The OPUS (Optoacoustic plus Ultrasound) system is a combination of a medical ultrasound scanner with a highrepetition rate, wavelength-tunable laser system and a suitable triggering interface to synchronize the laser and the ultrasound system. The pulsed laser generates an optoacoustic (OA), or photoacoustic (PA), signal which is detected by the ultrasound system. Alternatively, imaging in conventional ultrasound mode can be performed. Both imaging modes can be superimposed. The laser light is coupled into the tissue laterally, parallel to the ultrasound transducer, which does not require for any major modification to the transducer or the ultrasound beam forming. This was a basic requirement on the instrument, as the intention of the project was to establish the optoacoustic imaging modality as add-on to a conventional standard ultrasound instrument. We believe that this approach may foster the introduction of OA imaging as routine tool in medical diagnosis. Another key aspect of the project was to exploit the capabilities of OA imaging for quantitative analysis. The intention of the presented work is to summarize all steps necessary to extract the significant information from the PA raw data, which are required for the quantification of local absorber distributions. We show results of spatially resolved absorption measurements in scattering samples and a comparison of four different image reconstruction algorithms, regarding their influence on lateral resolution as well as on the signal to noise ratio for different sample depths and absorption values. The reconstruction algorithms are based on Fourier transformation, on a generalized 2D Hough transformation, on circular back-projection and the classical delay-and-sum approach which is implemented in most ultrasound scanners. Furthermore, we discuss the influence of a newly developed laser source, combining diode and flash lamp pumping. Compared to all-flash-lamp pumped systems it features a significantly higher pulse-to-pulse stability, which is required for sensitive and precise quantitative analyses.

We describe two laser optoacoustic imaging systems for breast cancer detection based on arrays of acoustic detectors operated manually in a way similar to standard ultrasonic breast imaging. The systems have the advantages of standard light illumination (regardless of the interrogated part of the breast), the ability to visualize any part of the breast, and convenience in operation. The first system could work in both ultrasonic and optoacoustic mode, and was developed based on a linear ultrasonic breast imaging probe with two parallel rectangular optical bundles. We used it in a pilot clinical study to provide for the first time demonstration that the boundaries of the tumors visualized on the optoacoustic and ultrasonic images matched. Such correlation of coregistered images proves that the objects on both images represented indeed the same tumor. In the optoacoustic mode we were also able to visualize blood vessels located in the neighborhood of the tumor. The second system was proposed as a circular array of acoustic transducers with an axisymmetric laser beam in the center. It was capable of 3D optoacoustic imaging with minimized optoacoustic artifacts caused by the distribution of the absorbed optical energy within the breast tissue. The distribution of optical energy absorbed in the bulk tissue of the breast was removed from the image by implementing the principal component analysis on the measured signals. The computer models for optoacoustic imaging using these two handheld probes were developed. The models included three steps: (1) Monte Carlo simulations of the light distribution within the breast tissue, (2) generation of optoacoustic signals by convolving N-shaped pressure signals from spherical voxels with the shape of individual transducers, and (3) back-projecting processed optoacoustic signals onto spherical surfaces for image reconstruction. Using the developed models we demonstrated the importance of the included spatial impulse response of the optoacoustic imaging system.

A real-time photoacoustic imaging system was designed and built. This system is based on a commercially available ultrasound imaging system. It can achieve a frame rate of 8 frames/sec. This system has been characterized in phantom experiments. In addition, vasculature in the hand of a human volunteer was imaged.

Existing techniques for total hemoglobin concentration [THb] monitoring are invasive and cannot be used continuously and in real time. We developed and built a novel, light-weight (2 lb), low-cost, optoacoustic system for noninvasive, accurate monitoring of [THb]. The system incorporates an optoacoustic probe designed for sensitive probing of blood vessels with high signal-to-noise ratio at low energy of laser diode pulses. We developed a new algorithm for accurate monitoring of [THb] in the radial artery with this system. We tested the system in human subjects with different [THb]. The studies confirmed the capability of the system to accurately monitor [THb].

Monitoring of cerebral venous oxygenation is critically important for management of patients with traumatic brain injury and cardiac surgery patients. At present, there is no technique for noninvasive, accurate monitoring of this important physiologic parameter. We built a compact optoacoustic system for noninvasive, accurate cerebral venous oxygenation monitoring using a novel optoacoustic probe and algorithm that allow for direct probing of sagittal sinus blood with minimal signal contamination from other tissues. We tested the system in large animal and clinical studies and identified wavelengths for accurate measurement of cerebral blood oxygenation. The studies demonstrated that the system may be used for noninvasive, continuous, and accurate monitoring of cerebral venous blood oxygenation.

We report experimental investigations of photoacoustic guidance of diffusive optical tomography for detection and characterization of optical contrast targets. The hybrid system combined an 8-source, 10-detector frequency domain DOT with a clinical reflection geometry probe. For the photoacoustic tomography (PAT) functionality, a high-energy 1×7 optical fiber delivery system illuminated a 2 cm central region for localization of absorptive targets. Two-dimensional PAT images along one central axis of the probe defined of regions of interest for a dual-zone mesh DOT imaging algorithm. PVC Plastisol phantom absorbers, 1 cm on a side, with absorption coefficients ranging from 0.075 to 0.23 cm^-1 were imaged at depths up to 2.5 cm. Pairs of absorbers simulating a multi-lobed heterogeneous tumor were also investigated. Without PAT guidance, the absorber location was not clear and lower contrast targets in the twoabsorber configurations were not distinguishable. With PAT guidance, the two targets were well resolved and the reconstructed absorption coefficients improved to within 15% of the true values.

Ovarian cancer has the highest mortality of all gynecologic cancers with a five-year survival rate of only 30%. Because current imaging techniques (ultrasound, CT, MRI, PET) are not capable of detecting ovarian cancer early, most diagnoses occur in later stages (III/IV). Thus many women are not correctly diagnosed until the cancer becomes widely metastatic. On the other hand, while the majority of women with a detectable ultrasound abnormality do not harbor a cancer, they all undergo unnecessary oophorectomy. Hence, new imaging techniques that can provide functional and molecular contrasts are needed for improving the specificity of ovarian cancer detection and characterization. One such technique is photoacoustic imaging, which has great potential to reveal early tumor angiogenesis through intrinsic optical absorption contrast from hemoglobin or extrinsic contrast from conjugated agents binding to appropriate molecular receptors. To better understand the cancer disease process of ovarian tissue using photoacoustic imaging, it is necessary to first characterize the properties of normal ovarian tissue. We have imaged ex-vivo ovarian tissue using a 3D co-registered ultrasound and photoacoustic imaging system. The system is capable of volumetric imaging by means of electronic focusing. Detecting and visualizing small features from multiple viewing angles is possible without the need for any mechanical movement. The results show strong optical absorption from vasculature, especially highly vascularized corpora lutea, and low absorption from follicles. We will present correlation of photoacoustic images from animals with histology. Potential application of this technology would be the noninvasive imaging of the ovaries for screening or diagnostic purposes.

The feasibility of transcranial imaging of neonatal brains with reflection mode photoacoustic technology has been explored. By using unembalmed infant skulls and fresh canine brains, experiments have been conducted to examine the ultrasound and light attenuation in the skull bone as well as consequent photoacoustic images through the skull. Mapping of blood vessels in a transcranial manner has been successfully achieved by employing the raster scan of a single-element transducer or a 2D PVDF array transducer. Experimental results indicate that noninvasive photoacoustic imaging of neonatal brain with a depth of 2 cm or more beneath the skull is feasible when working with near-infrared light. This study suggests that the emerging photoacoustic technology may become a powerful tool in the future for noninvasive diagnosis, monitoring and prognosis of disorders in prenatal or neonatal brains.

Photoacoustic spectroscopy has been shown to be able to discriminate between normal and atheromatous areas of arterial tissue in the visible range (410nm-680nm). However, at these wavelengths haemoglobin absorption is also very high. This makes it challenging to apply photoacoustic techniques using an intravascular probe, as a significant amount of the excitation light will be absorbed by the blood present in the artery. In this study we investigate the use of a wider range of excitation wavelengths (740-1800nm) for discriminating between normal arterial tissue and lipid rich plaques and minimise the effect of blood absorption. Special attention will be given to the near infra-red (NIR) wavelength range (900-1300nm) as in this region blood absorption is relatively weak and there are expected to be significant differences in the absorption spectrum of each tissue type. To investigate this, tissue samples were obtained and imaged at a range of wavelengths, the samples were illuminated first through water, then blood. This study demonstrated that the photoacoustic technique can discriminate between normal arterial tissue and lipid rich plaques, even when blood is present.

A 512-element photoacoustic tomography system for small animal imaging using a ring ultrasound array has been developed. The system features a 5 MHz piezocomposite transducer array formed into a complete circular aperture. Custom receiver electronics consisting of dedicated preamplifiers, 8:1 multiplexed post-amplifiers, and a 64-channel data acquisition module provide full tomographic imaging at up to 8 frames/second. We present details of the system design along with characterization results of the resolution, imaging volume, and sensitivity. Small animal imaging performance is demonstrated through images of mice brain vasculature at different depths and real-time spectroscopic scans. This system enables real-time tomographic imaging for functional photoacoustic studies for the first time.

In this work, we exploit the high depth and temporal resolutions of PAM to noninvasively image the blood-oxygenation dynamics of multiple cortex vessels in the mouse brain simultaneously in response to controlled hypoxic and hyperoxic challenges. The dark-field photoacoustic microscopy (PAM) technique was enhanced to image the cortex vasculature of the mouse brain in vivo using endogenous hemoglobin contrast with one second temporal resolution. The maximum values of about 20% with standard deviation ± 1.2% were found to vary significantly among the cortex vessels studied. The hypoxic response time to rise from 10 % to 90 % of maximum was 63 ± 6 sec. The reverse response time for this event was 16 ± 2 sec.

Noninvasive imaging of biological tissues using visible and near-infrared light may provide numerous insights into the underlying morphology or tissue function using a great variety of contrast and probing mechanisms. Nevertheless, mesoscopic-scale (i.e 1mm-1cm sized) living organisms remain largely inaccessible by current optical imaging methods. Depending on the optical properties of a particular object, light diffusion can significantly limit the resolution that can be achieved at depths beyond several hundred microns. To enable in-vivo optical contrast imaging of many important model organisms, such as insects, worms and similarly sized biological specimens, we have developed a multi-spectral optoacoustic tomography technique for high-resolution imaging of optically diffusive organisms and tissues. The method is capable of imaging at depths from sub-millimeter up to a centimeter range with a scalable spatial resolution on the order of magnitude of a few tenths of microns. Furthermore, we show for the first time that the technique is capable of resolving spatial distribution of fluorescent proteins inside intact opaque organisms, thus overcoming depth limitations of current fluorescence microscopy techniques.

We developed a 3D whole-body optoacoustic tomography system for applications in preclinical research on mice. The system is capable of generating images with resolution better than 0.6 mm. Two pulsed lasers, an Alexandrite laser operating at 755 nm and a Nd:YAG laser operating at 532 nm and 1064nm were used for light delivery. The tomographic images were obtained while the objects of study (phantoms or mice) were rotated within a sphere outlined by a concave arc-shaped array of 64 piezo-composite transducers. During the scan, the mouse was illuminated orthogonally to the array with two wide beams of light from a bifurcated fiber bundle. Illumination at 532 nm showed superficial vasculature, but limited penetration depth at this wavelength prevented the detection of deeper structures. Illumination at 755 and 1064 nm showed organs and blood vessels, respectively. Filtering of the optoacoustic signals using high frequency enhancing wavelets further emphasized the smaller blood vessels.

We have designed and built a prototype PCT (photoacoustic CT) scanner suitable for small animal imaging that acquires a sparse set of 128 photoacoustic, radial "projections" uniformly distributed over the surface of a hemisphere in response to optical absorption from a tunable, pulsed NIR (near-infrared) laser. Acquisition of a denser set of projections is achieved by rotating the hemispherical array about its vertical axis and acquiring additional, interleaved projections. Each detector in the array is a 3-mm diameter, piezo-composite with a center frequency of 5 MHz and 70% bandwidth. Spatial resolution is < 300 μm and nearly isotropic, owing to the array geometry. Preliminary results acquired at half of the allowable laser power and with no system optimizations show a low contrast sensitivity sufficient to detect a 350 nM concentration of a NIR-absorbing organic dye embedded in 12.5 mm of soft tissue. This scanner design will allow our group to take advantage of HYPR (HighlY constrained backPRojection) reconstruction techniques, which can significantly improve temporal (or spectral) resolution, without sacrificing signal-to-noise or spatial resolution. We will report how these accelerated reconstruction techniques can be implemented with this PCT scanner design. Using this approach, we may be able to achieve 100-ms temporal resolution for dynamic studies throughout a 20-mm-diameter imaging volume.

We present an in vivo reflection-mode photoacoustic microscopy system that performs B-scan imaging at 50 Hz with realtime beamforming and 3-D imaging of 166 B-scan frames at 1 Hz with post-beamforming. To our knowledge, this speed is currently the fastest in high frequency photoacoustic imaging. In addition, with a custom fiber based light delivery system, the imaging device is capable of performing handheld operation. Software for image processing and display with clinically user-friendly graphic user interface (GUI) is developed. The system has axial, lateral, and elevational resolutions of 25, 70, and 200 μm, respectively, and can image 3 mm deep in scattering biological tissue. Volumetric images of subcutaneous vasculature in murine are demonstrated in vivo. The system is anticipated to have potential clinical applications in skin melanoma detection due to its unique ability to image in realtime and to image anatomical sites inaccessible to other imaging systems.

Microvascular autoregulation is an intrinsic ability of vascular beds to compensate for the fluctuation in blood flow and tissue oxygen delivery. This function is crucial to maintaining the local metabolic activity. Here, using optical-resolution photoacoustic microscopy (OR-PAM), we clearly observed vasomotion and vasodilation in the intact mouse microcirculation in vivo in response to the changes in physiological state. Our results show that a significant lowfrequency vasomotion can be seen under hyperoxia but not hypoxia. Moreover, significant vasodilation is observed when the animal status is switched from hyperoxia to hypoxia. Our data show that arterioles have more pronounced vasodilation than venules.

Photoacoustic imaging and optical coherence tomography have complementary imaging contrasts. Photoacoustic imaging is sensitive to optical absorption, thus is able to generate detailed maps of deep microvasculature in vivo. Optical coherence tomography exploits the optical scattering contrast, and can provide real-time, micrometer-resolution imaging of tissue. We integrate an optical-resolution photoacoustic microscopy and a spectral-domain optical coherence tomography into a single system. Our preliminary experiments showed that it could be a valuable imaging tool for microcirculation studies in vivo.

We present the application of an optimized curved array photoacoustic tomographic imaging system, which can provide rapid, high-resolution photoacoustic imaging of small animal brains. The system can produce a B-mode, 90-degree field-of-view image at sub-200 μm resolution at a frame rate of ~1 frame/second when a 10-Hz pulse repetition rate laser is employed. By rotating samples, a complete 360-degree scan can be achieved within 15 seconds. In previous work, two-dimensional ex vivo mouse brain cortex imaging has been reported. In the current work, we report three-dimensional small animal brain imaging obtained with the curved array system. The results are presented as a series of two-dimensional cross-sectional images. Besides structural imaging, the blood oxygen saturation of the animal brain cortex is also measured in vivo. In addition, the system can measure the time-resolved relative changes in blood oxygen saturation level in the small animal brain cortex. Finally, ultrasonic gel coupling, instead of the previously adopted water coupling, is conveniently used in near-real-time 2D imaging.

We have developed a laser-scanning optical-resolution photoacoustic microscopy that can potentially be easily integrated with several existing optical microscopic modalities. During data acquisition, the ultrasonic transducer is kept stationary and only the laser light is raster-scanned by an x-y galvanometer scanner. In this configuration, the field-ofview is limited by the beam diameter of the ultrasonic detector, which is related to the active element size, numerical aperture, and the center frequency of the ultrasonic transducer. The spatial resolution is determined by the size of the optical focus. A lateral resolution of 7.8 μm and a circular field-of-view with a diameter of 6 mm were achieved in an optically clear medium. Using a laser system working at a pulse repetition rate of 1024 Hz, the data acquisition time for an image consisting of 256×256 pixels was less than two minutes. In vivo imaging of microvasculature in mouse ears were also achieved.

The ability to image polarization-selective tissue structures may provide valuable information on tissue anatomy, morphogenesis, and disease progression. So far, intensive light scattering in biological medium has limited implementation of polarization imaging to superficial tissue layers. We suggest overcoming the scattering problem using polarization-sensitive optoacoustic imaging. Due to intrinsically high spatial resolution and sensitivity of the method, it holds promise of becoming highly accurate modality for interrogation of small polarized structures deep in biological tissues. We show initial tomographic results in tissue-mimicking phantoms having polarization dichroism contrast.

We present a concept and system implementation for endoscopic photoacoustic microscopy that enables minimally invasive diagnosis of internal organs. The system incorporates an in-house made single element ultrasonic transducer for photoacoustic signal detection and an optical fiber for light delivery into a tubular probe with a mechanical scanning unit. The implemented probe size for the distal end is 4.2 mm in diameter and 48 mm in length. We evaluated the system's performance by imaging a carbon fiber in clear and turbid media. We also demonstrated its ability to image actual biological tissues and its endoscopic applicability, by imaging abdominal surfaces and a large intestinal tract of a rat ex vivo. This study shows the system's potential for in vivo optical biopsy of tissue abnormalities, such as tumors, developed in internal organs.

Noninvasive monitoring of blood parameters such as total hemoglobin concentration and saturation (oxygenation) is important for diagnostics in large populations of patients. We developed a novel optoacoustic array for monitoring of these variables in arteries and veins. The array allows for measurements without scanning, reduces the data acquisition time, and minimizes the influence of motion artifacts. The array combines a custom-made fiber-optic delivery system and multiple piezoelectric transducers. We tested the array in vitro and in vivo. The array design and materials allowed for sensitive measurement of optoacoustic signals without distortion and provided real time measurement of these parameters both in vitro and in vivo without scanning.

Photoacoustic microscopy and tomography are hybrid biomedical imaging technologies that provide optical absorption contrast with ultrasonic spatial resolution. To date, photoacoustic methods have provided little information about the optical scattering properties of tissues. Yet scattering is a key tissue parameter with diagnostic potential for a number of diseases. Moreover, quantitative knowledge of the optical scattering coefficient may prove valuable for improving quantitative estimates of blood oxygen saturation and other functional parameters. We present a new photoacoustic method that shows promise for sensing the local reduced scattering coefficient of tissues.

We demonstrated the focal point of a high-numerical-aperture (NA) ultrasonic transducer can be used as a virtual point detector. This virtual point detector detects omnidirectionally over a wide acceptance angle. It also combines a large active transducer surface and a small effective virtual detector size, thus the sensitivity is high compared with that of a real point detector, and the aperture effect is small compared with that of a finite size transducer. Phantom experiments are provided to demonstrate the applications of high-NA-based virtual point detectors in photoacoustic tomography and thermoacoustic tomography. The virtual point detector is also used to image the cerebral cortex of mice.

Photoacoustic imaging with a scanning, fixed focus receiver gives images with high resolution, without the need for reconstruction algorithms. However, the usually employed spherical ultrasound lenses have a limited focal depth that decreases with increasing lateral resolution due to the inverse relation between numerical aperture and Rayleigh length. In this study the use of an axicon detector is proposed, consisting of a conical surface onto which a piezoelectric polymer film is attached. The detector is characterized in simulations and in experiments, demonstrating the expected high resolution over an extended depth of focus. Simulated and experimental images reveal X-shaped artifacts that are due to the conical detector surface. Since the point spread function (PSF) of the detector is spatially invariant over the depth of field, a frequency domain deconvolution can be applied to the images. Although this clearly improves the image quality in simulations, the reduction of artifacts was not so efficient in experiments. However, the detector is able to produce images with accurate position and shape of objects. Moreover, the axicon transducer rejects signals from planar surfaces (e.g. the skin surface) and favors signals from small, isolated sources.

Currently two different types of integrating line sensors are used in photoacoustic tomography (PAT). Thin film piezoelectric polymer sensors (PVDF) are characterized by compactness, easy handling and the possibility to manufacture sensing areas with different shape. However, they are vulnerable to electrical disturbance and to scattered light from the illuminated sample. Also optical sensors are used as integrating line sensors in combination with some kind of interferometric setup. For example, one arm of a Mach-Zehnder interferometer or the cavity of a Fabry-Perot interferometer can be used as line detector. In both cases, the light wave either propagates freely in the liquid or is guided in an optical fiber. Such sensors are quite immune against noise sources described above and suitable for high bandwidth detection. One drawback is the limited mobility due to the complex arrangement of the setup. This study is focused on the comparison of the different implementations of line detectors, mainly on directivity and sensitivity. Shape and amplitude of signals generated by defined sources are compared among the various sensor types. While the shape of the signals recorded with the optical free beam detector matches quite well to the simulation the signals detected with the PVDF detector are affected by directivity effects. This causes a strong distortion of the signal shape depending on the incident angle of the acoustic wave. How these effects influence the reconstructed projection image is discussed.

Comprehensive characterization of optoacoustic transducers is achieved through the analysis of their frequency response using a procedure of measuring angular dependence of the transducer sensitivity to the ultrawide-band delta pulse. The testing was performed under standard repeatable operating conditions. Back-illumination of a blackened, acoustically matched, planar surface with a short laser pulse creates an acoustic impulse which was used as an ultrawide-band ultrasonic source. The bandwidth of such a source extends well over 10 MHz (6dB point at 16 MHz for illumination with a 16 ns pulse) and the low frequency roll-off is around 300 kHz. Analysis of the angular dependence of the frequency response yields invaluable directivity information about the detector under study, which in turn permits accurate forward and inverse problem models.

In atherosclerosis, tracking and locating the activity of macrophages that are highly involved in plaque development will help to identify the pathology of the disease. Intravascular photoacoustic (IVPA) imaging has shown potential to detect atherosclerosis and to determine plaque composition. Furthermore, using optical absorbers as contrast agents, IVPA can also be used for molecular imaging. In this paper, we study the feasibility of using gold nanoparticles as contrast agent for high sensitivity IVPA imaging of macrophages. The artery was modeled using a cylindrical tube made out of polyvinyl alcohol. Within the vessel wall, several compartments were made to mimic plaques. After incubating murine macrophages with 50 nm spherical gold nanoparticles overnight, macrophages loaded with particles were filled into the compartments of the arterial phantoms. Because of the plasmon resonance coupling of aggregated nanoparticles inside the macrophages, these macrophages can be detected by IVPA imaging using 680 nm wavelength. The sensitivity of the molecular IVPA imaging was tested using phantoms with different concentrations of nanoparticles and macrophages. Finally, to address the feasibility of in-vivo IVPA imaging with gold nanoparticles, the viability of the macrophages loaded with nanoparticles exposed to laser irradiation was studied. The results show that IVPA imaging can safely image macrophages loaded with gold nanoparticles with relatively high sensitivity.

We have developed a fast 3D photoacoustic imaging system based on a sparse array of ultrasound detectors and iterative image reconstruction. To investigate the high frame rate capabilities of our system in the context of rotational motion, flow, and spectroscopy, we performed high frame-rate imaging on a series of targets, including a rotating graphite rod, a bolus of methylene blue flowing through a tube, and hyper-spectral imaging of a tube filled with methylene blue under a no flow condition. Our frame-rate for image acquisition was 10 Hz, which was limited by the laser repetition rate. We were able to track the rotation of the rod and accurately estimate its rotational velocity, at a rate of 0.33 rotations-per-second. The flow of contrast in the tube, at a flow rate of 180 μL/min, was also well depicted, and quantitative analysis suggested a potential method for estimating flow velocity from such measurements. The spectrum obtained did not provide accurate results, but depicted the spectral absorption signature of methylene blue , which may be sufficient for identification purposes. These preliminary results suggest that our high frame-rate photoacoustic imaging system could be used for identifying contrast agents and monitoring kinetics as an agent propagates through specific, simple structures such as blood vessels.

Detection of non-radio-opaque foreign bodies can be difficult. Current imaging modalities employed for detection of foreign bodies include: X-ray computed tomography, magnetic resonance, and ultrasound. Successful diagnosis of the presence of foreign bodies is variable because of the difficulty of differentiating them from soft tissue, gas, and bone. We are applying laser-induced optoacoustic imaging to the detection of foreign bodies. Tissue-simulating phantoms containing various common foreign bodies have been constructed. Images of these phantoms were generated by two laser-based optoacoustic methods utilizing different detection modalities. A pre-commercial imager developed by Seno Medical Instruments (San Antonio), incorporated an ultrasound transducer to detect induced optoacoustic responses, while a laboratory-built imaging system utilized an optical probe beam deflection technique (PBDT) to detect the optoacoustic responses. The laboratory-built unit also included an optical parametric oscillator as the pump, providing tunable wavelength output to optimize the optoacoustic measurements by probing the foreign bodies at their maximum optical absorption. Results to date have been encouraging; both methodologies have allowed us to reconstruct successfully the image of foreign-body containing phantoms. In preliminary work the PBDT approach appeared to produce higher resolution than did the ultrasound detector, possibly because PBDT is not constrained by the lower bandwidth limit imposed on the ultrasound transducer necessary to increase imaging depth. During the research in progress, we will compare the optoacoustic images to those generated by MRI, CT, and ultrasound, and continue to improve the resolution of the technique by using multiple detection sensors, and to improve image contrast by scanning foreign bodies over a range of wavelengths.

We describe a new spectral approach for inversion of photoacoustic data with multi-wavelength pulsed laser illumination. Multi-spectral PAT provides a means of recovery of different chromophore concentrations and ultrasound velocity simultaneously and directly by incorporating prior spectral information into the image reconstruction process. It is demonstrated from simulation tests and small animal experiments that the multi-parameter recovery based on multispectral PAT is reliable and accurate. The reconstructed multiple parameter images may provide us a key tool to quantify physiological function, disease progression, or response to intervention.

For real-time optoacoustic imaging of the human body, a linear array transducer and reflection mode optical irradiation is preferably used. Experimental outcomes however revealed that such a setup results in significant image background, which prevents imaging structures at the ultimate depth limited only by optical attenuation and the signal noise level. Various sources of image background such as bulk tissue absorption, reconstruction artifacts, and backscattered ultrasound could be identified. We therefore developed a novel method which results in significantly reduced background and increased imaging depth. For this purpose, we acquire in parallel a series of optoacoustic and echo-ultrasound images while the tissue sample is gradually deformed by an externally applied force. Optoacoustic signals and background signals are differently affected by the deformation and can thus be distinguished by image processing. This method takes advantage of a combined optoacoustic/echo-ultrasound device and has a strong potential for improving real-time optoacoustic imaging of deep tissue structures.

Recently a realtime photoacoustic microscopy system has been demonstrated. Unfortunately, however, displayed B-scan images were sometimes difficult to interpret as there was little structural context. In this work, we provide structural context for photoacoustic microscopy images by adding ultrasound biomicroscopy as a complementary and synergistic modality. Our system uses a voice-coil translation stage capable of 1" lateral translation, and can operate in excess of 15 Hz for 1-cm translations, providing up to 30 ultrasound frames per second. The frame-rate of the photoacoustic acquisitions is limited by the 20-Hz pulse-repetition rate of the laser, but can be increased with a faster-repetition-rate laser. Data from the system is streamed in real time from a 2GS/s PCI data acquisition card to the host PC at rates as high as 200 MB/s. The system should prove useful for various in vivo studies, including combined ultrasound Doppler and photoacoustic imaging.

The absorption coefficients in most biological tissues range from 1 cm-1 to 10 cm-1, which produce photoacoustic signal with peak frequency lower than 5 MHz. However, the lower operating frequencies mean equivalently larger wavelengths, which are incapacitation to resolve smaller objects. In order to obtain the excellent performance of images in both sensitivity and resolution, this study discusses the design and fabrication of the dual frequencies photoacoustic transducer (DFPT) with 36°-rotated, Y-cut lithium niobate (LiNbO3) material. The DFPT had a diameter of 6 mm and comprised two concentric rings of equal area with center frequency of the outer and inner elements as 4.9 MHz and 14.8 MHz, respectively. Moreover, there was a 0.65 mm hole in the DFPT surface for insertion of an optical fiber, which solved conventional light-placing problem. The experiment was performed with an agarose phantom which mixed glass beads with various concentration of black stain to create different optical absorptions. The absorption coefficients of absorbers are 5.6 cm-1 and 11.8 cm-1, respectively. The mean amplitude between the two absorbers differs by 0.5 dB at band 0-5 MHz, while the difference increases to 5.9 dB at band 6-15 MHz. The results show that the DFPT not only provides high ultrasonic resolution but also enhances the contrast based on higher frequency subbands for photoacoustic imaging. The potential of improving the contrast between biological tissues and contrast agent with a significant higher absorption is revealed. In the future, the DFPT will be applied to in vivo investigation with gold nanoparticles. Bioconjugated gold nanoparticles have been used as a photoacoustic contrast agent as well as a molecular probe to target cancer cells in a small animal model. The denseness of targeted gold nanoparticles on tumor results in a higher absorptions, which means higher frequency signals comparing to those of surrounding tissues. Thus, DFPT will assist in recognizing the tumor region for a better diagnosis.

In recent years, some of the promised potential of biomedical photoacoustic imaging has begun to be realised. It has been used to produce good, three-dimensional, images of blood vasculature in mice and other small animals, and in human skin in vivo, to depths of several mm, while maintaining a spatial resolution of <100 μm. Furthermore, photoacoustic imaging depends for contrast on the optical absorption distribution of the tissue under study, so, in the same way that the measurement of optical spectra has traditionally provided a means of determining the molecular constituents of an object, there is hope that multiwavelength photoacoustic imaging will provide a way to distinguish and quantify the component molecules of optically-scattering biological tissue (which may include exogeneous, targeted, chromophores). In simple situations with only a few significant absorbers and some prior knowledge of the geometry of the arrangement, this has been shown to be possible, but significant hurdles remain before the general problem can be solved. The general problem may be stated as follows: is it possible, in general, to take a set of photoacoustic images obtained at multiple optical wavelengths, and process them in a way that results in a set of quantitatively accurate images of the concentration distributions of the constituent chromophores of the imaged tissue? If such an 'inversion' procedure - not specific to any particular situation and free of restrictive suppositions - were designed, then photoacoustic imaging would offer the possibility of high resolution 'molecular' imaging of optically scattering tissue: a very powerful technique that would find uses in many areas of the life sciences and in clinical practice. This paper describes the principal challenges that must be overcome for such a general procedure to be successful.

The photoacoustic technique can be used to quantify tissue absorption spectrum in a wide spectral range from visible to near infrared. As the photoacoustic signal intensity is proportional to tissue optical absorption coefficient and light fluence, it is important to know the local light fluence at the regional target in order to obtain the accurate absorption spectrum because the tissue optical properties including scattering and absorption are wavelength dependent and affect the distribution and intensity of light in the sample. In this work, an optical contrast agent has been employed to enhance the performance of spectroscopic photoacoustic technique. From the photoacoustic measurements with and without the contrast agent in a target tissue, the spectroscopic local light fluence in the tissue can be determined. Then a quantified measurement of the tissue optical absorption spectrum can be realized in a strong scattering medium without need to know the wavelength-dependent optical properties in the scattering medium. A commercially available dye which has strong absorption in the wavelength range of interest was used. The results of the spectroscopic photoacoustic measurements on fresh canine blood specimens in scattering media such as milk and chicken breast tissue have been presented. It was found that photoacoustic measurements after employing this new technique have an improved match with the standard absorption spectra of both oxygenated and deoxygenated blood.

Photoacoustic tomography is an emerging medical imaging modality based on the reconstruction of an initial internal pressure distribution from surface measurements of photoacoustic wave pulses over time. Current methods used for this image reconstruction assume that the propagation medium is acoustically non-attenuating. However, in soft biological tissue, the frequency dependent ultrasonic attenuation is sufficient to cause considerable distortion to photoacoustic waves, even over short propagation distances. This distortion introduces blurring artifacts into images reconstructed under the assumption of a lossless medium. Here, a general lossy wave equation applicable to biological media is developed for which an exact solution (formed in the wavenumber-frequency domain) is derived. Explicit consideration is given to sound speed dispersion which is shown to have a negligible effect on photoacoustic imaging. Given an initial pressure distribution, the developed model allows the complete pressure field within the domain to be computed at an arbitrary time without iteration. The computation relies only on the Fourier transform and a decaying time propagator dependent on the attenuation in the medium. This facilitates the fast calculation of pressure fields in two or three dimensions over large domains. The model is demonstrated through the simulation and reconstruction of an example pulse distribution in a lossy medium.

A modified MC convolution method for integration extension of MC simulation is developed for finite photon beam with random shape of translational or rotational invariance, which is proven consistent with the conventional convolution extension of MC simulation for normal incident finite beam. The method is applied to analyze the positions of fluence foci and ratios of fluence at the focus and surface which are two key factors in the application of dark-field confocal and some interesting points are presented including: 1) The fluence profile has a saddle-like shape with highest peak in the bright field and low valley near the surface and a second rise in the center of dark field which is defined as the effective optical focus; 2) Besides a little peak near zero inner radius, the ratio of fluences at the focus and surface increases linearly with the inner radius, suggesting the large inner radius more advantageous to image at the effective optical focus; 3) The position of effective optical foci deepens linearly with the increase of the inner radius, suggesting that to get a high quality image of deeper target, a dark-field with larger size is more beneficial. But the position of fluence foci are far away from the foci of geometrical laser beam in high scattering tissue, so aligning the foci of geometrical laser beam and acoustic transducer doesn't guarantee that effective optical focus is accurately overlapping with the acoustic focus. An MC simulation with integration extension presented in this paper maybe helpful to determine where the acoustic focus should be to maximize the SNR in tissue imaging; 4) incident angle makes little difference to ratio of fluences at the focus and surface and an incident angle between 30 and 50 degrees gives the highest fluence at the effective optical focus; 5) the depth of fluence focus is insensitive to the incident angle.

Plasmon resonant nanoparticles such as gold nanoshells and gold nanorods can be tuned to possess sharp interaction peaks in the near-infrared wavelength regions. These have great importance as contrast agents in photoacoustic imaging and as photothermal agents for therapeutic applications due to their high absorptions. While the optical properties of the particles are can be described using Mie theory and/or numerical methods such as the Discrete Dipole Approximation, discriminating between their optical absorption and scattering in experiments is not easy. In this paper we discuss for the first time a novel method based on a two-fiber spectrometer that allows measurement of the scattering and absorption coefficients of gold nanoparticles in solution. This technique, called Differential Path length Spectroscopy, has been developed earlier for measurement in highly diffusive media such as tissue. We demonstrate this concept on gold nanospheres and nanoshells of various sizes. We believe that this will develop into a fast and reliable method able to work on small samples (<1 ml) of nanoparticles to obtain scattering and absorbing spectra.

Previous research correcting for variable speed of sound in photoacoustic tomography (PAT) has used a generalized radon transform (GRT) model . In this model, the pressure is related to the optical absorption, in an acoustically inhomogeneous medium, through integration over non-spherical isochronous surfaces. This model assumes that the path taken by acoustic rays is linear and neglects amplitude perturbations to the measured pressure. We have derived a higher-order geometrical acoustics (GA) expression, which takes into account the first-order effect in the amplitude of the measured signal and higher-order perturbation to the travel times. The higher-order perturbation to travel time incorporates the effect of ray bending. Incorrect travel times can lead to image distortion and blurring. These corrections are expected to impact image quality and quantitative PAT. We have previously shown that travel-time corrections in 2D suggest that perceivable differences in the isochronous surfaces can be seen when the second-order travel-time perturbations are taken into account with a 10% speed of sound variation. In this work, we develop iterative image reconstruction algorithms that incorporate this higher-order GA approximation assuming that the speed of sound map is known. We evaluate the effect of higher-order GA approximation on image quality and accuracy.

Attenuation effects can be significant in photoacoustic tomography (PAT) since the measured pressure signals are broadband and ignoring them may lead to image artifacts and blurring. Previous work by our group had derived a method for modeling the attenuation effect and correcting for it in the image reconstruction. This was done by relating the ideal, unattenuated pressure signals to the attenuated pressure signals via an integral operator. In this work, we explore singularvalue decomposition (SVD) of a previously derived 3D integral equation that relates the Fourier transform of the measured pressure with respect to time and two spatial components to the 2D spatial Fourier transform of the optical absorption function. We find that the smallest singular values correspond to wavelet-like eigenvectors in which most of the energy is concentrated at times corresponding to greater depths in tissue. This allows us characterize the ill posedness of recovering absorption information from depth in an attenuating medium. This integral equation can be inverted using standard SVD methods and the optical absorption function can be recovered. We will conduct simulations and derive algorithm for image reconstruction using SVD of this integral operator.

We present results of investigations of the application of a priori information and sparse or limited-view algorithms to simultaneously improve imaging quality and timeresolution in photoacoustic tomography. Modified versions of existing MRI/CT algorithms such as constrained backprojection and keyhole imaging are evaluated as well as a new Wiener estimation methods for extrapolation of missing data from reference data sets. Simulations indicate the effectiveness of the approaches for accurate tracking of dynamic photoacoustic events for data sets with limited views (< 90 degrees) or tomographic views with up to 1/64 of the full data set. We present experimental data of contrast uptake and washout using a 512-element curved transducer with 1:8 electronic multiplexing that demonstrates high-resolution tomographic imaging with a temporal resolution of better than 150 milliseconds using these methods.

Ultrasound-modulated optical imaging is an emerging biodiagnostic technique which provides the optical spectroscopic signature and the spatial localization of an optically absorbing object embedded in a strongly scattering medium. The transverse resolution of the technique is determined by the lateral extent of ultrasound beam focal zone while the axial resolution is obtained by using short ultrasound pulses. The practical application of this technique is presently limited by its poor sensitivity. Moreover, any method to enhance the signal-to-noise ratio must satisfy the biomedical safety limits. In this paper, we propose to use a pulsed single-frequency laser source to raise the optical peak power applied to the scattering medium and to collect more ultrasonically tagged photons. Such a laser source allows illuminating the tissues mainly during the transit time of the ultrasonic wave. A single-frequency Nd:YAG laser emitting 500-μs pulses with a peak power superior to 100 W was used. Tagged photons were detected with a GaAs photorefractive interferometer characterized by a large optical etendue. When pumped by high intensity laser pulses, such an interferometer provides the fast response time essential to obtain an apparatus insensitive to the speckle decorrelation encountered in biomedical applications. Consequently, the combination of a large-etendue photorefractive interferometer with a high-power pulsed laser could allow obtaining both the sensitivity and the fast response time necessary for biomedical applications. Measurements performed in 30- and 60-mm thick optical phantoms made of titanium dioxide particles dispersed in sunflower oil are presented. Results obtained in 30- and 60-mm thick chicken breast samples are also reported.

Ultrasound-modulated optical imaging combines the good spatial resolution of ultrasonic waves (mm scale) and the spectroscopic properties of light to detect optically absorbing objects inside thick (cm scale) highly scattering media. Light propagating in a scattering medium can interact with an ultrasonic wave thereby being tagged by a frequency shift equal to the ultrasound frequency or its harmonics. In this paper, a confocal Fabry-Perot interferometer (CFPI) is used as a tunable spectral filter to detect selectively the ultrasound-tagged photons. The CFPI allows obtaining high spectral resolution (MHz scale) while maintaining a high light gathering power when compared to other spectroscopic devices of comparable resolution. The contrast between the tagged photons and the untagged photons can be further enhanced by cascading CFPI. Moreover, the fast response of the CFPI allows performing measurements within the speckle decorrelation time typically encountered in biomedical applications. In this paper, the use of a single-frequency laser emitting powerful optical pulses allows illuminating the scattering medium only during the transit time of the probing ultrasonic pulses. Consequently, the acoustic and the optical power are both concentrated in time to enhance the signal-to- noise ratio of the technique while remaining below the biomedical safety limits. The detection of optically absorbing objects (mm size) inside 30- and 60-mm thick scattering media is presented.

We have succeeded in implementing ring-shaped light illumination ultrasound-modulated optical tomography (UOT) in both transmission and reflection modes. These systems used intense acoustic bursts and a charge-coupled device camera-based speckle contrast detection method. By mounting an ultrasound transducer into an optical condenser, we can combine the illuminating light component with the ultrasound transducer. Thus, the UOT system is more clinically applicable than previous orthogonal mode systems. Furthermore, we have successfully imaged an ex vivo methyleneblue-dyed sentinel lymph node (SLN) embedded deeper than 12 mm, the mean depth of human sentinel lymph nodes, in chicken breast tissue. These UOT systems offer several advantages: noninvasiveness, nonionizing radiation, portability, cost-effectiveness, and possibility of combination with ultrasound pulse-echo imaging and photoacoustic imaging. One potential application of the UOT systems is mapping SLNs in axillary staging for breast cancer patients.

Acousto-optic imaging (AOI) is a dual-wave modality that combines ultrasound with diffuse light to achieve deep-tissue imaging of optical properties with the spatial resolution of ultrasound. Progress has been made in the detection of optically absorbing inhomogeneities in recent years, yet it remains a challenge for AOI to detect targets possessing low scattering contrast and to obtain quantitative measurement of optical properties at depth with high resolution. A new photorefractive crystal (PRC) based AOI system operating in the near-infrared optical wavelength was developed and optimized. Based on relative changes in the AOI response induced by different acoustic pressures, we now propose a new sensing and imaging modality, pressure contrast imaging (PCI), to enhance and quantify the detection of scattering inhomogeneities. It is demonstrated experimentally that the image contrast information obtained with the new approach is independent of the background light intensity and the details of the optical collection components and potentially allows for an accurate and quantitative characterization of the media's spatially dependent optical properties.

We have investigated the application of AO sensing for quantitative three-dimensional mapping of tissue-mimicking phantoms. An Intralipid phantom, which contains a turbid absorber, confined in a silicone tube, was used. Multiply scattered pulsed laser light was modulated by ultrasonic bursts focused in a predefined volume in the medium. By varying the delay time between ultrasound burst initiation and light pulse firing we could perform a scan in the ultrasound-propagation plane. By moving the ultrasound transducer, we could build up a volumetric map of modulation depth values. We have experimentally determined the acousto-optical modulation depth as a function of the absorption coefficient in phantom voxels of a few millimeters in size.

Ultrasound-modulated fluorescence from a fluorophore-quencher labeled microbubble system driven by a single ultrasound pulse was theoretically quantified by solving a modified Herring equation (for bubble oscillation), a two-energy-level rate equation (for fluorophore excitation), and a diffusion equation (for light propagation in tissue). The efficiency of quenching caused by fluorescence resonance energy transfer (FRET) between the fluorophore and the quencher was modulated when the microbubble oscillates in size driven by the ultrasound pulse. Both intensity- and lifetime-based imaging methods are discussed. An important finding in this study is that ultrasound-modulated fluorescent photons may be temporally separated from the un-modulated fluorescent photons if a super-short laser pulse is adopted. This result implies that the modulation efficiency may only be limited by thermal noise of the measurement system.

In vivo acousto-optic imaging promises to provide optical contrast at superior ultrasound spatial resolution. The main challenge is to detect ultrasound-modulated photons in the overwhelming presence of un-modulated photons. We have demonstrated in vitro detection of ultrasound-modulated photons with a variety of detection methods. Furthermore, we have detected ultrasound-modulated fluorescence offering potential for acousto-optic molecular imaging. Moreover, we have demonstrated the use of ultrasound microbubbles to significantly enhance the acousto-optic signal at the ultrasound frequency with the additional generation of higher order harmonic frequencies. Here the results from our various detection methods, ultrasound-modulated fluorescence, and enhancement with microbubbles are presented.

A new metallodielectric nanoparticle consisting of a silica core and silver outer cage was developed for the purpose of enhancing photoacoustic imaging contrast in pancreatic tissue. These nanocages were injected into an ex vivo porcine pancreas and imaged using a combined photoacoustic and ultrasound (PAUS) assembly. This custom-designed PAUS assembly delivered 800 nm light through a fiber optical light delivery system integrated with 128 element linear array transducer operating at 7.5 MHz center frequency. Imaging results prove that the nanocage contrast agents have the ability to enhance photoacoustic imaging contrast. Furthermore, the value of the combined PAUS imaging modality was demonstrated as the location of nanocages against background native tissue was evident. Future applications of these nanocage contrast agents could include targeting them to pancreatic cancer for enhancement of photoacoustic imaging for diagnosis and therapy.

Sentinel lymph node biopsy (SLNB), a less invasive alternative to axillary lymph node dissection (ALND), is routinely used in clinic for staging breast cancer. In SLNB, lymphatic mapping with radio-labeled sulfur colloid and/or blue dye helps identify the sentinel lymph node (SLN), which is most likely to contain metastatic breast cancer. Even though SLNB, using both methylene blue and radioactive tracers, has a high identification rate, it still relies on an invasive surgical procedure, with associated morbidity. In this study, we have demonstrated a non-invasive single-walled carbon nanotube (SWNT)-enhanced photoacoustic (PA) identification of SLN in a rat model. We have used single-walled carbon nanotubes (SWNTs) as a photoacoustic contrast agent to map non-invasively the sentinel lymph nodes (SLNs) in a rat model in vivo. We were able to identify the SLN non-invasively with high contrast to noise ratio (~90) and high resolution (~500 μm). Due to the broad photoacoustic spectrum of these nanotubes in the near infrared wavelength window we could easily choose a suitable light wavelength to maximize the imaging depth. Our results suggest that this technology could be a useful clinical tool, allowing clinicians to identify SLNs non-invasively in vivo. In the future, these contrast agents could be functionalized to do molecular photoacoustic imaging.

We demonstrated the ability to detect surface antigens, associated with pathogens, utilizing laser optoacoustic spectroscopy with antibody-coupled gold nanorods (GNR) as a contrast agent specifically targeted to the antigen of interest. The sensitivity of the technique has been assessed by determining the minimum detectable concentration of a surface antigen from biological samples. We compared the sensitivity and applicability of two different methods for detecting optoacoustic responses, using either optical or piezoelectric sensors. Biological samples were adsorbed to the inside walls of detachable, flat-bottomed plastic micro-wells, and then probed with appropriate antibodies conjugated with gold nanorods. If the target antigens were present, the antibody-nanoparticle conjugates were bound, while any nonadsorbed nanoparticles were washed out of the wells. Optoacoustic signals were generated from the bound nanorods using a pulsed pump laser at wavelengths corresponding to one of the peak absorptions of the nanorods. Optoacoustic responses were obtained from the samples using both detection modalities. The sensitivity, suitability, ease of use of each method were assessed and compared. Initial results indicate that optical detection gives comparable sensitivity as the piezoelectric method, and further enhancement of the detection sensitivity is possible for both methods. An advantage of the piezoelectric detection method is that it may be implemented in a more compact assembly, compared to the optical method, however, the optical method may be less sensitive to external electromagnetic and acoustic noise. Further evaluation will be required to refine these measurements. The results with both methods indicate that the use of antibody-targeted nanorod contrast agents, with laser-optoacoustic detection, is a promising technology for the development of rapid in vitro diagnostic tests.

We are detecting antigens (Ag), isolated from infectious organisms, utilizing laser optoacoustic spectroscopy and antibody-coupled gold nanorod (NR) contrast agents specifically targeted to the antigen of interest. We have detected, in clinical ocular samples, both Herpes Simplex Virus Type 1 and 2 (HSV-1 and HSV-2) . A monoclonal antibody (Ab) specific to both HSV-1 and HSV-2 was conjugated to gold nanorods to produce a targeted contrast agent with a strong optoacoustic signal. Elutions obtained from patient corneal swabs were adsorbed in standard plastic micro-wells. An immunoaffinity reaction was then performed with the functionalized gold nanorods, and the results were probed with an OPO laser, emitting wavelengths at the peak absorptions of the nanorods. Positive optoacoustic responses were obtained from samples containing authentic (microbiologically confirmed) HSV-1 and HSV-2. To obtain an estimate of the sensitivity of the technique, serial dilutions from 1 mg/ml to 1 pg/ml of a C. trachomatis surface Ag were prepared, and were probed with a monoclonal Ab, specific to the C. trachomatis surface Ag, conjugated to gold nanorods. An optoacoustic response was obtained, proportional to the concentration of antigen, and with a limit of detection of about 5 pg/ml. The optoacoustic signals generated from micro-wells containing albumin or saline were similar to those from blank wells. The potential benefit of this method is identify viral agents more rapidly than with existing techniques. In addition, the sensitivity of the assay is comparable or superior to existing colorimetric- or fluorometric-linked immunoaffinity assays.

Photoacoustic (PA) imaging is a rapidly developing imaging modality that can detect optical contrast agents with high sensitivity. While detectors in PA imaging have traditionally been single element ultrasound transducers, use of array systems is desirable because they potentially provide high frame rates to capture dynamic events, such as injection and distribution of contrast in clinical applications. We present preliminary data consisting of 40 second sequences of coregistered pulse-echo (PE) and PA images acquired simultaneously in real time using a clinical ultrasonic machine. Using a 7 MHz linear array, the scanner allowed simultaneous acquisition of inphase-quadrature (IQ) data on 64 elements at a rate limited by the illumination source (Q-switched laser at 20 Hz) with spatial resolution determined to be 0.6 mm (axial) and 0.4 mm (lateral). PA images had a signal-to-noise ratio of approximately 35 dB without averaging. The sequences captured the injection and distribution of an infrared-absorbing contrast agent into a cadaver rat heart. From these data, a perfusion time constant of 0.23 s^-1 was estimated. After further refinement, the system will be tested in live animals. Ultimately, an integrated system in the clinic could facilitate inexpensive molecular screening for coronary artery disease.

Plasmonic photothermal therapy is a new cancer thermotherapy method based on surface plasmon resonance of nanoparticles. It is important to measure the temperature during thermotherapy for safety and efficacy. In this study, we apply a photoacoustic (PA) method for real-time, non-invasive temperature measurements. In particular, this method can be effectively combined with a photothermal therapy system that we developed in parallel. The method is based on the fact that the PA pressure amplitude is linearly related to temperature. To explore its potential, a home-made, 20 MHz PA transducer was used, in which an optical fiber was inserted in its center for emitting laser pulses while the PA signal was simultaneously detected. Continuous wave (CW) laser was used to heat the subject, including both phantoms and mice. The temperature of the region of interest was also measured by a fine-needle thermal couple. Results show that the temperature was linearly proportional to the PA signal with good correlation with the CW laser irradiation. The in vivo study also demonstrated potential of this technique.

Thermoacoustic contrast under RF illumination is a function of electrical conductivity and applied electric field, as well as mechanical and thermal properties. An array of phantom objects complement our RF testbed which illuminates with a controlled E field. All rely on electrical conductivity to generate TCT contrast. Inexpensive and easily fabricated resolution phantoms are highly conductive small inclusions with similar acoustic and thermal properties as background water bath. Tissue mimicking phantoms developed for microwave imaging have acoustic and dielectric properties similar to fat and muscle. Thermal properties are measured to completely quantify expected TCT contrast for experimental corroboration.

Thermoacoustic signal excitation is a function of intrinsic tissue properties and illuminating electric field. We have designed a water-filled testbed propagating a controlled electric field with respect to pulse shape, power, and polarization. Illuminating with a known and carefully controlled E field will enable quantitative measurement of the thermoacoustic contrast mechanism.

Minimally invasive thermal therapy is being investigated as an alternative cancer treatment. It involves heating tissues to greater than 55°C over a period of a few minutes, which results in tissue coagulation. Optoacoustic (OA) imaging is a new imaging technique that involves exposing tissues to pulsed light and detecting the acoustic waves that are generated. In this study, adult bovine liver tissue samples were heated using continuous wave laser energy for various times, then scanned using an optoacoustic imaging system. Large optoacoustic signal variability was observed in the native tissue prior to heating. OA signal amplitude increased with maximum tissue temperature achieved, characterized by a correlation coefficient of 0.63. In this study we show that there are detectable changes in optoacoustic signal strength that arise from tissue coagulation, which demonstrates the potential of optoacoustic technology for the monitoring of thermal therapy delivery.

Optoacoustic imaging is a relatively novel biomedical imaging modality that relies on the absorption of light to create pressure transients that can be detected ultrasonically. In most scientific communications, the source of tissue contrast has been described as primarily optical. However, the thermomecahnical properties of tissue, as expressed through the Gruneisen coefficient, also affect the optoacoustic signal. To investigate the effect of thermomechanical tissue properties short pulses (~ 6.5 ns) from an optical parametric oscillator at 750 nm were used to irradiate coagulated and uncoagulated tissue-mimicking albumen phantoms, to emulate normal tissue and tissue that has been heated. The phantoms respond to the laser-induced stress by thermoelastic expansion. This thermomechanical behavior of the samples was assessed using an interferometric system capable of measuring transient displacements with a temporal resolution of less than 10 ns and a spatial resolution of < 10 nm. The experimental measurement allowed determination of the Gruneisen coefficient which is an important thermo-mechanical sample property that can affect generation of optoacoustic signals. An increase in the value of Gruneisen coefficient of 65% was measured when phantoms were coagulated compared to uncoagulated phantoms, consistent with the stiffening of the tissue mimicking material. This suggests that for thermal therapy the changes in the Gruneisen coefficient are also an important source of optoacoustic contrast.

Our recent efforts are to develop a system for the detection of extremely low number of prostate cancer cells tagged to gold nanorods. Such a system provides an attractive platform to detect the early metastasis. By monitoring the metastatic prostate cancer cells, the physicians are provided with more time to act and design the treatment and also provide better hope for the prostate cancer patients. We developed an optical flowmetry system which employs photoacoustic excitation coupled with an optical transducer capable of determining the presence of cells within the circulating system in vitro.The particles were tagged to gold nanoparticles at Dr.Katti's lab. This provided the required optical contrast to detect the individual prostate cancer cells. Detection trials resulted in a detection threshold of the order of a couple of individual cells in the detection volume, thus validating the effectiveness of the proposed mechanism.

Melanoma is the deadliest form of skin cancer. Although the initial malignant cells are removed, it is impossible to determine whether or not the cancer has metastasized until a secondary tumor forms that is large enough to detect with conventional imaging. Photoacoustic detection of circulating melanoma cells in the bloodstream has shown promise for early detection of metastasis that may aid in treatment of this aggressive cancer. When blood is irradiated with energy from an Nd:YAG laser at 532 nm, photoacoustic signals are created and melanoma cells can be differentiated from the surrounding cells based on waveforms produced by an oscilloscope. Before this can be used as a diagnostic technique, however, we needed to investigate several parameters. Specifically, the current technique involves the in vitro separation of blood through centrifugation to isolate and test only the white blood cell layer. Using this method, we have detected a single cultured melanoma cell among a suspension of white blood cells. However, the process could be made simpler if the plasma layer were used for detection instead of the white blood cell layer. This layer is easier to obtain after blood separation, the optical difference between plasma and melanoma cells is more pronounced in this layer than in the white blood cell layer, and the possibility that any stray red blood cells could distort the results is eliminated. Using the photoacoustic apparatus, we detected no melanoma cells within the plasma of whole blood samples spiked with cultured melanoma cells.

As in MRI, orientation of the patient relative to field lines significantly impacts signal generated. Oblong inclusions generate far less thermoacoustic signal when oriented with long axis perpendicular to E field lines of radiofrequency excitation. We present simulated and low-power measured results below.

The resolution in photoacoustic imaging is limited by the acoustic bandwidth and therefore by acoustic attenuation, which can be substantial for high frequencies. This effect is usually ignored for photoacoustic reconstruction but has a strong influence on the resolution of small structures. The amount of information about the interior of samples, which can be gained in general by the detection of optical, thermal, or acoustical waves on the sample surface, is essentially influenced by the propagation from its excitation to the surface. Scattering, attenuation, and thermal diffusion cause an entropy production which results in a loss of information of propagating waves. Using a model based time reversal method, it was possible to partly compensate acoustic attenuation in photoacoustic imaging. To examine this loss of information in more detail, we have restricted us to "thermal waves" in one dimension, which can be realized experimentally by planar layers. Simulations using various boundary conditions and experimental results are compared. Reconstruction of the initial temperature profile from measurement data is performed by various regularization methods, the influence of the measurement noise (fluctuations) on the information loss during reconstruction is shown to be equal to the entropy production during wave propagation.

Coronary atherosclerosis is a complex disease accompanied by the development of plaques in the arterial wall. Since the vulnerability of the plaques depends on their composition, the appropriate treatment of the arteriosclerosis requires a reliable characterization of the plaques' geometry and content. The intravascular ultrasound (IVUS) imaging is capable of providing structural details of the plaques as well as some functional information. In turn, more functional information about the same plaques can be obtained from intravascular photoacoustic (IVPA) images since the optical properties of the plaque's components differ from that of their environment. The combined IVUS/IVPA imaging is capable of simultaneously detecting and differentiating the plaques, thus determining their vulnerability. The potential of combined IVUS/IVPA imaging has already been demonstrated in phantoms and ex-vivo experiments. However, for in-vivo or clinical imaging, an integrated IVUS/IVPA catheter is required. In this paper, we introduce two prototypes of integrated IVUS/IVPA catheters for in-vivo imaging based on a commercially available single-element IVUS imaging catheter. The light delivery systems are developed using multimode optical fibers with custom-designed distal tips. Both prototypes were tested and compared using an arterial mimicking phantom. The advantages and limitations of both designs are discussed. Overall, the results of our studies suggest that both designs of integrated IVUS/IVPA catheter have a potential for in-vivo IVPA/IVUS imaging of atherosclerotic plaques.

Photoacoustic molecular imaging is an emerging technology offering non-invasive high resolution imaging of the molecular expressions of a disease using a photoacoustic imaging agent. Here we demonstrate for the first time the utility of single walled carbon nanotubes (SWNTs) as targeted imaging agents in living mice bearing tumor xenografts. SWNTs were conjugated with polyethylene-glycol-5000 connected to Arg-Gly-Asp (RGD) peptide to target the α_vβ₃ integrin that is associated with tumor angiogenesis. In-vitro, we characterized the photoacoustic spectra of the particles, their signal linearity and tested their uptake by α_vβ₃-expressing cells (U87MG). The photoacoustic signal of SWNTs was found not to be affected by the RGD conjugation to the SWNTs and was also found to be highly linear with concentration (R² = 0.9997 for 25-400nM). The cell uptake studies showed that RGD-targeted SWNTs gave 75% higher photoacoustic signal than non-targeted SWNTs when incubated with U87MG cells. In-vivo, we measured the minimal detectable concentration of SWNTs in living mice by subcutaneously injecting SWNTs at increasing concentrations. The lowest detectable concentration of SWNTs in living mice was found to be 50nM. Finally, we administered RGDtargeted and non-targeted SWNTs via the tail-vein to U87MG tumor-bearing mice (n=4 for each group) and measured the signal from the tumor before and up to 4 hours post-injection. At 4 hours post-injection, tumors of mice injected with RGD-targeted SWNTs showed 8 times higher photoacoustic signal compared with mice injected with non-targeted SWNTs. These results were verified ex-vivo using a Raman microscope that is sensitive to the SWNTs Raman signal.

Ultrasound microscopy uses high frequency (>40 MHz) ultrasound to produce high resolution images. For high resolution microscopy, broadband ultrasound generation and detection is necessary. Because high frequency ultrasound experiences significant absorption loss that results in weaker signals, it is desirable to focus the energy for microscopy applications, which also results in higher lateral resolution. In this work, thermo-elastically generated ultrasound was brought into a tight focus by shining a ns laser pulse onto a thin metal film-coated concave surface. For ultrasound detection, we use polymer microring resonators which have high frequency and wide band response. We experimentally obtained spatial and temporal characteristics of focused ultrasound by optical generation and detection. The 3-dB spot width of the focused ultrasound is ~50 μm. By frequency filtering over 40~100 MHz, 21 μm width is obtained. The temporal profile is close to the time-derivative of laser pulse waveform. Frequency domain analysis for the signal shows that high frequency loss mechanism of our system is dominated by angular directivity of the microring detector. The issues to improve high frequency response are discussed.

An acceleration of angiogenesis in the adventitial vasa-vasorum is usually associated with vulnerable, thin-cap fibroatheroma in atherosclerotic plaques. Angiogenesis creates microvasculature too small to be detected and differentiated using conventional imaging techniques. However, by using spectroscopic photoacoustic imaging, we take advantage of the wavelength-dependent optical absorption properties of blood. We used a vessel-mimicking phantom with micro blood vessels. The phantom was imaged with intravascular photoacoustic imaging across a range of wavelengths. The image intensities were cross-correlated with the known absorption spectra of blood. The resulting cross-correlation image was able to reveal the location of the artificial blood vessels differentiated from non-blood vessel components.

Photothermal spectroscopy is a powerful tool to investigate the optical absorption and thermal characteristics of a sample. The photo-thermal effect, that is the basis for photothermal spectroscopy, is the conversion of optical energy into heat. Photothermal spectroscopy is implemented in a variety of methods. Biomedical imaging applications commonly implement the Photo-Thermo-Acoustic (PTA) method, that is based on measuring the acoustic pressure wave that propagates due to the photothermal effect, caused by absorption of energy from a heating laser. This research demonstrates photothermal elastic displacement measurement using a coherent confocal microscope, as a first step towered pothothermal spectroscopy. The high accuracy of the interferometer, that is the heart of the coherent confocal microscope, in detecting small changes in position makes it intrinsically adequate to measure the thermoelastic expansion of the sample that results from the photothermal process. In this research Polyvinyl-Chloride Plastisol (PVCP) samples, constructed with different absorption coefficients, were tested using different heating light fluences. The results are compared against an approximate theoretical model and are found to be in good agreement. The Opto-Photo-Thermo-Elastic (Optical detection of elastic displacement changes due to the phototheraml process) technique is demonstrated in this research as a first step toward extending the capability of confocal microscopes to image deeper into tissues than is presently possible, and to detect new modes of contrast.

Recently, we have developed multiphoton excitation-induced photoacoustic imaging for thick tissues employing a 1064-nm nanosecond pulsed laser. The combination of multiphoton excitation and photoacoustic imaging improves the depth resolution. To apply the multiphoton-photoacoustic imaging for precise investigation in living tissues, it is important to enhance only the photoacoustic signals induced by multiphoton excitation, because the generation of multiphotonphotoacoustic signals is less efficient than that of one-photon photoacoustic signals. In this study, we investigated the relation between the signal intensity and the thermophysical properties of various solutions of fluorescent dyes in multiphoton-photoacoustic imaging. We found that the signal intensity is proportional to the coefficient of thermal expansion divided by the specific heat of the solvent. Thus thermophysical properties are also important, together with absorption properties, in enhancing the multiphoton-photoacoustic signal. Based on our findings, we propose the use of gold nanoparticles surrounded by fluorescent dyes as contrast agents. Rhodamine B, which is employed in fluorescent dyes, selectively evokes the two-photon absorption. In addition, because gold nanoparticles have a small specific heat, the multiphoton-photoacoustic signal generated is strong due to effective photon-to-heat conversion. We conclude that this combination allows deeper observation in living tissues by multiphoton-photoacoustic imaging.

Photoacoustic tomography is an imaging technique based on the reconstruction of distribution of acoustic pressure, generated by the absorption of short laser pulses in biological tissues. The detected ultrasound signals can be represented by the convolution of the structure of objects, the laser pulse, and the impulse response of the ultrasound detectors. Detector's wideband response is essential for imaging reconstruction of multiscale objects by utilizing a range of characteristic acoustic wavelengths. Optical detection of ultrasound has the advantage of realizing high-frequency widebandwidth ultrasound detection. Previously we have demonstrated a polymer microring resonator based ultrasound detector with flat spectral response from dc to high frequency, over 90 MHz at -3-dB. By using a reconstruction algorithm to simulate the photoacoustic tomography of microspheres of different sizes, we compared the imaging performance of the microring resonators and piezoelectric transducers. Due to the broadband response, the former was able to faithfully detect both the boundaries that are characteristics of high spatial frequencies and the inner structure consisting primarily of low spatial frequency components. Piezoelectric transducers can only preserve one of the two aspects, depending on the choice of detector's central frequency. Experimental results demonstrate the benefit of broadband response of polymer microring resonators.

To obtain efficient antibacterial photodynamic effect in traumatic injuries such as burns, depth-resolved dosimetry of photosensitizer is required. In this study, we performed dual-wavelength photoacoustic (PA) measurement for rat burned skins injected with a photosensitizer. As a photosensitizer, methylene blue (MB) or porfimer sodium was injected into the subcutaneous tissue in rats with deep dermal burn. The wound was irradiated with red (665 nm or 630 nm) pulsed light to excite photosensitizers and green (532 nm) pulsed light to excite blood in the tissue; the latter signal was used to eliminate blood-associated component involved in the former signal. Acoustic attenuation was also compensated from the photosensitizer-associated PA signals. These signal processing was effective to obtain high-contrast image of a photosensitizer in the tissue. Behaviors of MB and porfimer sodium in the tissue were compared.

Rod shaped gold nanoparticles are synthesized using cetyltriammonium bromide (CTAB) as a major component of growth solutions. This surfactant is toxic to cells, but is at the moment unavoidable when monodisperse and high yield nanorods are to be synthesized. CTAB is found coating side walls of the nanoparticles and plays a role in maintaining colloidal stability. It may be displaced using thiolated PEG which is non-toxic to cells. Here we report on systematic studies of cell viability of such PEGylated nanorods on an SKBR3 cell-line using the MTS assay. These PEGylated particles are characterized using electron microscopy, optical spectroscopy and zeta potential measurements. It is expected that such treatment will be crucial in making nanorods compatible for in vivo biomedical applications.

Ultrasound modulated optical tomography (UOT) is a hybrid technique which combines optical contrast with ultrasound resolution and has shown some potential for early cancer detection, functional and molecular imaging. However, one current problem with this technique is the weak optical modulation signal strength and consequently low Signal-to-Noise Ratio (SNR). In this study, the effect of increasing the amplitude of the ultrasound induced particle displacement on the UOT signal is investigated using a Monte Carlo simulation tool. The simulation software was validated against those reported in the literature and good agreement was achieved. The simulated amplitude of particle displacement was varied from 0.1 to 500nm . The results showed a significant increase in UOT signal with particle displacement for low displacements, followed by saturation when displacement increased beyond a certain level. Further simulations were performed to investigate the saturation by changing the optical wavelength from 400nm to 600nm . The results show that the UOT signal saturates at lower particle displacements for smaller wavelengths. This suggests that the phase variations along a photon path can be large enough to cause cancellation as particle displacement increases. This study is part of ongoing efforts to improve the SNR of UOT through using the large particle displacements created by high amplitude ultrasound and radiation forces.

Photoacoustic imaging systems that utilize small numbers of detectors and iterative reconstruction methods require sensitive calibration of the detector array. For each voxel-detector pair, this includes the time-of-flight, fullwidth-half-maximum, and signal amplitude. The objective of this work was to develop a photoacoustic point source which emitted signal uniformly in all directions such that these features can be precisely characterized to more accurately provide an estimate of the shape and position of an acoustic signal in the imaging volume. The source was placed equidistant from acoustic detectors at different zenith and azimuthal angles from a reference position where the acoustic signal could be captured and analyzed. In the zenith direction, the signal decreased in strength by approximately 32% over the range of angles (up to 67.5°). However, in the azimuthal direction, the signal varied substantially as the source was rotated in a stationary axial position indicating imperfections over the source surface that were created during the fiber polishing procedure. The source was used to characterize time-of-flight, full-width-half-maximum, and signal amplitude at a multitude of locations within the imaging volume. While characterization maps obtained with the point source provided reasonable results, the quality of the source could be improved by constructing a truly hemispherical tip on the fiber optic.

We report here a novel breast cancer scanner using microwave and light excitation and ultrasound detection. This combined thermoacoustic and photoacoustic tomography scanner is a nonionizing low cost system that can potentially provide high-resolution, dual contrast (microwave and light absorption) three dimensional images of the breast. Front breast compression will be used in this scanner to alleviate patient discomfort, experienced in side breast compression during traditional X-ray mammography. This scanner will use dry instead of gel ultrasonic coupling. We have also developed a carbon nanotube-based contrast agent for both thermoacoustic and photoacoustic imaging. In the future, targeted molecular photoacoustic and thermoacoustic imaging should be possible using this contrast agent.

We present a multi-wavelength semiconductor laser source for photoacoustic imaging. We discuss the abilities of the system and its limitations. In detail we analyze how this laser diode system might be used to increase the spectral contrast of ultrasonic systems. In a first test set-up we prove in principle the spectral sensitivity of this device.

Optical resolution photoacoustic microscopy (OR-PAM) possesses optical resolution and reveals endogenous optical absorption contrast, promising to be a valuable tool for in vivo microvascular imaging. In laser dermatology, OR-PAM can provide fruitful structural and functional information about the targeted microvascular lesions, such as their threedimensional (3D) morphology, precise location inside the tissue, and blood oxygenation within single vessels, which will facilitate accurate diagnosis and proper treatment. More importantly, the advantages of noninvasiveness and measurement consistency also permit OR-PAM to monitor the healing process of the laser-surgical wound noninvasively. In this work, we employed OR-PAM to monitor the healing process of microvascular lesions induced by nanosecond-pulsed laser. Our results indicate that OR-PAM could be a very useful tool in laser dermatology and laser microsurgery.

Photoacoustic imaging for biomedical applications has seen significant growth during the past few years. Despite its coherent nature, it possesses a unique advantage to produce images devoid of speckle artifacts. The reason responsible for this salient feature has not been addressed so far. We found this is a direct result of its extraordinary absorption contrast. Our discovery is explained using simulations based on a practical photoacoustic imaging system.

Photoacoustic imaging of living subjects offers high spatial resolution at increased tissue depths compared to purely optical imaging techniques. We have recently shown that intravenously injected single walled carbon nanotubes (SWNTs) can be used as targeted photoacoustic imaging agents in living mice using RGD peptides to target α_vβ₃ integrins. We have now developed a new targeted photoacoustic imaging agent based on SWNTs and Indocyanine Green (SWNT-ICG) with absorption peak at 780nm. The photoacoustic signal of the new imaging agent is enhanced by ~20 times as compared to plain SWNTs. The particles are synthesized from SWNT-RGD that noncovalently attach to multiple ICG molecules through pi-pi stacking interactions. Negative control particles had RAD peptide instead of RGD. We measured the serum stability of the particles and verified that the RGD/RAD conjugation did not alter the particle's absorbance spectrum. Finally, through cell uptake studies with U87MG cells we verified that the particles bind selectively to α_vβ₃ integrin. In conclusion, the extremely high absorption of the SWNT-ICG particles shows great promise for high sensitivity photoacoustic imaging of molecular targets in-vivo. This work lays the foundations for future in-vivo studies that will use the SWNT-ICG particles as imaging agents administered systemically.

Sentinel lymph node biopsy (SLNB) has been widely performed and become the standard procedure for axillary staging in breast cancer patients. In current SLNB, identification of SLNs is prerequisite, and blue dye and/or radioactive colloids are clinically used for mapping. However, these methods are still intraoperative, and especially radioactive colloids based method is ionizing. As a result, SLNB is generally associated with ill side effects. In this study, we have proposed near-infrared Au nanocages as a new tracer for noninvasive and nonionizing photoacoustic (PA) SLN mapping in a rat model as a step toward clinical applications. Au nanocages have great features: biocompatibility, easy surface modification for biomarker, a tunable surface plasmon resonance (SPR) which allows for peak absorption to be optimized for the laser being used, and capsule-type drug delivery. Au nanocage-enhanced photoacoustic imaging has the potential to be adjunctive to current invasive SLNB for preoperative axillary staging in breast cancer patients.

Recently, there has been a growing interest in the development of photoacoustic flow measuring methods aimed to study microvascular blood flow in deep tissue. Here, we describe a method of M-mode photoacoustic flow imaging, where a photoacoustic microscope equipped with a high repetition rate pulsed dye laser is used. As a demonstration, we studied the flow of a diluted suspension of carbon glassy particles in a small tube, and extracted the flow profile. Potentially, the method can be applied to detect the blood flow in microvessels either directly or by injecting optically absorbing particles.

Ultrasound-modulated optical microscopy (UOM) based on a long-cavity confocal Fabry-Perot interferometer (CFPI) [J.Biomed.Opt. 13(5), 0504046, (2008)] is used for real time detection of multiply scattered light modulated by high frequency (30 MHz) ultrasound pulses propagating in an optically strongly scattering medium. In this article, we use this microscope to study the dependence of ultrasound-modulated optical signals on the optical absorption of objects embedded about 3 mm deep in tissue mimicking phantoms. These results demonstrate that the dependence is nearly linear. Most importantly, we imaged blood vasculature and melanin in highly scattering tissue samples from a mouse and a rat. Thus UOM can be used to study the morphology of blood vasculature and blood-associated functional parameters, such as oxygen saturation.