue classification. For brain tissue recognition, a simulation experiment employing hybrid correlator architecture has been carried out. We have used Wavelet modified maximum average correlation hight (MACH) filter for hybrid correlator. Mexican-hat wavelet has used to synthesize the wavelet MACH filter for simulation experiment.

In this study, we introduce a new reconstruction method developed to reduce the finite aperture effect in photoacoustic tomography with finite-aperture detectors. The finite aperture effect and degradation in tangential resolution result from the spatial impulse response of the finite-size flat transducer. The proposed method is based on a linear, discrete model of the photoacoustic tomography system in matrix formalism. Using this model, a spatiotemporal deconvolution filter designed in minimum mean square error sense is used to compensate the spatial impulse responses associated with a finite-size flat transducer at each imaging point; thus restoration of the tangential resolution can be achieved retrospectively. The performance of the proposed reconstruction method is verified using simulation data. Compared with that reconstructed by the backprojection algorithm, the proposed method provides uniform tangential resolution over the imaging area while retaining the radial resolution because the full geometry of the flat transducer, instead of the simplified point-detector approximation is taken into consideration.

Photoacoustic microscopy (PAM) provides excellent image contrast based on optical absorption. Microchip lasers are attractive optical sources for PAM, as they are compact and provide nanosecond pulse durations at several kHz repetition rates. However, spectroscopic imaging is not possible with microchip lasers due to their fixed wavelength output. We are investigating multispectral PAM with a supercontinuum source based on a photonic crystal fiber (PCF) pumped with a microchip laser. The Q-switched Nd:YAG microchip laser produces 0.6 ns duration pulses at 1064 nm with 8 uJ of energy at a 6.6 kHz repetition rate. These pulses are sent through 7 meters of PCF with a 5 um diameter core and a zero dispersion wavelength of 1040 nm. The supercontinuum is sent through a tunable band-pass filter before being focused into the object. Photoacoustic detection is performed with a 25 MHz spherically focused f/2 transducer. En-face imaging experiments were performed on ink phantoms. Images are acquired at seven different wavelengths from 575 to 875 nm. A simple discriminant analysis of the multispectral photoacoustic data produces images that clearly distinguish the different absorbing regions of the sample. These preliminary results suggest the potential of the supercontinuum PCF source for multispectral PAM.

Blood brain barrier (BBB) prevents most of the drug from transmitting into the brain tissue and decreases the treatment performance for brain disease. One of the methods to overcome the difficulty of drug delivery is to locally increase the permeability of BBB with high-intensity focused ultrasound. In this study, we have investigated the feasibility of photoacoustic microscopy of focused-ultrasound induced BBB opening in a rat model in vivo with gold nanorods (AuNRs) as a contrast agent. This study takes advantage of the strong near-infrared absorption of AuNRs and their extravasation tendency from BBB opening foci due to their nano-scale size. Before the experiments, craniotomy was performed on rats to provide a path for focused ultrasound beam. Localized BBB opening at the depth of about 3 mm from left cortex of rat brains was achieved by delivering 1.5 MHz focused ultrasound energy into brain tissue in the presence of microbubbles. PEGylated AuNRs with a peak optical absorption at ~800 nm were then intravenously administered. Pre-scan prior to BBB disruption and AuNR injection was taken to mark the signal background. After injection, the distribution of AuNRs in rat brains was monitored up to 2 hours. Experimental results show that imaging AuNRs reveals BBB disruption area in left brains while there are no changes observed in the right brains. From our results, photoacoustic imaging plus AuNRs shows the promise as a novel monitoring strategy in identifying the location and variation of focused-ultrasound BBB-opening in a rat model.

We built a photoacoustic tomographic (PAT) imaging system by scanning a single detector (φ 3.5 mm) made of piezoelectric copolymer poly(vinylidene difluoride-trifluoroethylene), P(VDF-TrFE), which had been fabricated for diagnostic photoacoustic measurement of cartilage tissues in our group. The PAT images of a phantom were obtained at two excitation wavelength of 687.5 nm and 795 nm. The phantom was made of agar including a black hair and agarose gels dissolving indocyanine green (ICG) and methylene blue (MB). Laser pulses (685-900 nm) were generated from a Ti:Sappire tunable laser to excite ICG and MB molecules. The PAT image at 687.5 nm shows signals due to all absorption sources. This is good agreement with dimension of the phantom. The PAT image at 795 nm shows a strong signal due to the ICG-dyed gel and almost no signal due to the MB-dyed gel. This result indicated that absorption sources were extracted by excitation wavelength according to their absorption spectra. The signal/noise ratio of the PAT images were compared between the P(VDF-TrFE) transducer in our group and a PZT transducer (Parametrics V309, 5 MHz, φ 12.7 mm) which is commercially available. The P(VDF-TrFE) transducer was more sensitive by 9 times (120 times per area) than the PZT transducer. By using this imaging system with a P(VDF-TrFE) transducer which is highly sensitive in a wide frequency range, we will achieve frequency analysis of the PAT images to associate photoacoustic waveforms with physical properties of sample tissues.

The purpose of this study is to map non-invasively sentinel lymph nodes (SLNs) and lymphatic vessels of rats in vivo using FDA-approved indocyanine green (ICG) and two non-ionizing imaging modalities: volumetric spectroscopic photoacoustic (PA) imaging, which measures optical absorption, and planar fluorescence imaging, which measures fluorescent emission. SLNs and lymphatic vessels were clearly visible after a 0.2 ml-intradermal-injection of 1 mM ICG in both imaging systems. We also imaged deeply positioned lymph nodes in vivo by layering biological tissues on top of rats. These two modalities, when used together with ICG, have the potential to map SLNs in axillary staging and to study tumor metastasis in breast cancer patients.

In the present study, we evaluate the applicability of ex-vivo photoacoustic imaging (PAI) in organs of small animals. We used photoacoustic tomography (PAT) to visualize infarcted areas within mouse hearts and compared it to other imaging techniques (MRI and μCT). In order to induce ischemia an in-vivo ligation of the Ramus interventricularis anterior (RIVA, left anterior descending, LAD) was performed on nine wild type C41 mice. After varying survival periods the mice were sacrificed. The hearts were excised and immediately transferred into a formaldehyde solution for conservation. Various wavelengths in the visible and near infrared region (500 nm - 1000 nm) had been tested to find the best representation of the ischemic regions. Samples were illuminated with nanosecond laser pulses delivered by an Nd:YAG pumped optical parametric oscillator. Ultrasound detection was achieved by an optical Mach-Zehnder interferometer working as an integrating line detector. For acoustic coupling the samples were located inside a water tank. The voxel data are computed from the measurement data by a Fourier-domain based reconstruction algorithm, followed by a sequence of inverse Radon transforms. Results clearly show the capability of PAI to detect pathological tissue and the possibility to produce three-dimensional images with resolutions well below 100 μm. Different wavelengths allow the representation of structure inside an organ or on the surface even without contrast enhancing tracers.

Over the last decade fluorescent reporter technologies (both fluorescent probes and proteins) have become a very powerful imaging tool in everyday biomedical research. Multispectral optoacoustic tomography (MSOT) is an emerging imaging technology that can resolve fluorophore concentration in small animals situated in deep tissue by multispectral acquisition and processing of optoacoustic signals. In this work, we study the optimum operating conditions of MSOT in imaging fluorescence activity in small animals. The performance of various fluorochromes / fluorescent proteins is examined and it is shown that the new infrared fluorescent protein is an order of magnitude brighter than the red ones. Finally, wavelength reduction after principle component analysis shows, that accurate unmixing and 3D reconstruction of the distribution of fluorochromes is possible only with 2 or 3 wavelengths.

This study demonstrates a method for measuring the optical absorption cross-sections (σ_a) of Au-Ag nanocages and Au nanorods using photoacoustic (PA) sensing. PA signals are directly proportional to the absorption coefficient (μ_a) of the nanostructure. For each type of nanostructure, we first obtained μa from the PA signal by benchmarking against a linear calibration curve (PA signal vs. μ_a) derived from a set of methylene blue solutions with different concentrations. We then calculated σ_a by dividing the μ_a by the corresponding concentration of the Au nanostructure. Additionally, we obtained the extinction cross-section (σ_e, sum of absorption and scattering cross-sections) from the extinction spectrum recorded using a conventional UV-vis-NIR spectrometer. From the measurements of σ_a and σ_e, we were able to easily derive both the absorption and scattering cross-sections for each type of gold nanostructure. This method can potentially provide the optical absorption and scattering properties of gold nanostructures and other types of nanomaterials.

The use of ultrasonic tagging of multiple scattered photons within turbid media for tomographic imaging is typically performed using optical detection in transmission mode. This study aimed to optimize the detection of the acousto-optic (AO) signal in cylindrical geometry, with a view to using the technique to measure blood oxygenation in the internal jugular vein of infants in the future. In our experiments, homogeneous phantoms of multiple transport scattering coefficients were constructed for the described geometry mimicking the infant neck. The optical source was systematically repositioned at different angles relative to the optical detector and the resulting AO signal was measured. The experimental results were also compared to focused ultrasound AO Monte Carlo (MC) simulation results. It was found that the optimal modulation depth and noise variance were highly dependent on the overlap region between the optical path length of the optical source-detector pair and the ultrasound focal zone. Therefore the optimal positions for both the optical and ultrasound probes could be estimated from both experimental and simulation results for a given geometry.

Photoacoustic imaging is a hybrid imaging modality capable of producing contrast similar to optical imaging techniques but with increased penetration depth and resolution in turbid media by encoding the information as acoustic waves. In general, it is important to characterize system performance by parameters such as sensitivity, resolution, and contrast. However, system characterization can extend beyond these metrics by implementing advanced analysis via singular value decomposition. A method was developed to experimentally measure a matrix that represented the imaging operator for the system. Analysis of the imaging operator was done via singular value decomposition so that the capability of the system to reconstruct objects and the inherent system sensitivity to those objects could be understood. The results provided by singular value decomposition were compared to simulations performed on an ideal system with matching transducer arrangement and defined object space.

Ultrasound imaging can provide excellent resolution at reasonable depths while retaining the advantages of being nonionizing, cost-effective and portable. However, the contrast in ultrasound imaging is limited, and various ultrasoundbased techniques such as photoacoustic (PA) and magneto-motive ultrasound (MMUS) imaging have been developed to augment ultrasound imaging. Photoacoustic imaging enhances imaging contrast by visualizing the optical absorption of either tissue or injected contrast agents (e.g., gold or silver nanoparticles). MMUS imaging enhances the sensitivity and specificity of ultrasound based on the detection of magnetic nanoparticles perturbed by an external magnetic field. This paper presents integrated magneto-photo-acoustic (MPA) imaging - a fusion of complementary ultrasound-based imaging techniques. To demonstrate the feasibility of MPA imaging, porcine ex-vivo tissue experiments were performed using a dual contrast (magnetic/plasmonic) agent. Spatially co-registered and temporally consecutive ultrasound, photoacoustic, and magneto-motive ultrasound images of the same cross-section of tissue were obtained. Our ex-vivo results indicate that magneto-photo-acoustic imaging can be used to detect magnetic/plasmonic nanoparticles with high resolution, sensitivity and contrast. Therefore, our study suggests that magneto-photo-acoustic images can identify the morphological properties, molecular information and complementary functional information of the tissue.

A photoacoustic tomography (PAT) imaging system based on a sparse 2D array of detector elements and an iterative image reconstruction algorithm has been proposed, which opens the possibility for high frame-rate 3D PAT. The efficacy of this PAT implementation is highly influenced by the choice of the reconstruction algorithm. In recent years, a variety of new reconstruction algorithms have been proposed for medical image reconstruction that have been motivated by the emerging theory of compressed sensing. These algorithms have the potential to accurately reconstruct sparse objects from highly incomplete measurement data, and therefore may be highly suited for sparse array PAT. In this context, a sparse object is one that is described by a relatively small number of voxel elements, such as typically arises in blood vessel imaging. In this work, we investigate the use of a gradient projection-based iterative reconstruction algorithm for image reconstruction in sparse-array PAT. The algorithm seeks to minimize an 1-norm penalized least-squares cost function. By use of computer-simulation studies, we demonstrate that the gradient projection algorithm may further improve the efficacy of sparse-array PAT.

Purpose: The purpose of this study is to determine the feasibility of using photacoustic CT spectroscopy(PCT-s) to track a near infrared dye conjugated with trastuzumab in vivo. Materials and Methods: An animal model was developed which contained both high and low Her2 expression tumor xenografts on the same mouse. The tumors were imaged at multiple wavelengths (680- 950nm) in the PCT scanner one day prior to injection of the near infrared conjugated probe. Baseline optical imaging data was acquired and the probe was then injected via the tail vein. Fluorescence data was acquired over the next week, PCT spectroscopic data was also acquired during this timeframe. The mice were sacrificed and tumors were extirpated and sent to pathology for IHC staining to verify Her2 expression levels. The optical fluorescence images were analyzed to determine probe uptake dynamics. Reconstructed PCT spectroscopic data was analyzed using IDL routines to deconvolve the probe signal from endogenous background signals, and to determine oxygen saturation. Results: The location of the NIR conjugate was able to be identified within the tumor utilizing IDL fitting routines, in addition oxygen saturation, and hemoglobin concentrations were discernible from the spectroscopic data. Conclusion: Photacoustic spectroscopy allows for the determination of in vivo tumor drug delivery at greater depths than can be determined from optical imaging techniques.

Metallic nanoparticles have been widely used in a variety of imaging and therapeutic applications due to their unique optical properties in the visible and near-infrared (NIR) regions - for example, various plasmonic nanoparticles are used for molecular photoacoustic imaging and photothermal therapy. However, there are concerns that these agents may not be safe under physiological conditions, because these nanoparticles are not biodegradable, could accumulate and, therefore, could be toxic long-term. We investigate the feasibility of using biodegradable gold nanoclusters as a contrast agent for highly sensitive photoacoustic imaging. The size of these biodegradable nanoclusters, consisting of sub-5 nm primary gold particles and a biodegradable polymer binder, is less than 100 nm. Due to plasmon coupling, these nanoclusters are characterized by a broad extinction spectrum that extends to the near infrared (NIR) spectral range. Photoacoustic imaging of tissue models containing inclusions with different concentrations of nanoparticles was performed using a tunable pulsed laser system. The results indicate that the biodegradable nanoclusters, comprised of small gold nanoparticles, can be used as contrast agents in photoacoustic imaging.

A photoacoustic tomography (PAT) method that employs a sparse two-dimentional (2D) array of detector elements has recently been employed to reconstruct images of simple objects from highly incomplete measurement data. However, there remains an important need to understand what type of object features can be reliably reconstructed from such a system. In this work, we numerically compute the singular value decomposition (SVD) of different system matrices that are relevant to implementations of sparse-array PAT. For a given number and arrangement of measurement transducers, this will reveal the type of object features that can reliably be reconstructed as well as those that are invisible to the imaging system.

Purpose: The purpose of this study is to use PCT spectroscopy scanner to monitor the hemoglobin concentration and oxygen saturation change of living mouse by imaging the artery and veins in a mouse tail. Materials and Methods: One mouse tail was scanned using the PCT small animal scanner at the isosbestic wavelength (796nm) to obtain its hemoglobin concentration. Immediately after the scan, the mouse was euthanized and its blood was extracted from the heart. The true hemoglobin concentration was measured using a co-oximeter. Reconstruction correction algorithm to compensate the acoustic signal loss due to the existence of bone structure in the mouse tail was developed. After the correction, the hemoglobin concentration was calculated from the PCT images and compared with co-oximeter result. Next, one mouse were immobilized in the PCT scanner. Gas with different concentrations of oxygen was given to mouse to change the oxygen saturation. PCT tail vessel spectroscopy scans were performed 15 minutes after the introduction of gas. The oxygen saturation values were then calculated to monitor the oxygen saturation change of mouse. Results: The systematic error for hemoglobin concentration measurement was less than 5% based on preliminary analysis. Same correction technique was used for oxygen saturation calculation. After correction, the oxygen saturation level change matches the oxygen volume ratio change of the introduced gas. Conclusion: This living mouse tail experiment has shown that NIR PCT-spectroscopy can be used to monitor the oxygen saturation status in living small animals.

Purpose: The purpose of this study is to calibrate the PCT scanner to quantify the hemoglobin status utilizing a blood flow phantom. Materials and Methods: A blood circulation system was designed and constructed to control the oxygen saturation and hemoglobin concentration of blood. As a part of the circulation system, a 1.1mm FEP tube was placed in the center of imaging tank of PCT scanner as the imaging object. Photoacoustic spectra (690-950 nm) was acquired for different hemoglobin concentrations (CtHb) and oxygen saturation levels (SaO2), where the formers was formed by diluting blood samples with PBS and the latter by mixing blood with gases at different oxygen content. Monte Carlo simulations were performed to calculate the photon energy depositions in the phantom tube, which took into account photon losses in water and blood. A Kappa value which represents the energy transfer efficiency of hemoglobin molecule was calculated based on the PCT measurement and simulation result. The final SaO2 value of each blood sample was calculated based on the PCT spectrum and Kappa value. These oxygen saturation results were compared with co-oximeter measurements to obtain systematic errors. Results and Conclusion: The statistic error of calculating Kappa value from hemoglobin concentration experiment was less than 5%. The systematic error between PCT spectra analysis and co-oximeter analysis for hemoglobin oxygen saturation was -4.5%. These calibration techniques used to calculate Kappa and hemoglobin absorption spectra would be used in hypoxia measurements in tumors as well as for endogenous biomarkers studies.

Understanding the tumor microenvironment is critical to characterizing how cancers operate and predicting how they will eventually respond to treatment. The mouse window chamber model is an excellent tool for cancer research, because it enables high resolution tumor imaging and cross-validation using multiple modalities. We describe a novel multimodality imaging system that incorporates three dimensional (3D) photoacoustics with pulse echo ultrasound for imaging the tumor microenvironment and tracking tissue growth in mice. Three mice were implanted with a dorsal skin flap window chamber. PC-3 prostate tumor cells, expressing green fluorescent protein (GFP), were injected into the skin. The ensuing tumor invasion was mapped using photoacoustic and pulse echo imaging, as well as optical and fluorescent imaging for comparison and cross validation. The photoacoustic imaging and spectroscopy system, consisting of a tunable (680-1000nm) pulsed laser and 25 MHz ultrasound transducer, revealed near infrared absorbing regions, primarily blood vessels. Pulse echo images, obtained simultaneously, provided details of the tumor microstructure and growth with 100-μm3 resolution. The tumor size in all three mice increased between three and five fold during 3+ weeks of imaging. Results were consistent with the optical and fluorescent images. Photoacoustic imaging revealed detailed maps of the tumor vasculature, whereas photoacoustic spectroscopy identified regions of oxygenated and deoxygenated blood vessels. The 3D photoacoustic and pulse echo imaging system provided complementary information to track the tumor microenvironment, evaluate new cancer therapies, and develop molecular imaging agents in vivo. Finally, these safe and noninvasive techniques are potentially applicable for human cancer imaging.

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

Advances in high-resolution imaging have permitted microscopic observations within the brains of living animals. Applied to Alzheimer's disease (AD) mouse models, multiphoton microscopy has opened a new window to study the real-time appearance and growth of amyloid plaques. Here, we report an alternative technology-optical-resolution photoacoustic microscopy (OR-PAM)-for in vivo imaging of amyloid plaques in a transgenic AD mouse model. In vivo validation using multiphoton microscopy shows that OR-PAM has sufficient sensitivity and spatial resolution to identify amyloid plaques in living brains. In addition, with dual-wavelength OR-PAM, the three-dimensional morphology of amyloid plaques and the surrounding microvasculature are imaged simultaneously through a cranial window. In vivo transcranial OR-PAM imaging of amyloid plaques is highly likely once the imaging parameters are optimized.

Both iris fluorescein angiography (IFA) and indocyanine green angiography (ICGA) provide ophthalmologists imaging tools in studying the microvasculature structure and hemodynamics of the anterior segment of the eye in normal and diseased status. However, a non-invasive, endogenous imaging modality is preferable for the monitoring of hemodynamics of the iris microvasculature. We investigated the in vivo, label-free ocular anterior segment imaging with photo-acoustic microscopy (PAM) in mouse eyes. We demonstrated the unique advantage of endogenous contrast that is not available in both IFA and ICGA. The laser radiation was maintained within the ANSI laser safety limit. The in vivo, label-free nature of our imaging technology has the potential for ophthalmic applications.

In this study, we report on using multi-wavelength photoacoustic microscopy to image hemodynamic changes of total hemoglobin concentration (HbT) (i.e., blood volume) and oxygenation (SO2) in rat brain cortex vessels with electrical stimulation. Electrical stimulation of the rat left forelimb was applied to evoke changes in vascular dynamics of the rat somatosensory cortex. The applied current pulses were with a pulse frequency of 3 Hz, pulse duration of 0.2 ms, and pulse amplitude of 5 mA, respectively. The imaging target of rat brains was demarcated at AP 0 - -2.5 mm and ML ± 6 mm with respect to bregma. HbT changes were probed by images acquired at 570 nm, a hemoglobin isosbestic point while SO₂ changes were imaged by those acquired at 560 nm or 600 nm and their derivatives, which were normalized to those with 570 nm wavelengths. Correlation between the electrical stimulation paradigm and images acquired at 570, 560, and 600 nm in contralateral and ipsilateral vasculature was statistically analyzed, showing that the HbT and SO₂ changes revealed by multi-wavelength photoacoustic images spatially correlated with contralateral vasculature.

Ovarian cancer has a five-year survival rate of only 30%, which represents the highest mortality of all gynecologic cancers. The reason for that is that the current imaging techniques are not capable of detecting ovarian cancer early. Therefore, new imaging techniques, like photoacoustic imaging, that can provide functional and molecular contrasts are needed for improving the specificity of ovarian cancer detection and characterization. Using a coregistered photoacoustic and ultrasound imaging system we have studied thirty-one human ovaries ex vivo, including normal and diseased. In order to compare the photoacoustic imaging results from all the ovaries, a new parameter using the RF data has been derived. The preliminary results show higher optical absorption for abnormal and malignant ovaries than for normal postmenopausal ones. To estimate the quantitative optical absorption properties of the ovaries, additional ultrasound-guided diffuse optical tomography images have been acquired. Good agreement between the two techniques has been observed. These results demonstrate the potential of a co-registered photoacoustic and ultrasound imaging system for the diagnosis of ovarian cancer.

Ultrasound imaging suffers from poor sensitivity (~50%) and specificity in detecting small foreign bodies in tissue. Hence, alternative imaging methods are needed. Photoacoustic (PA) imaging takes advantage of strong optical absorption contrast and high ultrasonic resolution. This work employed a PA imaging system to detect foreign bodies in biological tissues. To achieve deep penetration, we used near-infrared light and a 5-MHz spherically focused ultrasonic transducer. PA images were obtained from objects (glass, wood, cloth, plastic, and metal) embedded in chicken tissue. The location and size of the targets from the PA images agreed well with those of the actual samples. Objects were imaged more than 1 cm deep. Spectroscopic PA imaging was also performed on the objects. These results suggest PA imaging can potentially be a useful intraoperative imaging tool to identify foreign bodies and discriminate viable tissues in wounded patients.

A 3-D optoacoustic imaging system was used to visualize thermal lesions produced in excised tissue specimens and in vivo mice using high intensity focused ultrasound (HIFU). A 7.5 MHz surgical, focused transducer with a radius of curvature of 35 mm and an aperture diameter of 23 mm was used to generate HIFU. A pulsed laser, which could operate at 755 nm and 1064 nm, was used to illuminate excised tissue and mice using a bifurcated fiber bundle resulting in two wide beams of light. Tomographic images were obtained while the specimens were rotated within a sphere outlined by a concave arc-shaped array of 64 piezo-composite transducers. These images were then combined to reconstruct 3-D volume images (voxel resolution 0.5 mm), which were acquired before and after HIFU exposure. Optoacoustic images acquired at 1064 nm provided visualization of HIFU lesions. The lesion in excised tissue was indicated by an increase in the optoacoustic signal; the in vivo lesion was indicated by a decrease in the optoacoustic signal. The location and the extent of the lesions were confirmed upon dissection. The discrepancy between the ex vivo and the in vivo results might be attributed to the different effective thermal deposition in the two cases. These preliminary results demonstrate the potential of optoacoustic imaging to assess and monitor the progress of HIFU therapy.

For photoacoustic imaging in reflection geometry, the front-face reflectivity of the ultrasound transducers imposes different boundary conditions on the light fluence distribution inside the tissue. Understanding and characterizing the boundary effects on the fluence distribution is critical for optimizing the light illumination and therefore the signal-tonoise ratio (SNR) of the photoacoustic measurements. Monte Carlo (MC) simulations were performed to quantify the fluence distribution under different reflectivity boundary conditions and the results were validated by experiments. It is demonstrated that the light fluence obtained due to a highly reflective boundary, for instance, white-colored transducer face, is higher and more uniformly distributed than that of lower reflectivity boundaries such as red, gray, or black face colors. Both simulations and experiments were performed with near-infrared (NIR) light as the illumination source.

A new approach for implementing pulsed excitation enables time-resolved characterization of flow, using the photoacoustic Doppler effect. The method yields two-dimensional maps of the Doppler shift vs. axial position of flowing absorbing particles. It takes advantage of the unique flexibility and accuracy of external modulation which offers excellent control over the parameters of the pulsed optical excitation. The experimental setup comprised a CW tunable laser source operating in the fiber optic communications band (1510-1620nm) followed by an electro-optic modulator, electronically driven by an arbitrary waveform generator. Using the technique the flow of a suspension of carbon particles in a C-flex tube was measured over a wide range of velocities from 18 mm/sec up to 200mm/sec.