This PDF file contains the front matter associated with SPIE Proceedings Volume 8581, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Detection of tumor metastases in the lymphatic system is essential for accurate staging of various malignancies, however fast, accurate and cost-effective intra-operative evaluation of the nodal status remains difficult to perform with common available medical imaging techniques. In recent years, numerous studies have confirmed the additional value of superparamagnetic iron oxide dispersions (SPIOs) for nodal staging purposes, prompting the clearance of different SPIO dispersions for clinical practice. We evaluate whether a combination of photoacoustic (PA) imaging and a clinically approved SPIO dispersion, could be applied for intra-operative nodal staging. Metastatic adenocarcinoma was inoculated in Copenhagen rats for 5 or 8 days. After SPIO injection, the lymph nodes were photoacoustically imaged both in vivo and ex vivo whereafter imaging results were correlated with MR and histology. Results were compared to a control group without tumor inoculation. In the tumor groups clear irregularities, as small as 1 mm, were observed in the PA contrast pattern of the nodes together with an decrease of PA response. These irregularities could be correlated to the absence of contrast in the MR images and could be linked to metastatic deposits seen in the histological slides. The PA and MR images of the control animals did not show these features. We conclude that the combination of photoacoustic imaging with a clinically approved iron oxide nanoparticle dispersion is able to detect lymph node metastases in an animal model. This approach opens up new possibilities for fast intra-operative nodal staging in a clinical setting.

We report on findings from the clinical feasibility study of the Imagio^TM. Breast Imaging System, which acquires two-dimensional opto-acoustic (OA) images co-registered with conventional ultrasound using a specialized duplex hand-held probe. Dual-wavelength opto-acoustic technology is used to generate parametric maps based upon total hemoglobin and its oxygen saturation in breast tissues. This may provide functional diagnostic information pertaining to tumor metabolism and microvasculature, which is complementary to morphological information obtained with conventional gray-scale ultrasound. We present co-registered opto-acoustic and ultrasonic images of malignant and benign tumors from a recent clinical feasibility study. The clinical results illustrate that the technology may have the capability to improve the efficacy of breast tumor diagnosis. In doing so, it may have the potential to reduce biopsies and to characterize cancers that were not seen well with conventional gray-scale ultrasound alone.

There remains an urgent need to develop effective photoacoustic computed tomography (PACT) image recon- struction methods for use with acoustically inhomogeneous media. Transcranial PACT brain imaging is an im- portant example of an emerging imaging application that would benefit greatly from this. Existing approaches to PACT image reconstruction in acoustically heterogeneous media are limited to weakly varying media, are computationally burdensome, and/or make impractical assumptions regarding the measurement geometry. In this work, we develop and investigate a full-wave approach to iterative image reconstruction in PACT for media possessing inhomogeneous speed-of-sound and mass density distributions. A key contribution of the work is the formulation of a procedure to implement a matched discrete forward and backprojection operator pair, which facilitates the application of a wide range of modern iterative image reconstruction algorithms. This presents the opportunity to employ application-specific regularization methods to mitigate image artifacts due to mea- surement data incompleteness and noise. Our results establish that the proposed image reconstruction method can effectively compensate for acoustic aberration and reduces artifacts in the reconstructed image.

We have developed a real-time (8~30 fps) photoacoustic (PA) imaging system with a linear-array transducer for burn diagnosis. In this system, PA signals originating from blood in the noninjured tissue layer located under the injured tissue layer are detected. The phantom study showed that thin light absorbers embedded in the tissue-mimicking scattering medium at depths of > 3 mm can be imaged with high contrast. The diagnostic experiments using rat burn models showed good agreements between the injury depths (zones of stasis) indicated by PA imaging and those by histological analysis. These results demonstrate the potential usefulness of the present system for clinical burn diagnosis.

Recently we demonstrated the feasibility of Optical-Resolution Photoacoustic Micro-Endoscopy (OR-PAME) using an image guide fiber. However, the use of an ultrasound transducer for signal collection limited useful applications. We demonstrate detection of OR-PAM signals using an external array transducer in order to make endoscopic imaging practical for clinical use for the first time. The array system is able to visualize the placement of the image-guide fiber using pulse-echo ultrasound then switch to an OR-PAME acquisition mode. Photoacoustic signals are captured by a Verasonics ultrasound system using an L7-4 linear array transducer. A high-repetition-rate 532-nm fiber laser was used as the excitation source. This light was focused and raster­ scanned into a 800m-diameter image-guide fiber bundle consisting of 30,000 individual fiber elements. The operator finds the end of the endoscope using a flash ultrasound imaging mode, then captures endoscopic data by clicking a button. This activates the motion of scanning mirrors into the end of the image guide, and engages an endoscopic capture sequence. Endoscopic data are used to form a maximum amplitude image by simply taking the maximum of the absolute value of the signal across the 64 center channel lines used for capture. Using this technique, we have captured images of carbon fibers with a resolution of 6 microns at an SNR of greater than 30dB. Electronic focusing is expected to improve the SNR. The use of an ultrasound array transducer for both endoscope guidance and data collection allows for a much smaller endoscope footprint while opening up clinical possibilities.

We performed a photoacoustic endoscopic imaging study of melanoma tumor growth in a nude rat in vivo. After inducing the tumor at the colorectal wall of the animal, we monitored the tumor development in situ by using a photoacoustic endoscopic system. This paper introduces our experimental method for tumor inoculation and presents imaging results showing the morphological changes of the blood vasculature near the tumor region according to the tumor progress. Our study could provide insights for future studies on tumor development in small animals.

The difficulty in delineating brain tumor margins is a major obstacle in the path toward better outcomes for patients with brain tumors. Current imaging methods are often limited by inadequate sensitivity, specificity and spatial resolution. Here we show that a unique triplemodality magnetic resonance imaging - photoacoustic imaging - Raman imaging nanoparticle (termed here MPR nanoparticles), can accurately help delineate the margins of brain tumors in living mice both preoperatively and intraoperatively. The MPRs were detected by all three modalities with at least a picomolar sensitivity both in vitro and in living mice. Intravenous injection of MPRs into glioblastoma-bearing mice led to MPR accumulation and retention by the tumors, with no MPR accumulation in the surrounding healthy tissue, allowing for a noninvasive tumor delineation using all three modalities through the intact skull. Raman imaging allowed for guidance of intraoperative tumor resection, and a histological correlation validated that Raman imaging was accurately delineating the brain tumor margins. This new triple-modality– nanoparticle approach has promise for enabling more accurate brain tumor imaging and resection.

Identifying neovascularity, i.e. angiogenesis, as a feature of inflammatory arthritis, can help in early diagnosis and treatment monitoring of this disease. Photoacoustic tomography (PAT), as a hybrid imaging modality, relies on intrinsic differences in the optical absorption among the tissues being imaged. Since blood has highly absorbing chromophores including both oxygenated and deoxygenated hemoglobin, PAT holds potential in identifying early angiogenesis associated with inflammatory joint diseases. In this study, we used PAT to identify the changes in the development of inflammatory arthritis, through the study on a well-established adjuvant-induced arthritis (AIA) rat model. Imaging at two different wavelengths, 1064 nm and 532 nm, revealed that there was a significant signal enhancement in the ankle joints of the arthritis affected rats when compared to the normal control group. Histological analysis of both the normal and the arthritic rats correlated well with the imaging findings. The results from this study suggest that the emerging PAT technology could become a new tool for clinical management of inflammatory joint diseases.

pH plays a critical role in many aspects of cell and tissues physiology. Lower pH is also a typical characteristic of arthritic joints and tumor tissues. These pH anomalies are also exploited in different drug delivery mechanisms. Here we present, a new method of pH sensing in vivo using spectroscopic photoacoustic measurements facilitated by pH sensitive nanosensors. The nanosensors consist of Seminaphtharhodafluor (SNARF), a pH sensitive dye, encapsulated in a specially designed polyacrylamide hydrogel matrix with a hydrophobic core. The photoacoustic intensity ratio between the excitation wavelengths of 585nm and 565nm increases in the pH range from 6.0 to 8.0 and is used to determine the pH of the local environment. These nanosensors are biodegradable, biocompatible, have a long plasma lifetime and can be targeted to any type of cells or tissues by surface modification using proper targeting moieties. The encapsulation of the dye prevents the interaction of the dye with proteins in plasma and also reduces the dye degradation. The SNARF dye in its free form loses 90% of its absorbance in presence of albumin, a protein found in abundance in plasma, and this has severely limited its adaptation to in vivo environments. In comparison, the SNARF nanosensors lose only 16% of their absorbance in the same environment. We employ these nanosensors to demonstrate the feasibility of pH sensing in vivo through photoacoustic measurements on a rat joint model.

We explored the potential of an emerging laser-based technology, photoacoustic imaging (PAI), for bladder cancer diagnosis through high resolution imaging of microvasculature in the interior bladder tissues. Images of ex vivo canine bladders demonstrated the excellent ability of PAI to map three-dimensional microvasculature in optically scattering bladder tissues. By comparing the results from human bladder specimens affected by cancer to those from the normal control, the feasibility of PAI in differentiating malignant from benign bladder tissues was explored. The reported distinctive morphometric characteristics of tumor microvasculature can be seen in the images from cancer samples, suggesting that PAI may allow in vivo assessment of neoangiogenesis that is closely associated with bladder cancer generation and progression. By presenting subsurface morphological and physiological information in bladder tissues, PAI, when performed in a similar way to that in conventional endoscopy, provides an opportunity for improved diagnosis, staging and treatment guidance of bladder cancer.

Due to the wide use of animals for human disease studies, small animal whole-body imaging plays an increasingly important role in biomedical research. Currently, none of the existing imaging modalities can provide both anatomical and glucose metabolic information, leading to higher costs of building dual-modality systems. Even with image coregistration, the spatial resolution of the metabolic imaging modality is not improved. We present a ring-shaped confocal photoacoustic computed tomography (RC-PACT) system that can provide both assessments in a single modality. Utilizing the novel design of confocal full-ring light delivery and ultrasound transducer array detection, RC-PACT provides full-view cross-sectional imaging with high spatial resolution. Scanning along the orthogonal direction provides three-dimensional imaging. While the mouse anatomy was imaged with endogenous hemoglobin contrast, the glucose metabolism was imaged with a near-infrared dye-labeled 2-deoxyglucose. Through mouse tumor models, we demonstrate that RC-PACT may be a paradigm shifting imaging method for preclinical research.

Optoacoustic imaging offers the unique capability of simultaneous excitation of a three-dimensional (volumetric) region with a single interrogating laser pulse. In this way, three-dimensional imaging with single-shot illumination is theoretically achievable, which in principle allows the visualization of dynamic events at a high frame rate mainly limited by the pulse repetition rate of the laser. Simultaneous acquisition of optoacoustic signals at a set of points surrounding the imaging sample is however required for this purpose, which is hampered by several technical limitations related to lack of appropriate ultrasound detection technology, digital sampling and processing capacities. Also, a convenient reconstruction algorithm must be selected to accurately image the distribution of the optical absorption from the acquired signals. Specifically, the resolution and quantitativeness of the images depend on the reconstruction procedure employed. Herein we describe an accurate three-dimensional model-based optoacoustic reconstruction algorithm based on a convenient discretization of the analytical solution of the forward model. Subsequent algebraic inversion is done with the LSQR algorithm. The performance of the algorithm is showcased by reconstructing an excised mouse heart with a custom made three-dimensional optoacoustic imaging system. In this system, 256 optoacoustic signals corresponding to single-shot excitation are simultaneously collected with an array of ultrasonic transducers disposed on a spherical surface, which allows three-dimensional imaging at a frame rate of 10 Hz.

A photoacoustic (PA) and ultrasound (US) dual modality system for imaging human peripheral joints is introduced. The system utilizes a commercial US unit for both US control imaging and PA signal acquisition. Preliminary in vivo evaluation of the system on normal volunteers revealed that this system can recover both the structural and functional information of intra- and extra-articular tissues. Presenting both morphological and pathological information in joint, this system holds promise for diagnosis and characterization of inflammatory joint diseases such as rheumatoid arthritis.

In this work, we introduce a novel three-dimensional imaging system for in vivo high-resolution anatomical and functional whole-body visualization of small animal models developed for preclinical or other type of biomedical research. The system (LOUIS-3DM) combines a multi-wavelength optoacoustic and ultrawide-band laser ultrasound tomographies to obtain coregistered maps of tissue optical absorption and acoustic properties, displayed within the skin outline of the studied animal. The most promising applications of the LOUIS-3DM include 3D angiography, cancer research, and longitudinal studies of biological distribution of optoacoustic contrast agents (carbon nanotubes, metal plasmonic nanoparticles, etc.).

We propose the further development of the optical detection setup towards photoacoustic (PA) and ultrasound (US) dual-modality section imaging. Both imaging modalities use optical generation and detection of ultrasound waves. A onesided chrome coated concave cylindrical optical lens is used as target to induce acoustic signals for US imaging and as acoustic mirror that forms acoustic images. By probing the temporal evolution of the acoustic images with an optical beam perpendicular to the acoustic axis and simultaneously rotating the object, data for reconstruction of PA and US slice images are acquired. All acoustic signals are excited optically via the thermoelastic effect using laser pulses coming from the same laser system.

Photoacoustic Doppler flowmetry as well as Doppler ultrasound were performed in acoustic resolution regime on tubes filled with flowing blood with indocyanine green (ICG) at different concentrations. The photoacoustic excitation utilized a pair of directly-modulated fiber-coupled 830nm laser-diodes, modulated with either CW or tone-bursts for depthresolved measurements. The amplitude of the Doppler peak in photoacoustic Doppler measurements was found to be proportional to the ICG concentration. Photoacoustic Doppler was measured in ICG at human safe concentrations, but not in whole blood. Comparing the results between the two modalities implied that using a wavelength with higher optical absorption may improve the photoacoustic signal in blood.

Optical excitation based imaging modalities, with aim to image structures deep inside the scattering medium, suffer from quantification problem. We propose a methodology to solve the problem of non-invasively mapping the fluence in optically heterogeneous medium without the need of prior knowledge of its optical properties. We present a theoretical model of our concept and provide proof of principle with Monte Carlo simulations. Simulation results show that it is possible to measure the local light fluence in highly scattering medium in absolute terms. Furthermore, we performed an experiment to validate the concept as a strategy to measure local fluence in relative manners. We used reflection mode acousto optics (AO) in our experiment, and showed that with this method we can measure local light fluence (in relative term) in highly scattering medium.

Quantitative photoacoustic tomography involves the construction of a photoacoustic image from surface measurements of photoacoustic wave pulses and the recovery of the optical properties of the imaged region. This is a nonlinear, ill-posed inverse problem, for which model-based inversion techniques have been proposed. Here, the radiative transfer equation is used to model the light propagation, and the acoustic propagation and image reconstruction are included. In other words, the full quantitative inversion is tackled. Since Newton-based minimisations are impractical when dealing with three-dimensional images, an adjoint-assisted gradient-based inversion was used as a practical alternative to determining the optical coefficients.

Genetically expressed contrast agents are of great interest in the life sciences as they allow the study of structure and function of living cells and organisms. However, many commonly used fluorescent proteins present disadvantages when used in mammalian organisms, such as low near-infrared absorption and photostability. In this study, a variety of genetically expressed fluorescent proteins and novel chromoproteins were evaluated using photoacoustic spectroscopy. The results showed that chromoproteins provide stronger photoacoustic signals, better spectral stability, and exhibit less photobleaching than fluorescent proteins.

Gold nanorods exhibit intense optical absorbance in the near-infrared region of principal interest for applications in biomedical optics, which evokes their use to improve contrast in photoacoustic imaging and selective photothermolysis of cancer. However their limited photostability remains a drawback of practical concern. In particular, when GNRs are irradiated with nanosecond laser pulses in resonance with their plasmon oscillations, there may occur phenomena like reshaping into spherical particles, as well as fragmentation at higher optical fluences, which result into dramatic modifications of their optical absorption bands and substantial loss of photoacoustic conversion efficiency. In this contribution we present an experimental investigation of stability and photoacoustic conversion efficiency from gold nanorods embedded in biomimetic scaffolds.

A new technique using pulsed laser heating of a nanocomposite contrast agent resulting in local bubble formation and concomitant harmonic generation in a scattered probe ultrasound (US) beam is proposed to increase specific contrast in both US imaging and laser-induced photoacoustic (PA) imaging. The composite combines an emulsion bead core with amphiphilic gold nanospheres (GNSs) assembled at the interface. Clustered GNSs result in a broadened absorption spectrum in the near infrared range (700-1000 nm) compared to the typical 520 nm peak of distributed GNSs, enabling their use at depth in tissue. Illuminating the composite with a pulsed laser with appropriately chosen parameters heats the composite through optical absorption by the GNSs and results in a phase transition of the emulsion bead to form a transient bubble. By delivering a probe US pulse simultaneously, or immediately after the laser pulse is delivered, harmonic signals are produced in the scattered US beam. The results show that a residual signal created by subtracting a US signal from the simultaneous US/laser probing signal of the emulsion bead sample is 1.7 dB higher than the laser alone generated PA signal and 20 dB higher than the PA signal of a control homogeneous GNSs dispersion with the same optical absorption, indicating the nonlinear contrast enhancement from bubble dynamics. The proposed technique of local activation of this designed contrast agent can be used to dramatically enhance both the specificity and sensitivity of integrated US/PA molecular imaging.

The ultrasound detector is the heart of a photoacoustic imaging system. In photoacoustic imaging of the breast there is a requirement to detect tumors located a few centimeters deep in tissue, where the light is heavily attenuated. Thus a sensitive ultrasound transducer is of crucial importance. As the frequency content of photoacoustic waves are inversely proportional to the dimensions of the absorbing structures, and in tissue can range from hundreds of kHz to tens of MHz, a broadband ultrasound transducer is required centered on an optimum frequency. A single element piezoelectric transducer structurally consists of the active piezoelectric material, front- and back-matching layers and a backing layer. To have both high sensitivity and broad bandwidth, the materials, their acoustic characteristics and their dimensions should be carefully chosen. In this paper, we present design considerations of an ultrasound transducer for imaging the breast such as the detector sensitivity and frequency response, which guides the selection of active material, matching layers and their geometries. We iterate between simulation of detector performance and experimental characterization of functional models to arrive at an optimized implementation. For computer simulation, we use 1D KLM and 3D finite-element based models. The optimized detector has a large-aperture possessing a center frequency of 1 MHz with fractional bandwidth of more than 80%. The measured minimum detectable pressure is 0.5 Pa, which is two orders of magnitude lower than the detector used in the Twente photoacoustic mammoscope.

Piezoelectric ultrasound (US) transducers are at the heart of almost any ultrasonic medical imaging probe. However, their sensitivity and reliability severely degrade in applications requiring high frequency (>20 MHz) and small element size (<0.1 mm). Alternative technologies such as capacitive micromachined ultrasound transducers (CMUT) and optical sensing and generation of ultrasound are being investigated. In this paper we present our first steps in developing optical micromachined ultrasound transducers (OMUT) technology. OMUTs rely on microfabrication techniques to construct micron-size air cavities capped by an elastic membrane. The membrane functions as the active ultrasound transmitter and receiver. We will describe the design and testing of prototype OMUT devices which implement a receive-only function. The cavity detector is an optical cavity which its top mirror is deflected under the application of pressure. The intensity of a reflected light beam is highly sensitive to displacement of the top membrane if the optical wavelength is at near-resonance condition. Therefore, US pulses can be detected by recording the reflected light intensity. The sensitivity of the device depends on the mechanical properties of the top membrane and optical characteristics of the optical cavity. The device was fabricated using SU8 as a structural material and gold as a mirror. We have developed a new bonding method to fabricate a sealed, low roughness, high quality optical cavity. The 60μm cavity with the 8.5 μm top membrane is tested in water with 25MHz ultrasound transducer. The NEP of the device for bandwidth of 28MHz was 9.25kPa. The optical cavity has a finesse of around 23.

We present results of a novel and highly sensitive technique for the optical detection of ultrasound using the selective storage of frequency shifted photons in an inherently highly efficient and low noise atomic frequency comb (AFC) based quantum memory. The ultrasound ‘tagged’ optical sidebands are absorbed within a pair of symmetric AFCs, generated via optical pumping in a Pr³+:Y₂SiO₅ sample (tooth separation Δ = 150 kHz, comb finesse f_c ~ 2 and optical depth αL ~ 2), separated by twice the ultrasound modulation frequency (1.5 MHz) and centered on either side of a broad spectral pit (1.7 MHz width) allowing transmission of the carrier. The stored sidebands are recovered with 10-20% efficiency as a photon echo (as defined by the comb parameters), and we demonstrate a record 49 dB discrimination between the sidebands and the carrier pulse, high discrimination being important for imaging tissues at depth. We further demonstrate detector limited discrimination (~29 dB) using a highly scattered beam, confirming that the technique is immune to speckle decorrelation. We show that it also remains valid in the case of optically thin samples, and thus represents a significant improvement over other ultrasound detection methods based on rare-earth-ion-doped crystals. These results strongly suggest the suitability of our technique for high-resolution non-contact real-time imaging of biological tissues.

A novel photoacoustic thermometric method is presented for simultaneously imaging cells and sensing their temperature. With 3 seconds per frame imaging speed, a temperature resolution of 0.2 °C was achieved in a photo-thermal cell heating experiment. Compared to other approaches, the photoacoustic thermometric method has the advantage of not requiring custom-developed temperature-sensitive biosensors. This feature should facilitate the conversion of single-cell thermometry into a routine lab tool and make it accessible to a much broader biological research community.

Recently, a number of optical imaging modalities have achieved single molecule sensitivity, including photothermal imaging, stimulated emission microscopy, ground state depletion microscopy, and transmission microscopy. These optical techniques are based on optical absorption contrast, extending single-molecule detection to non-fluorescent chromophores. Photoacoustics is a hybrid technique that utilizes optical excitation and ultrasonic detection, allowing it to scale both the optical and acoustic regimes with 100% sensitivity to optical absorption. However, the sensitivity of photoacoustics is limited by thermal noise, inherent in the medium itself in the form of acoustic black body radiation. In this paper, we investigate the molecular sensitivity of photoacoustics in the context of the thermal noise limit. We show that single molecule sensitivity is achievable theoretically at room temperature for molecules with sufficiently fast relaxation times. Hurdles to achieve single molecule sensitivity in practice include development of detection schemes that work at short working distance, <100 microns, high frequency, <100 MHz, and low loss, <10 dB.

Achieving real-time photoacoustic (PA) tomography typically requires massive ultrasound transducer arrays and data acquisition (DAQ) electronics to receive PA waves simultaneously. In this paper, we report the first demonstration of a photoacoustic tomography (PAT) system using optical fiber-based parallel acoustic delay lines (PADLs). By employing PADLs to introduce specific time delays, the PA signals (on the order of a few micro seconds) can be forced to arrive at the ultrasonic transducers at different times. As a result, time-delayed PA signals in multiple channels can be ultimately received and processed in a serial manner with a single-element transducer, followed by single‐channel DAQ electronics. Our results show that an optically absorbing target in an optically scattering medium can be photoacoustically imaged using the newly developed PADL-based PAT system. Potentially, this approach could be adopted to significantly reduce the complexity and cost of ultrasonic array receiver systems.

Almost all known photoacoustic image reconstruction algorithms are based on the assumption that the acoustic waves leave the object (the imaged region) after a finite time. This assumption is fulfilled if the measurements are made in free space and reflections from the detectors are negligible. However, when the object is surrounded by acoustically hard detectors arrays (and/or by additional acoustic mirrors), the acoustic waves will bounce around in such a reverberant cavity many times (in the absence of absorption, forever). This paper proposes fast reconstruction algorithms for the measurements made from the walls of a rectangular reverberant cavity. The algorithms are tested using numerical simulations.

Ocular imaging plays a key role for the diagnosis of various ocular diseases. In this work, we have developed an ocular imaging system based on the photoacoustic tomography. This system has successfully imaged the entire eye of a mouse, from its iris to the retina region, and the imaging is label-free and non-invasively. The resolution of this system reaches several micron meters, allowing the study of microstructures in various ocular tissues. Our system has the potential to be a powerful non-invasive imaging method for the ophthalmology.

Semiconductor light sources, such as laser diodes or light emitting diodes (LEDs) could provide an inexpensive and compact alternative to traditional Q-switched lasers for photoacoustic imaging. So far, only laser diodes ^1-3 operating in the 750 to 905nm wavelength range have been investigated for this purpose. However, operating in the visible wavelength range (400nm to 650nm) where blood is strongly absorbent (<10cm^-1) and water absorption is weak (<0.01cm^-1) could allow for high contrast photoacoustic images of the superficial vasculature to be achieved. High power laser diodes (<10Watt peak power) are however not available in this wavelength range. High power LEDs could be a potential alternative as they are widely available in the visible wavelength range (400nm to 632nm) and relatively cheap. High power LEDs are generally operated in continuous wave mode and provide average powers of several Watts. The possibility of over driving them by tens of times their rated current when driven at a low duty cycle (<1%), offers the prospect of achieving similar pulse energies (tens of μJ) to that provided by high peak power pulsed laser diodes. To demonstrate the possibility of using high power LEDs as an excitation source for biomedical applications, single point measurements were implemented in a realistic blood vessel phantom. A four colour device was also used to demonstrate the possibility of using LEDs for making spectroscopic measurements. It was shown that when driving all four wavelengths at once, the generated photoacoustic signal could be used to design a filter in order to improve the SNR of the photoacoustic signals generated at each individual wavelength. The possibility of acquiring multiwavelength data sets simultaneously when using Golay excitation methods was also demonstrated. This preliminary study demonstrated the potential for using high power LEDs as an inexpensive and compact excitation source for biomedical photoacoustics.

Time-reversed ultrasonically encoded (TRUE) optical focusing focuses light beyond one transport mean free path by phase-conjugating the ultrasonically tagged light. However, in previous works, only a small portion of the tagged light was phase-conjugated by using a photorefractive Bi₁₂SiO₂₀ crystal, due to its small active area (1x1 cm²). In this work, we report high-efficiency TRUE focusing using a large-area photorefractive polymer (5x5 cm²), which demonstrated ~40 times increase in focused energy. Further, we imaged absorbers embedded in a turbid sample of thickness of ~12 transport mean free paths.

X-ray induced photoacoustic tomography, also called X-ray acoustic computer tomography (XACT) is investigated in this paper. Short pulsed (μs-range) X-ray beams from a medical linear accelerator were used to generate ultrasound. The ultrasound signals were collected with an ultrasound transducer (500 KHz central frequency) positioned around an object. The transducer, driven by a computer-controlled step motor to scan around the object, detected the resulting acoustic signals in the imaging plane at each scanning position. A pulse preamplifier, with a bandwidth of 20 KHz–2 MHz at −3 dB, and switchable gains of 40 and 60 dB, received the signals from the transducer and delivered the amplified signals to a secondary amplifier. The secondary amplifier had bandwidth of 20 KHz–30 MHz at −3 dB, and a gain range of 10–60 dB. Signals were recorded and averaged 128 times by an oscilloscope. A sampling rate of 100 MHz was used to record 2500 data points at each view angle. One set of data incorporated 200 positions as the receiver moved 360°. The x-ray generated acoustic image was then reconstructed with the filtered back projection algorithm. The twodimensional XACT images of the lead rod embedded in chicken breast tissue were found to be in good agreement with the shape of the object. This new modality may be useful for a number of applications, such as providing the location of a fiducial, or monitoring x-ray dose distribution during radiation therapy.

Photoacoustic imaging employing molecular overtone vibration as contrast mechanism opens a new avenue for deep tissue imaging with chemical bond selectivity. Here, we demonstrate vibration-based photoacoustic tomography with an imaging depth on the centimeter scale. To provide sufficient pulse energy at the overtone transition wavelengths, we constructed a compact, barium nitrite crystal-based Raman laser for excitation of 2^nd overtone of C-H bond. Using a 5-ns Nd:YAG laser as pumping source, up to 105 mJ pulse energy at 1197 nm was generated. Vibrational photoacoutic spectroscopy and tomography of phantom (polyethylene tube) immersed in whole milk was performed. With a pulse energy of 47 mJ on the milk surface, up to 2.5 cm penetration depth was reached with a signal-to-noise ratio of 12.

Photoacoustic Thermal Diffusion Flowmetry (PA-TDF) utilizes photothermal heating and photoacoustic temperature monitoring to measure the tissue heat clearance time constants from which blood velocity can be inferred. We extended our study of PA-TDF to tissue-mimicking phantoms with vessels at various diameters, configurations and depths and experimentally verified the relations between the estimated time constants and the vessels and the illuminating beam dimensions. We also demonstrated, for the first time, depth-resolved PA-TDF measurement using tone-burst photoacoustic excitation. The excitation utilized two fiber-coupled 830nm laser diodes, one induced slow temperature oscillations and the other induced the PA excitation.

The high optoacoustic resolution at depths beyond the diffusive limit of light stems from the low scattering of sound, as compared to photons, within biological tissues. However, some biological samples contain strongly mismatched tissues such as bones or lungs that generally produce acoustic reflections and scattering, and image distortion is consequently produced by assuming an acoustically homogeneous medium. We describe herein a statistical procedure to modify the reconstruction algorithms in order to avoid such distortion. The procedure is based on weighting the contribution of the collected optoacoustic signals to the reconstruction with the probability that they are not affected by reflections or scattering. A rough estimation of such probability by considering an area enclosing the sample allows significantly reducing the artefacts associated to acoustic distortion. Furthermore, the available structural information of the imaging sample can be incorporated in the estimation of the distortion probability, in a way that a further improvement in the quality of the reconstructed images is achieved. The benefit of the reconstruction procedure described herein is showcased by reconstructing tissue mimicking phantoms containing air-gaps. In all cases, the image artefacts produced when no weighting is done are significantly reduced.

Hypoxia is a condition where a region of tissue has less than adequate oxygen. It is of particular importance in tumor biology, as the hypoxic core of tumors has been shown to impede the effectiveness of many therapies. We demonstrate a novel method for oxygen sensing in vivo, based on the photoacoustic lifetime measurement of an oxygen sensitive probe. The experimental results derived from the main artery in the rat tail indicated that the lifetime of the probe, quantified by the photoacoustic measurement, shows a good linear relationship with the blood oxygenation level in the targeted artery.

Hemodynamic responses of the brain to hypoxia or ischemia are one of the major interests in neurosurgery and neuroscience. In this study, we performed real-time transcutaneous PA imaging of the rat brain that was exposed to a hypoxic stress and investigated depth-resolved responses of the brain, including the hippocampus. A linear-array 8ch 10-MHz ultrasonic sensor (measurement length, 10 mm) was placed on the shaved scalp. Nanosecond, 570-nm and 595- nm light pulses were used to excite PA signals indicating cerebral blood volume (CBV) and blood deoxygenation, respectively. Under spontaneous respiration, inhalation gas was switched from air to nitrogen, and then reswitched to oxygen, during which real-time PA imaging was performed continuously. High-contrast PA signals were observed from the depth regions corresponding to the scalp, skull, cortex and hippocampus. After starting hypoxia, PA signals at 595 nm increased immediately in both the cortex and hippocampus for about 1.5 min, showing hemoglobin deoxygenation. On the other hand, PA signals at 570 nm coming from these regions did not increase in the early phase but started to increase at about 1.5 min after starting hypoxia, indicating reactive hyperemia to hypoxia. During hypoxia, PA signals coming from the scalp decreased transiently, which is presumably due to compensatory response in the peripheral tissue to preserve blood perfusion in the brain. The reoxygenation caused a gradual recovery of these PA signals. These findings demonstrate the usefulness of PA imaging for real-time, depth-resolved observation of cerebral hemodynamics.

Oxygen plays a key role in the energy metabolism of living organisms. Any imbalance in the oxygen levels will affect the metabolic homeostasis and lead to pathophysiological diseases. Hypoxia, a status of low tissue oxygen, is a key factor in tumor biology as it is highly prominent in tumor tissues. However, clinical tools for assessing tissue oxygenation are limited. The gold standard is polarographic needle electrode which is invasive and not capable of mapping (imaging) the oxygen content in tissue. We applied the method of photoacoustic lifetime imaging (PALI) of oxygen-sensitive dye to small animal tissue hypoxia research. PALI is new technology for direct, non-invasive imaging of oxygen. The technique is based on mapping the oxygen-dependent transient optical absorption of Methylene Blue (MB) by pump-probe photoacoustic imaging. Our studies show the feasibility of imaging of dissolved oxygen distribution in phantoms. In vivo experiments demonstrate that the hypoxia region is consistent with the site of subcutaneously xenografted prostate tumor in mice with adequate spatial resolution and penetration depth.

We report the development of photoacoustic microscopy capable of video-rate high-resolution in-vivo imaging in deep tissue. A lightweight photoacoustic probe is made of a single-element broadband ultrasound transducer, a compact photoacoustic beam combiner, and a bright-field light delivery system. Focused broadband ultrasound detection provides a 44-μm lateral resolution and a 28-μm axial resolution. A multimode optical fiber is used to deliver laser pulses. The bright-field light delivery system can effectively improve the illumination efficiency. The photoacoustic probe weighs less than 40 grams and is mounted on a voice-coil scanner to acquire 40 cross-sectional images per second over several-mm range. The fast speed can effectively improve imaging throughput, reduce motion artifacts, and enable the visualization of highly dynamic biomedical processes. High-resolution micro-vascular imaging is successfully demonstrated.

Blood flow measurements have been demonstrated using the acoustic resolution mode of photoacoustic sensing. This is unlike previous flowmetry methods using the optical resolution mode, which limits the maximum penetration depth to approximately 1mm. Here we describe a pulsed time correlation photoacoustic Doppler technique that is inherently flexible, lending itself to both resolution modes. Doppler time shifts are quantified via cross-correlation of pairs of photoacoustic waveforms generated in moving absorbers using pairs of laser light pulses, and the photoacoustic waves detected using an ultrasound transducer. The acoustic resolution mode is employed by using the transducer focal width, rather than the large illuminated volume, to define the lateral spatial resolution. The use of short laser pulses allows depth-resolved measurements to be obtained with high spatial resolution, offering the prospect of mapping flow within microcirculation. Whilst our previous work has been limited to a non-fluid phantom, we now demonstrate measurements in more realistic blood-mimicking phantoms incorporating fluid suspensions of microspheres flowing along an optically transparent tube. Velocities up to 110 mm/s were measured with accuracies approaching 1% of the known velocities, and resolutions of a few mm/s. The velocity range and resolution are scalable with excitation pulse separation, but the maximum measurable velocity was considerably smaller than the value expected from the detector focal beam width. Measurements were also made for blood flowing at velocities up to 13.5 mm/s. This was for a sample reduced to 5% of the normal haematocrit; increasing the red blood cell concentration limited the maximum measurable velocity so that no results were obtained for concentrations greater than 20% of a physiologically realistic haematocrit. There are several possible causes for this limitation; these include the detector bandwidth and irregularities in the flow pattern. Better results are obtained using a detector with a higher centre frequency and larger bandwidth and tubes with a narrower diameter.

Photoacoustic imaging is exquisitely sensitive to blood and can infer blood oxygenation based on multispectral images. In this work we present multispectral real-time 3D photoacoustic imaging of blood phantoms. We used a custom-built 128-channel hemispherical transducer array coupled to two Nd:YAG pumped OPO laser systems synchronized to provide double pulse excitation at 680 nm and 1064 nm wavelengths, all during a triggered series of ultrasound pressure measurements lasting less than 300 μs. The results demonstrated that 3D PAI is capable of differentiating between oxygenated and deoxygenated blood at high speed at mm-level resolution.

This paper investigates the application of the vessel filter proposed by Frangi et al., [MICCAI, LNCS vol. 1496, pp. 130-137, 1998] to photoacoustic images of the vasculature. The filter works by classifying the eigenvalue decomposition of the local Hessian matrix at each image voxel to find tubular structures in the image. A detailed analysis of the algorithm is provided, and the effect of the filters on photoacoustic images is studied using numerical and experimental phantoms. In particular, the impact of the filter on image resolution, feature preservation, and noise is discussed. The vessel filter is then applied to photoacoustic images of the vasculature in mice. The classical Hessian filter is shown to be highly effective at removing noise and highlighting vessels, at the expense of reducing the sharpness of vessel edges.

Gold-nanorods incorporated with microbubbles (AuMBs) were introduced as a photoacoustic/ultrasound dual- modality contrast agent in our previous study. The application can be extended to theragnosis purpose. With the unique physical characteristics of AuMBs, we propose an enhanced delivery method for the encapsulated particles. For example, laser thermotherapy mediated by plasmonic nanoparticles can be made more effective by using microbubbles as a targeted carrier and acoustic cavitation for enhanced sonoporation. The hypothesis was experimentally tested. Firts, these AuMBs first act as molecular probes with binding to specific ligands. The improved targeting efficacy was macroscopically observed by an ultrasound system. The extended retention of targeted AuMB was observed and recorded for 30 minutes in a CT-26 tumor bearing mouse. Secondly, cavitation induced by time-varying acoustic field was also applied to disrupt the microbubbles and cause increased transient cellular permeability (a.k.a., sonoporation). Multimodal optical microscope based on a Cr:forsterite laser was used to directly observe these effects. The microscope can acquired third-harmonic generation (THG) and two-photon fluorescent (2PF) signals produced by the AuMBs. In vitro examination shows approximately a 60% improvement in terms of fluorescence signals from the cellular uptake of gold nanoparticles after sonoporation treatment. Therefore, we conclude that the controlled release is feasible and can further improve the therapeutic effects of the nanoparticles.

Existing optical techniques for in vivo measurement of blood flow velocity are not quite applicable for determination of velocity of individual cells or nanoparticles. A time-of-flight photoacoustic (PA) technique can solve this problem by measuring the transient PA signal width, which is related to the cell velocity passing the laser beam. This technique was demonstrated in vivo using an animal (mouse) model by estimating the velocity of nanoparticles, and red and white blood cells labeled with conjugated gold nanorods (GNRs) in the bloodstream. Here we describe the features and the parameters of novel modifications to the PA time-of-flight method and its new application for real-time monitoring of circulating tumor cells (CTCs), such as B16F10 melanoma. This method provided, for the first time, identification of rolling CTCs in analogy to rolling white blood cells and CTC aggregates. Specifically, monitoring of PA signal widths from CTCs in mouse ear microvessels revealed double maxima in peak-width histograms associated with the fast moving portion of CTCs in central flow and slowly rolling CTCs in analogy to white blood cells. We also developed a two-parameter plot representing PA peak amplitude and peak widths. This method allowed identification of fast-moving individual CTCs, CTC aggregates, and rolling CTCs. The discovery of rolling CTCs in relatively large blood vessels indicates a higher probability of CTC extravasations, further increasing the possibility of metastasis through rolling mechanism in addition to mechanical capturing of CTCs in small vessels.

Compared with single-focus optical-resolution photoacoustic microscopy (OR-PAM), multifocal OR-PAM utilizes both multifocal optical illumination and an ultrasonic array transducer, significantly increasing the imaging speed. Here we present a reflection-mode multifocal OR-PAM system based on a microlens array that provides multiple foci and an ultrasonic array transducer that receives the excited photoacoustic waves from all foci simultaneously. By using a customized microprism to reflect the incident laser beam to the microlens array, we align the multiple optical foci confocally with the focal zone of the ultrasonic array transducer. Experiments show our reflection-mode multifocal ORPAM system is capable of imaging microvessels in vivo, and it can image a 9 mm x 5 mm x 2.5 mm volume at 16 μm lateral resolution in ~4 min, limited by the signal multiplexing ratio and laser pulse repetition rate.

By offering images with high spatial resolution and unique optical absorption contrast, optical-resolution photoacoustic microscopy (OR-PAM) has gained increasing attention in biomedical research. Recent developments in OR-PAM have improved its imaging speed, but have sacrificed either the detection sensitivity or field of view or both. We have developed a wide-field fast-scanning OR-PAM by using a water-immersible MEMS scanning mirror (MEMS-ORPAM). Made of silicon with a gold coating, the MEMS mirror plate can reflect both optical and acoustic beams. Because it uses an electromagnetic driving force, the whole MEMS scanning system can be submerged in water. In MEMS-ORPAM, the optical and acoustic beams are confocally configured and simultaneously steered, which ensures uniform detection sensitivity. A B-scan imaging speed as high as 400 Hz can be achieved over a 3 mm scanning range. A diffraction-limited lateral resolution of 2.4 μm in water and a maximum imaging depth of 1.1 mm in soft tissue have been experimentally determined. Using the system, we imaged the flow dynamics of both red blood cells and carbon particles in a mouse ear in vivo. By using Evans blue dye as the contrast agent, we also imaged the flow dynamics of lymphatic vessels in a mouse tail in vivo. The results show that MEMS-OR-PAM could be a powerful tool for studying highly dynamic and time-sensitive biological phenomena.

It is a challenge to non-invasively visualize in vivo the neovascularization in a three-dimensional (3D) scaffold with high spatial resolution and deep penetration depth. Here we used photoacoustic microscopy (PAM) to chronically monitor neovascularization in an inverse opal scaffold implanted in a mouse model for up to six weeks. The neovasculature was observed to develop gradually in the same mouse. These blood vessels not only grew on top of the implanted scaffold but also penetrated into the scaffold. The PAM system offered a lateral resolution of ~45 μm and a penetration depth of ~3 mm into the scaffold/tissue construct. By using the 3D PAM data, we further quantified the vessel area as a function of time.

In this paper we demonstrate a multi-wavelength optical resolution photoacoustic microscopy system for both phantom and in vivo imaging. Using a 1ns pulse width, 40-kHz repetition-rate ytterbium-doped fiber laser and a 3m single-mode polarization maintaining fiber, we produced numerous Raman-shifted wavelength peaks at with pulse energies between 100 and 400nJ per peak. Peak wavelengths were selected by using 10-nm linewidth bandpass filters. The capabilities of this system is shown by creating C-scan photoacoustic images of carbon fiber networks, 200μm dye-filled tubes, and Swiss Webster mouse ears at several wavelengths. Functional imaging potential was confirmed by assessing tubes filled with varying concentrations of two different dyes.

Blood pulse wave velocity (PWV) is an important indicator for vascular stiffness. In this letter, we present electrocardiogram-synchronized photoacoustic microscopy for in vivo noninvasive quantification of the PWV in the peripheral vessels of mice. Interestingly, strong correlation between blood flow speed and ECG were clearly observed in arteries but not in veins. PWV is measured by the pulse travel time and the distance between two spot of a chose vessel, where simultaneously recorded electrocardiograms served as references. Statistical analysis shows a linear correlation between the PWV and the vessel diameter, which agrees with known physiology. Keywords: photoacoustic microscopy, photoacoustic spectroscopy, bilirubin, scattering medium.

Förster resonance energy transfer (FRET) provides fluorescence signals sensitive to intra- and inter-molecular distances in the 1-10 nm range. Widely applied in the optical imaging environment, FRET enables visualization of physicochemical processes in molecular interactions and conformation changes. We reported photoacoustic imaging of FRET, based on non-radiative decay that produces heat and subsequent acoustic waves. The experimental results show that photoacoustic imaging offers better penetration into scattering biological tissue. Through its ability to three-dimensionally image tissue with scalable resolution, photoacoustic microscopy provides a beneficial biomedical tool to broaden the in vivo application of the FRET technique.

Optical-resolution photoacoustic microscopy (OR-PAM) becomes a premier microscopic imaging tool in biomedicine because it provides agent-free optical absorption information in tissues. By tightly focusing light to a spot, the fine lateral resolution can be achieved in OR-PAM. The focal spot size is typically determined by the numerical aperture of the used objective lens. Here, we demonstrate focus-free OR-PAM using a Bessel beam generator. In this approach, no objective lens is required. We have photoacoustically imaged a carbon fiber with a diameter of ~6 μm, and the measured lateral resolution was ~6-7 μm. Beneficially, the complexities of the existing OR-PAM systems can be greatly relieved.

It has been studied that a potential marker to obtain prognostic information about bladder cancer is tumor neoangiogenesis, which can be quantified by morphometric characteristics such as microvascular density. Photoacoustic microscopy (PAM) can render sensitive three-dimensional (3D) mapping of microvasculature, providing promise to evaluate the neoangiogenesis that is closely related to the diagnosis of bladder cancer. To ensure good image quality, it is desired to acquire bladder PAM images from its inside via the urethra, like conventional cystoscope. Previously, we demonstrated all-optical PAM systems using polymer microring resonators to detect photoacoustic signals and galvanometer mirrors for laser scanning. In this work, we build a miniature PAM system using a microelectromechanical systems (MEMS) scanning mirror, demonstrating a prototype of an endoscopic PAM head capable of high imaging quality of the bladder. The system has high resolutions of 17.5 μm in lateral direction and 19 μm in the axial direction at a distance of 5.4 mm. Images of printed grids and the 3D structure of microvasculature in animal bladders ex vivo by the system are demonstrated.

Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The multimodal system may provide complementary structural and functional information to the fluorescently labeled tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits, pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR spectrum.

Photoacoustics is a hybrid imaging modality that is based on the detection of acoustic waves generated by absorption of pulsed light by tissue chromophors. In current research, this technique uses large and costly photoacoustic systems with a low frame rate imaging. To open the door for widespread clinical use, a compact, cost effective and fast system is required. In this paper we report on the development of a small compact handset pulsed laser probe which will be connected to a portable ultrasound system for real-time photoacoustic imaging and ultrasound imaging. The probe integrates diode lasers driven by an electrical driver developed for very short high power pulses. It uses specifically developed highly efficient diode stacks with high frequency repetition rate up to 10 kHz, emitting at 800nm wavelength. The emitted beam is collimated and shaped with compact micro optics beam shaping system delivering a homogenized rectangular laser beam intensity distribution. The laser block is integrated with an ultrasound transducer in an ergonomically designed handset probe. This handset is a building block enabling for a low cost high frame rate photoacoustic and ultrasound imaging system. The probe was used with a modified ultrasound scanner and was tested by imaging a tissue mimicking phantom.

Thermoacoustic (TA) imaging provides a novel contrast mechanism that may enable visualization of cancerous lesions which are not robustly detected by current imaging modalities. Prostate cancer (PCa) is the most notorious example. Imaging entire prostate glands requires 6 cm depth penetration. We therefore excite TA signal using submicrosecond VHF pulses (100 MHz). We will present reconstructions of fresh prostates imaged in a well-controlled benchtop TA imaging system. Chilled glycine solution is used as acoustic couplant. The urethra is routinely visualized as signal dropout; surgical staples formed from 100-micron wide wire bent to 3 mm length generate strong positive signal.

This study investigates the feasibility of characterizing the microstructures within a biological tissue by analyzing the frequency spectrum of the photoacoustic signal from biological tissue. Hypotheses are derived from theoretical analyses on the relationship between the dimensions as well as concentrations of the optical absorbing sources within the region-of- interest and the linear model fitted to the power spectra of photoacoustic signals. The hypotheses are afterwards validated, following the procedures of ultrasound spectrum analysis, by simulations and experiments with phantoms fabricated by embedding the optically absorbing polyethylene microspheres in porcine gelatin. Quantitatively comparable simulation and experiment results substantiated that photoacoustic spectrum analysis could be a potential tool for characterizing microstructures and optical properties in biological samples.

We introduce a multichannel optical fiber based detector for photoacoustic imaging. By using in-house produced photodetectors and relative low-cost components from telecommunication industries we were able to reduce the costs for one channel significantly compared to previous setups. The estimated cost for one channel (without sampling device) is below 800 €. The self-made balanced photodetector for 1550 nm achieves a gain of 100 dB, a -3dB bandwidth of 45 MHz and a maximum signal-to-noise-ratio of 48 dB. We present a four channel annular detector array based on optical fiber Mach-Zehnder interferometers. Photoacoustic imaging is demonstrated by measuring photoacoustic signals of a black polyethylene microsphere.

Unique features in co-registered ultrasound and photoacoustic images of ex vivo ovarian tissue are introduced, along with the hypotheses of how these features may relate to the physiology of tumors. The images are compressed with wavelet transform, after which the mean Radon transform of the photoacoustic image is computed and fitted with a Gaussian function to find the centroid of the suspicious area for shift-invariant recognition process. In the next step, 24 features are extracted from a training set of images by several methods; including features from the Fourier domain, image statistics, and the outputs of different composite filters constructed from the joint frequency response of different cancerous images. The features were chosen from more than 400 training images obtained from 33 ex vivo ovaries of 24 patients, and used to train a support vector machine (SVM) structure. The SVM classifier was able to exclusively separate the cancerous from the non-cancerous cases with 100% sensitivity and specificity. At the end, the classifier was used to test 95 new images, obtained from 37 ovaries of 20 additional patients. The SVM classifier achieved 76.92% sensitivity and 95.12% specificity. Furthermore, if we assume that recognizing one image as a cancerous case is sufficient to consider the ovary as malignant, then the SVM classifier achieves 100% sensitivity and 87.88% specificity.

In this work, we investigated the principle of the two-photon absorption (TPA) detection with a loss modulation technique, and first demonstrated the existence of two-photon photoacoustics ultrasound excited by a femtosecond high repetition rate laser. By using the AO modulation with different modulation frequencies, we successfully create the beating of the light signal when the two arms of the beams are both spatial and temporal overlapping. The pulse train of the femtosecond laser causes the narrow band excitation, providing the frequency selectivity and sensitivity. Moreover, the pulse energy is no more than 15nJ/pulse, which is at least 3 orders of magnitude smaller than that of the nanosecond laser, and therefore prevents the thermal damage of the sample. With the help of lock-in detection and a low noise amplifier, we can separate the signal of two-photon absorption from one-photon absorption. We used an ultrasonic transducer to detect the response of the sample, and verified the existence of the two-photon photoacoustics ultrasound generating by the femtosecond laser. Several contrast agents, such as the black carbon solution, the fluorescence dye and the nano-particles, were used in the experiment. In the end, we demonstrated the application, two photo-acoustic imaging, which provides the high spatial resolution (<10μm) and large penetration depth (~1mm), to the simulated biological tissue. This is a milestone to develop the two-photon photoacoustics microscopy, which, in principle, has the great potential to achieve the in vitro and in vivo high resolution deep tissue imaging.

The characteristic microstructures in biological tissues could be used to differentiate tissue types, such as tumor vs. normal tissue. The spatial resolution of classical photoacoustic tomography (PAT) mainly depends on the wavelengths of the detected ultrasonic signals. In order to present the very detailed microstructures in a biological sample, the receiving bandwidth of the PAT system needs to be extremely wide. Another challenge in detecting the high frequency signals associated with microstructures is the strong acoustic attenuation which increases quadratically with ultrasound frequency. In this study, we propose a novel photoacoustic spectral analysis (PSA) technique which evaluates the microstructures in tissues by analyzing the spectral parameters of detected photoacoustic signals. Experimental result verified that, using a limited 1-5 MHz working bandwidth, PSA could effectively differentiate two melanoma-mimicking phantoms containing different microstructures (49 μm and 199 μm absorber sizes respectively). In comparison, since the physical scales of the microstructures are too small and beyond the spatial resolution of the PAT system, classical tomographic imaging could not differentiate the two phantoms. The findings from this study suggest that the proposed PSA technique could help distinguish different tissue types, by evaluating the characteristic microstructures in tissues, without relying on the detection of high frequency signals which is extremely challenging when the target object is deep.

Acoustic-resolution photoacoustic microscopy (AR-PAM) with dark-field confocal illumination enables unique high-resolution visualization of chromophores in tissue, such as microvasculatures, within depths of a few millimeters. However, most current systems are bulky and use complex optical components for illumination, thus requiring highly sensitive alignment. In this study, we developed a compact alignment-free acoustic-resolution photoacoustic imaging system with simple fiber illumination. Four optical fibers were placed in four directions around a high-frequency (30-MHz) ultrasound sensor attached with the high-numerical-aperture acoustic lens. The setting angle of the fibers were determined to form a dark field on the tissue surface under the acoustic lens and for the four light beams from the fibers to be combined near the focal point of the acoustic lens, i.e., at a depth of around 1.2 mm in the tissue. The acoustic lens and output ends of the fibers were capped with an acoustically and optically transparent engineering plastic sheet, whose surface can be directly placed and scanned on the tissue surface with ultrasound gel. The diameter and height of this imaging head were as small as 32 mm and 27 mm respectively. The phantom study showed that the lateral signal spreading was 120 μm, which agreed well with the theoretical value of 112 μm. With the system, we attempted to image vasculatures in the rat skin, demonstrating high-contrast visualization of the blood vessels of a few hundred micrometers in diameter in the tissue.

Most biological chromophores and molecules relax primarily through non-radiative processes; therefore, mapping of relaxation time related to non-rediative process can be a potential indicator of tissue status. In order to map relative nonradiative relaxation time, modulated tone-burst light is used to generate photoacoustic signals. Then nonradiative relaxation time is indicated by the amplitude decay rate as modulation frequency increases. The results show that although blood is an optically weak absorber at 808 nm, by using this method a significant enhancement of contrast-tonoise ratio of a blood target compared to pulsed photoacoustic imaging at this wavelength is achieved.

The axial resolution of photoacoustic microscopy (PAM) is much lower than its lateral resolution, which resolves down to the submicron level. Here we achieved so far the highest axial resolution of 7.6 μm by using a commercial 125 MHz ultrasonic transducer for signal detection, followed by the Wiener deconvolution for signal processing. The axial resolution was validated by imaging two layers of red ink in a wedge shape. Melanoma cells were imaged ex vivo with high axial resolution. Compared with a PAM system with a 50 MHz ultrasonic transducer, our high-axial-resolution PAM system resolved the blood vessels in mouse ears in vivo much more clearly in the depth direction.

For years, ultrasound-modulated optical tomography (UOT) has been proposed to image optical contrasts deep inside turbid media (such as biological tissue) at an ultrasonic spatial resolution. The reported imaging depth so far, however, has been limited, preventing this technique from finding broader applications. In this work, we present our latest experimental explorations that push UOT to clinically useful imaging depths, achieved through optimizing from different aspects. One improvement is the use of a large aperture fiber bundle, which more effectively collects the diffused light, including both ultrasound-modulated and unmodulated portions, from the turbid sample and then sends it to the photorefractive material. Another endeavor is employment of a large aperture photorefractive polymer film for demodulating the ultrasound-induced phase modulation. Compared with most UOT detection schemes, the polymer film based setup provides a much higher etendue as well as photorefractive two-beam-coupling gain. Experimentally, we have demonstrated enhanced sensitivity and have imaged through tissue-mimicking samples up to 9.4 cm thick at the ultrasonically-determined spatial resolutions.

Photoacoustics (PA) enables optical-absorption imaging such as functional imaging with depth information by combining ultrasonics and optics. The issue with PA is to increase the signal-to-noise ratio (SNR) because excitation light is attenuated with depth. Repeating irradiation degrades the frame rate, and it will be worse to acquire multispectral information. In previous research, we proposed the m-sequence family such as gold codes, which provide better SNR images than the ensemble average. However, the sending procedure is so complex that it was essential to send sequences twice aperiodically since the negative codes of bipolar sequences must be sent separately, and the decoding artifacts of those bipolar sequences cannot be minimized in aperiodic sending, unlike periodic sending. This study proposes periodic and unipolar m-sequences (PUM): a unipolar sequence consisting of {1, 0}, selected from positive codes of bipolar msequences. Signals can be enhanced by decoding periodically sent PUM using bipolar sequences, and there are no coding artifacts at all for a single wavelength. Moreover, in multispectral simultaneous irradiation, the crosstalk in PUM remains low, which is inherited from m-sequences. We demonstrated that PUM’s improved SNR is superior to that of aperiodic m-sequence family codes or orthogonal Golay codes. Furthermore, the frame-rate, which is normally limited by acoustic time-of-flight, can be maximized up to the pulse repetition frequency since the decoding start point can be set in any code in periodic irradiation.

We report the development of a combined dual-modal photoacoustic and optical coherence tomography (PA-OCT) system using a single near-infrared (NIR) supercontinuum laser source which can provide both optical absorption and scattering contrasts simultaneously. By using a small sized pulsed Nd:YAG microchip laser and a photonic crystal fiber, we fabricated a pulsed broadband supercontinuum source from 600 to 1700 nm. Under the same optical hardware system, intrinsically registered PA and OCT images are acquired in a single scanning. In order to demonstrate feasibility of our system, we successfully acquired the PA and OCT images of black and white hairs images at the same time. The black hair was detected in both PA and OCT images, while the white hair appeared only in the OCT image. This result suggests the potential of compact, cost-effective, and simple dual-modal PA-OCT system. Moreover, we believe that this approach will be a key point for commercialization and clinical translation.

We introduce here a temporally constrained image reconstruction algorithm for fast dynamic imaging of the spatial distribution of tissue parameters such as oxy-hemoglobin, HbO2, or deoxy-hemoglobin, Hb, and their derived parameters, e.g., HbT or StO2. An unknown spatial-temporal distribution of the tissue parameter is represented by a combination of basis functions where bases are predefined and their coefficients are unknown. The performance of the new algorithm is evaluated using experimental studies with dynamic imaging of vascular disease in foot. The results show that the temporally constrained algorithm leads to 26- fold acceleration in the image reconstruction as compared to more traditional methods that have to reconstruct all time frames data sequentially.

Studying the functional hemodynamic roles of individual cerebral cortical arterioles in maintaining both the structure and function of cortical regions during and after brain stroke in small animals is an important issue. Recently, functional photoacoustic microscopy (fPAM) has been proved as a reliable imaging technique to probe the total hemoglobin concentration (HbT), cerebral blood volume (CBV) and hemoglobin oxygen saturation (SO₂) in single cerebral blood vessels of rats. Here, we report the application of fPAM associated with electrophysiology recordings to investigating functional hemodynamic changes in single cortical arterioles of rats with electrical forepaw stimulation after photo-induced ischemic stroke. Because of the weak optical focusing nature of our fPAM system, photo-induced ischemic stroke targeting single cortical arterioles can be easily conducted with simple adaptation. Functional HbT, CBV and SO₂ changes associated with the induced stroke in selected arterioles from the anterior cerebral artery system were imaged with 36 x 65-μm spatial resolution. Experimental results showed that after photo-occlusion of a single arteriole, the functional changes of nearby arterioles in cerebral cortex only can be observed immediately after the stroke. After a few minutes of stroke onset, there are no significant functional changes under the forepaw stimulation, suggesting that alternate blood flow routes are not actively recruited. The fPAM with electrophysiology recordings complements existing imaging techniques and has the potential to offer a favorable tool for explicitly studying cerebral hemodynamics in small animal models of photo-indcued ischemic stroke.

The recently introduced remote photoacoustic imaging technique allows measurement of photoacoustic signals on nonplanar surfaces without the need for a water bath or coupling agent. Hereby, photoacoustically generated ultrasonic displacements are detected without physical contact to the sample by utilizing laser interferometric techniques. In this work we adapted different algorithms to allow reconstruction on non-planar surfaces and evaluate them on experimental and simulated data. Experimental data were obtained using a remote photoacoustic setup based on two-wave mixing in a photorefractive crystal. Ultrasonic displacements were acquired on flat and non-flat surfaces. Three-dimensional reconstruction of simulated and real measurement data is shown with synthetic aperture focusing technique, Fourier domain synthetic aperture focusing technique, and spectral-domain time reversal algorithms. For the synthetic aperture focusing technique and the time reversal algorithm the surface morphology is taken into account. It is demonstrated that artifacts can occur if the surface is not considered. For the experimental data the shape of the surface is obtained from optical coherence tomography or by a priori knowledge.

A method to reconstruct the optical properties in the inhomogeneous medium from the photoacoustic (PA) signal is discussed. The forward modeling of the propagations of the excitation light and of the photoacoustic pressure is carried out with the finite element method. The inverse problem is formulated with a linear equation relating the optical properties and time-domain PA signals. By solving the inverse problem, the distribution of the optical properties in the optically inhomogeneous medium is reconstructed. The measurement noise and the mismatches between the actual measurement conditions and the forward model cause artifacts in the reconstructed image. To reduce the artifacts and to obtain high resolved reconstructed image, we uses the l_p sparsity regularization method which minimizes the l_p norm of the solution of the inverse problem. It is demonstrated by some numerical simulations that the regularization method using the l_p norm obtains a sparse distribution of the changes in the optical properties. The images reconstructed with the l_p sparsity regularization are compared those with truncated singular value decomposition.

In this work, Fourier transform based analytical solution to photoacoustic wave equation is obtained for an optically absorbing spherical object warmed up by a pulsed laser for rectangular and Gaussian radial profiles by treating the temporal profile of the laser as Gaussian. The photoacoustic signal is investigated as a function of time for different locations outside the spherical object. An expression including the dependency of the laser parameters on the photoacoustic signal is presented.

Most reconstruction algorithms for photoacoustic tomography, like back-projection or time-reversal, work ideally for point-like detectors. For real detectors, which integrate the pressure over their finite size, it was shown that images reconstructed by back-projection or time-reversal show some blurring. Iterative reconstruction algorithms using an imaging matrix can take the finite size of real detectors directly into account, but the numerical effort is significantly higher compared to the use of direct algorithms. For spherical or cylindrical detection surfaces the blurring caused by a finite detector size is proportional to the distance from the rotation center (“spin blur”) and is equal to the detector size at the detection surface. In this work we use deconvolution algorithms to reduce this type of blurring on simulated and on experimental data. Experimental data were obtained on a plastisol cylinder with 6 thin holes filled with an absorbing liquid (OrangeG). The holes were located on a spiral emanating from the center of the cylinder. Data acquisition was done by utilization of a piezoelectric detector which was rotated around the plastisol cylinder.

The Grüneisen parameter of tissue is a constitutive parameter in photoacoustic tomography. Here, we applied photoacoustic spectrometry (PAS) to directly measure the Grüneisen parameter. In our PAS system, laser pulses at wavelengths between 460 and 1600 nm were delivered to tissue samples, and photoacoustic signals were detected by a 20 MHz flat water-immersion ultrasonic transducer. By fitting photoacoustic spectra to light absorption spectra, we found that the Grüneisen parameter was 0.73 for porcine subcutaneous fat tissue, and 0.15 for oxygenated bovine red blood cells at room temperature (24°C).

Noninvasive detection of both bilirubin concentration and its distribution is important for disease diagnosis. Here we implemented photoacoustic microscopy (PAM) to detect bilirubin distribution. We first demonstrate that our PAM system can measure the absorption spectra of bilirubin and blood. We also image bilirubin distributions in tissuemimicking samples, both without and with blood mixed. Our results show that PAM has the potential to quantitatively image bilirubin in vivo for clinical applications.

In current photoacoustic tomography (PAT) systems, ultrasound transducer arrays and multi-channel data acquisition (DAQ) electronics are used to receive the PA signals. To achieve real-time PA imaging, massive 1D or even 2D transducer arrays and large number of DAQ channels are necessary. As a result, the ultrasound receiver becomes very complex, bulky and also costly. In this paper, we report the development of novel micromachined silicon acoustic delay line systems, which are expected to provide a new approach to address the above issue. First, fundamental building block structures of the acoustic delay line systems were designed and fabricated. Their acoustic properties were characterized with ultrasound and photoacoustic measurements. Second, two different acoustic delay line systems (parallel and serial) were designed and fabricated using advanced micromachining processes to ensure compact size, high accuracy, and good repeatability. The transmission of multiple acoustic signals in the acoustic delay line systems were studied with ultrasound experiment. Experimental results show that the silicon acoustic delay line systems can guide multiple channels of acoustic signals with low loss and distortion. With the addition of a set of suitable time delays, the time-delay acoustic signals arrived at a single-element transducer at different times and were unambiguously received and processed by the following DAQ electronics. Therefore, the micromachined silicon acoustic delay line systems could be used to combine multiple signal channels into a single one (without the involvement of electronic multiplexing), thereby reducing the complexity and cost of the ultrasound receiver for real-time PAT application.

The employment of contrast agents in photoacoustic imaging has gained significant attention within the past few years for their biomedical applications. In this study, the use of silica-coated superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPION) was investigated as a contrast agent in biomedical photoacoustic imaging. SPIONs have been widely used as Food-and-Drug-Administration (FDA)-approved contrast agents for magnetic resonance imaging (MRI) and are known to have an excellent safety profile. Using our frequency-domain photoacoustic correlation technique (“the photoacoustic radar") with modulated laser excitation, we examined the effects of nanoparticle size, concentration and biological medium (e.g. serum, sheep blood) on its photoacoustic response in turbid media (intralipid solution). Maximum detection depth and minimum measurable SPION concentration were determined experimentally. The detection was performed using a single element transducer. The nanoparticle-induced optical contrast ex vivo in dense muscular tissues (avian pectus) was evaluated using a phased array photoacoustic probe and the strong potential of silicacoated SPION as a possible photoacoustic contrast agent was demonstrated. This study opens the way for future clinical applications of nanoparticle-enhanced photoacoustic imaging in cancer therapy.

This photoacoustic volume imaging (PAVI) system is designed to study breast cancer detection and diagnosis in the mammographic geometry in combination with automated 3D ultrasound (AUS). The good penetration of near-infrared (NIR) light and high receiving sensitivity of a broad bandwidth, 572 element, 2D PVDF array at a low center-frequency of 1MHz were utilized with 20 channel simultaneous acquisition. The feasibility of this system in imaging optically absorbing objects in deep breast tissues was assessed first through experiments on ex vivo whole breasts. The blood filled pseudo lesions were imaged at depths up to 49 mm in the specimens. In vivo imaging of human breasts has been conducted. 3D PAVI image stacks of human breasts were coregistered and compared with 3D ultrasound image stacks of the same breasts. Using the designed system, PAVI shows satisfactory imaging depth and sensitivity for coverage of the entire breast when imaged from both sides with mild compression in the mammographic geometry. With its unique soft tissue contrast and excellent sensitivity to the tissue hemodynamic properties of fractional blood volume and blood oxygenation, PAVI, as a complement to 3D ultrasound and digital tomosynthesis mammography, might well contribute to detection, diagnosis and prognosis for breast cancer.

A technique based on acoustically modulated laser speckle has been demonstrated which can quantify and classify 25 colored papers, even when they are hidden 5 mm behind an opaque slab barrier with a thickness of 5 mm and a reduced scattering coefficient of 1.8 mm^-1. A small vibration at 200 Hz was induced on the colored paper by attaching it to the central diaphragm of a loudspeaker. Two He-Ne lasers (green at 543 nm and red at 633 nm) illuminated the slab surface sequentially. Although the slab blocked most of the incoming light, a small proportion of light penetrated through, interacted with the vibrating colored paper and backscattered, causing a time-varying speckle pattern on the slab surface. A consumer grade digital camera was used to capture the speckle pattern from which the speckle contrast difference was calculated and shown to be indicative of the color of the hidden object. Using the speckle contrast difference measured at 543 nm and 633 nm, the nearest neighbor classification algorithm was employed to classify the 25 hidden colors (formed by different percentages of base colors magenta and cyan), achieving an accuracy of 72%. This work has demonstrated that the acoustically modulated laser speckle technique can increase the sensitivity of spectroscopic measurements in a deeper region, which has the potential to be translated into clinical applications such as cerebral oxygenation measurement in which a superficial layer (skull) is present.

This paper presents an in vitro study in photoacoustic evaluation of laser controlled drug release by using gold nanostructure agents. Laser controlled drug release has been studied and reported by using high intensity nanosecond laser source and optical absorbing gold nanostructures. With the same nanosecond laser source in low intensity, photoacoustic imaging technique can be employed to evaluate the laser controlled drug release, which makes it promising to combine low energy photoacoustic imaging/evaluation with high energy laser controlled drug delivery in a non-radiation, low cost and minimum modified theranostic platform. As an effort to test the feasibility, gold nanoparticles and gold nanorods which have strong absorption of laser specifically in 531 nm and 808nm to trig the drug release in carriers were evaluated in high energy laser treatment and low energy photoacoustic imaging. 20%-50% drop of the intensity in the photoacoustic images has been observed in gold nanoparticles solutions and gold nanorods solutions, after being treated by high energy laser. This study suggests that it is possible to evaluate the high energy laser controlled drug release with low energy photoacoustic imaging method in a non-complex theranostic platform.

To view the individual cells and ambient microvasculature simultaneously will be helpful to study tumor angiogenesis and microenvironments. To achieve this, two molecular contrast mechanisms were exploited simultaneously by integrating two imaging modalities, confocal fluorescence microscopy (CFM) and photoacoustic microscopy (PAM). These share the same scanning optical path and laser source. The induced photoacoustic (PA) signal was detected by a highly sensitive needle hydrophone; while the back-traveling fluorescent photons emitted from the same sample were collected by an avalanche photodetector. Experiments on ex vivo rat bladders were conducted. The CFM image depicted the shape and size of the individual cells successfully. Besides large polygonal umbrella cells, some intracellular components can also be discerned. With the CFM image presenting morphologic cellular information in the bladder wall, the PAM image provides the complementary information, based on the endogenous optical absorption contrast, of the microvascular distribution inside the bladder wall, from large vessels to capillaries. Such multimodal imaging provides the opportunity to realize both histological assay and characterization of microvasculature using one imaging setup. This approach offers the possibility of comprehensive diagnosis of cancer in vivo.

This paper presents an ex vivo study in imaging high-intensity focused ultrasound induced tissue denaturation with multispectral photoacoustic approach. Beef tissues treated by both water bath and high-intensity focused ultrasound were imaged and evaluated by photoacoustic imaging method, where light in multiple optical wavelengths between 700nm and 900nm is applied. Tissue denaturation after being treated by water bath and high-intensity focused ultrasound has been observed in multispectral photoacoustic images. The denaturation is more striking in relatively shorter optical wavelength photoacoustic images than in relatively longer optical wavelength photoacoustic images. This study suggests that multispectral photoacoustic imaging method is promising in the evaluation of tissue denaturation induced by high- intensity focused ultrasound treatment.

The ability to collect data in real time is important in all biological imaging modalities that aim to image dynamic processes. Photoacoustic Microscopy (PAM) is a rapidly growing biomedical imaging technique that is often used to image microvasculature and melanoma, and is capable of fully rendering three-dimensional images. However, due to the bi-polar nature of the PAM signal, post processing through demodulation is required to accurately display morphological data. Typically, demodulation requires post processing of the data, limiting its use in real-time applications. This results in many PAM systems displaying data through maximum amplitude projection (MAP) images, completely ignoring the axial dimension of their scans and throwing away useful data. We overcome this processing limit by utilizing a configurable integrated circuit known as a Field Programmable Gate Array (FPGA). The FPGA allows us to perform quadrature demodulation of the photoacoustic signal as it is being collected. The result is a PAM system capable of producing continuous, morphologically accurate B-scans and volumes at a rate limited only by the repetition rate of the laser. This allows us to generate accurately rendered volumes at the same speed as MAP images. With a 100 KHz actively q-switched laser we are able to generate 200 by 200 pixel b-scans at a rate of 500 Hz. The imaging potential of the system has been demonstrated in volumes of human hair phantoms and chick embryo vasculature. This system is capable of 50 x 50 x 50 volume stacks processed and displayed at better than video rate.

Photoacoustic computed tomography (PACT) is an emerging imaging technique which is based on the acoustic detection of optical absorption from tissue chromophores, such as oxy-hemoglobin and deoxy-hemoglobin. An important application of PACT is functional brain imaging of small animals. The conversion of light to acoustic waves allows PACT to provide high resolution images of cortical vasculatures through the intact scalp. Here, PACT was utilized to study the activated areas of the mouse brain during forepaw and hindpaw stimulations. Temporal PACT images were acquired enabling computation of hemodynamic changes during stimulation. The stimulations were performed by trains of pulses at different stimulation currents (between 0.1 to 2 mA) and pulse repetition rates (between 0.05 Hz to 0.01Hz). The response at somatosensory cortex-forelimb, and somatosensory cortex-hindlimb, were investigated. The Paxinos mouse brain atlas was used to confirm the activated regions. The study shows that PACT is a promising new technology that can be used to study brain functionality with high spatial resolution.

This paper presents a real-time transvaginal photoacoustic imaging probe for imaging human ovaries in vivo. The probe consists of a high-throughput (up to 80%) fiber-optic 1 x 19 beamsplitters, a commercial array ultrasound transducer, and a fiber protective sheath. The beamsplitter has a 940-micron core diameter input fiber and 240-micron core diameter output fibers numbering 36. The 36 small-core output fibers surround the ultrasound transducer and delivers light to the tissue during imaging. A protective sheath, modeled in the form of the transducer using a 3-D printer, encloses the transducer with array of fibers. A real-time image acquisition system collects and processes the photoacoustic RF signals from the transducer, and displays the images formed on a monitor in real time. Additionally, the system is capable of coregistered pulse-echo ultrasound imaging. In this way, we obtain both morphological and functional information from the ovarian tissue. Photoacousitc images of malignant human ovaries taken ex vivo with the probe revealed blood vascular and networks that was distinguishable from normal ovaries, making the probe potential useful for characterizing ovarian tissue.

The use of near-infrared wavelengths for photoacoustic (PA) imaging takes advantage of the relatively low inherent absorption of tissues and has encouraged the development of agents which show high contrast in this range. Here, we describe the modification of a commercially available PA imaging system (Vevo LAZR, VisualSonics, Toronto) to take advantage of the 532nm and 1064nm wavelengths inherent in the generation of the currently tuneable range of 680 to 970nm and in the use of these two wavelengths to assess contrast agents. The photoacoustic imaging system generated light from a Nd/YAG laser modified to extract the 532 and 1064nm wavelengths in addition to its OPO-derived tuneable range (680 - 970 nm) and deliver this light through a fiber integrated into a linear array transducer (LZ400, VisualSonics). Gold nanorods (UT Austin), carbon nanotubes (Stanford U), DyLight 550 (Thermo Fisher) and blood were imaged in a phantom (PE20 tubing) and in a hindlimb subcutaneous tumor in vivo to determine their photoacoustic signal intensity at all wavelengths. In the phantom and in vivo, all agents caused an enhancement of the photoacoustic signal at their respective peak absorbance wavelengths. These results show that the 532nm and 1064nm wavelengths could prove useful in biomedical imaging due to the contrast agents customized for them. The 1064nm wavelength in particular has the advantage of having very low generation of endogenous signal in vivo, making agents tuned to this wavelength ideal for targeted contrast imaging.

This study focuses on a multimodal imaging technique that integrates both structural and functional information using a priori ultrasound (US) information to assist near-infrared (NIR) diffuse optical tomography (DOT). Up to date, handheld systems that integrates DOT and US have been demonstrated. Our system is designed to be fully-automated and non-contact. Our aim is to build an interface, in which the optical source and detector fibers will rotate around the breast together with the US transducer. However, in this study we built a prototype system, which rotated the phantom and kept the transducers stationary for simplicity. Simulation and experimental studies were performed using a variety of sourcedetector configurations. The reconstruction results were compared with and without US a priori information. To collect the a priori US information, the multi-modality agar phantom was rotated 360° using a computer controlled rotational stage. The multi-modality phantom had an inclusion that had both optical absorption and US contrast. 360 US images were collected in 1° increments covering the entire phantom volume. The DOT data was also collected while the phantom is rotated with particular source-detector configurations. These results have shown that when the detectors were π /8 apart, and the phantom is rotating at π /16 increments with a total of 32 views provide the optimum image reconstruction. As expected, US a priori information further improved the quantification accuracy.

Photoacoustic microscopy, as an imaging modality, has shown promising results in imaging angiogenesis and cutaneous malignancies like melanoma, revealing systemic diseases including diabetes, hypertension, tracing drug efficiency and assessment of therapy, monitoring healing processes such as wound cicatrization, brain imaging and mapping. Clinically, photoacoustic microscopy is emerging as a capable diagnostic tool. Parameters of lasers used in photoacoustic microscopy, particularly, pulse duration, energy, pulse repetition frequency, and pulse-to-pulse stability affect signal amplitude and quality, data acquisition speed and indirectly, spatial resolution. Lasers used in photoacoustic microscopy are typically Q-switched lasers, low-power laser diodes, and recently, fiber lasers. Significantly, the key parameters cannot be adjusted independently of each other, whereas microvasculature and cellular imaging, e.g., have different requirements. Here, we report an integrated fiber laser system producing nanosecond pulses, covering the spectrum from 600 nm to 1100 nm, developed specifically for photoacoustic excitation. The system comprises of Yb-doped fiber oscillator and amplifier, an acousto-optic modulator and a photonic-crystal fiber to generate supercontinuum. Complete control over the pulse train, including generation of non-uniform pulse trains, is achieved via the AOM through custom-developed field-programmable gate-array electronics. The system is unique in that all the important parameters are adjustable: pulse duration in the range of 1-3 ns, pulse energy up to 10 μJ, repetition rate from 50 kHz to 3 MHz. Different photocoustic imaging probes can be excited with the ultrabroad spectrum. The entire system is fiber-integrated; guided-beam-propagation rendersit misalignment free and largely immune to mechanical perturbations. The laser is robust, low-cost and built using readily available components.

The simultaneous photoacoustic assessment of oxygen saturation and red blood cell aggregation is presented. Aggregation was induced on porcine red blood cells using Dextran-70 at multiple hematocrit levels. Samples were exposed to 750 nm and 1064 nm for each hematocrit and aggregate size in order to compute the oxygen saturation. As the size of the aggregate increased, the photoacoustic signal amplitude increased monotonically. The same trend was observed for increasing hematocrit at each aggregation level. The oxygen saturation of aggregated samples was 30% higher than non-aggregated samples at each hematocrit level. This suggests that the presence of red blood cell aggregates impairs the release of oxygen to the surrounding environment. Such a result has important implications for detecting red blood cell aggregation non-invasively and making clinical decisions based on the simulatenous assessment of oxygen saturation.

The majority of optoacoustic reconstruction algorithms are based on the assumption that the speed of sound within the imaging sample is constant and equal to the speed of sound in the coupling medium, typically water. However, small speed of sound changes between different organs and structures are common in actual samples. The variations in the speed of sound within biological tissues are usually below 10% with respect to the speed of sound in water. Under these circumstances, the acoustic wave propagation can be modeled as acoustic rays and the main effect of the acoustic heterogeneities is the time-shifting of the optoacoustic signals. Herein, we describe a model-based reconstruction algorithm capable of accounting for such small speed of sound variations. It is based on modifying the integration curve in the forward optoacoustic model according to the time-shifting produced by differences in the speed of sound. The forward model is then discretized and inverted algebraically by means of the LSQR algorithm. The algorithm was tested experimentally with tissue-mimicking agar phantoms containing glycerine to simulate a higher speed of sound than water. The improvement in the image quality as compared to the results obtained by assuming a uniform speed of sound is discussed in this work.

Like ultrasound endoscopy, photoacoustic endoscopy (PAE) could become a valuable addition to clinical practice due to its deep imaging capability. Results from our recent in vivo transesophageal endoscopic imaging study on rabbits demonstrate the technique’s capability to image major organs in the mediastinal region, such as the lung, trachea, and cardiovascular systems. Here, we present various features from photoacoustic images from the mediastinal region of several rabbits and discuss possible clinical contributions of this technique and directions of future technology development.

A photoacoustic technique for monitoring absorber size and concentration is presented. The technique relies on analyzing the power spectra of the radio-frequency signals and taking into account the receiving transducer response in order to remove system dependencies. By normalizing the power spectra, parameters derived from ultrasound tissue characterization (spectral slope and midband fit) can be obtained. Tissue mimicking phantoms were constructed using black polystyrene beads of various sizes and concentrations as absorbers. The spectral slope decreased by 0.63 dB/MHz when the size of the particle increased from 1 μm to 10 μm at every bead concentration. The midband fit was ~4 dB higher for the 10 μm particle and increased linearly with concentration. These results suggest that photoacoustic radiofrequency spectroscopy (PA-RFS) can potentially monitor changes in absorber size and concentration thus improving the ability of photoacoustic imaging to distinguish structural tissue variations.

Laser-tissue interaction during laser surgery can be classified into two biophysical processes: tissue removal in the focal zone of the laser beam and heating in the surrounding tissue. In order to ensure a precise cut and minimal collateral thermal damage, the surgeon has to control several parameters, such as power, repetition rate and fiber movement velocity. In this study we propose utilizing optoacoustics for providing the necessary real-time feedback of cutting and heating processes. A single Q-switched Nd-YAG laser (532nm, 4 KHz, 18 W, pulse duration 7.6ns) was used for ablation and generation of optoacoustic signals in fresh bovine tissue samples. Both shockwaves, generated due to tissue removal, as well as normal optoacoustic responses from the surrounding tissue were detected using a single 10MHz piezoelectric transducer. It has been observed that rapid reduction in the shockwave amplitude occurs as more material is being removed from the focal zone, indicating decrease in cutting efficiency of the laser beam, whereas gradual decrease in the optoacoustic signal likely corresponds to coagulation around the ablation crater. Further heating of surrounding tissue leads to carbonization accompanied by a significant shift of spectral components of the optoacoustic signal. Our results hold promise for real-time monitoring of cutting efficiency and collateral thermal damage during laser surgery.

Clutter noise is an important challenge in photocoustic (PA) and ultrasound (US) imaging as they degrade the image quality. In this paper, the short-lag spatial coherence (SLSC) imaging technique is used to reduce clutter and side lobes in PA images. In this technique, images are obtained through the spatial coherence of PA signals at small spatial distances across the transducer aperture. The performance of this technique in improving image quality and detecting point targets is compared with a conventional delay-and-sum (DAS) beamforming technique. A superior contrast, contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR) are observed when SLSC imaging is employed. Point spread function of point targets shows an improved spatial resolution and reduced side lobes when compared with DAS beamforming. Also shown is the impact of increasing the number of frames on which SLSC is applied. The results show that contrast, CNR, and SNR are improved with increasing number of frames.

Photoacoustic tomography (PAT) is a hybrid imaging method, which combines ultrasonic and optical imaging modalities, in order to overcome their respective weaknesses and to combine their strengths. It is based on the reconstruction of optical absorption properties of the tissue from the measurements of a photoacoustically­ generated pressure field. Current methods consider laser excitation, under thermal and stress confinement assumptions, which leads to the generation of a propagating pressure field. Conventional reconstruction tech­ niques then recover the initial pressure field based on the boundary measurements by iterative reconstruction algorithms in time- or Fourier-domain. Here, we propose an application of a new sensing principle that allows for efficient and non-iterative reconstruction algorithm for imaging point absorbers in PAT. We consider a closed volume surrounded by a measurement surface in an acoustically homogeneous medium and we aim at recovering the positions and the amount of heat absorbed by these absorbers. We propose a two-step algorithm based on proper choice of so-called sensing functions. Specifically, in the first step, we extract the projected positions on the complex plane and the weights by a sensing function that is well-localized on the same plane. In the second step, we recover the remaining z-location by choosing a proper set of plane waves. We show that the proposed families of sensing functions are sufficient to recover the parameters of the unknown sources without any discretization of the domain. We extend the method for sources that have joint-sparsity; i.e., the absorbers have the same positions for different frequencies. We evaluate the performance of the proposed algorithm using simulated and noisy sensor data and we demonstrate the improvement obtained by exploiting joint sparsity.

We demonstrated the potential of carbon nanoparticles (CNPs) as exogenous contrast agents for both thermoacoustic (TA) tomography (TAT) and photoacoustic (PA) tomography (PAT). In comparison to deionized water, the CNPs provided a four times stronger signal in TAT at 3 GHz. In comparison to blood, The CNPs provided a much stronger signal in PAT over a broad wavelength range of 450-850 nm. Specifically, the maximum signal enhancement in PAT was 9.4 times stronger in the near-infrared window of 635-670 nm. In vivo blood-vessel PA imaging was performed non-invasively on a mouse femoral area. The images, captured after the tail vein injection of CNPs, show a gradual enhancement of the optical absorption in the vessels by up to 230%. The results indicate that CNPs can be potentially used as contrast agents for TAT and PAT to monitor the intravascular or extravascular pathways in clinical applications.

In this paper a new method is proposed to classify vascular tissues in the range from normal to different degrees of abnormality based on the Photo-Acoustic (PA) signals generated by different categories of vasculatures. The classification of the vasculatures is achieved based on the statistical features of the photoacoustic radiofrequency (RF) signals such as energy, variance, and entropy in the wavelet domain. A feature vector for each category of vasculature is provided and the distance between feature vectors are computed as the measure of similarity between vasculatures. The distances are mapped in two-dimensional space depicting the proximities of the different categories of the vasculatures. The method proposed in this paper can help both detecting abnormal tissues and monitoring the treatment progress by measuring the similarity between vascular tissues in different stages of treatment. The method is applied to simulated data as well as in vivo data from tumor bearing mice to detect cancer treatment effects.

Photoacoustic microscopy (PAM), whose spatial resolution and penetration depth are both scalable, has made great progress in recent years. According to their different lateral resolutions, PAM systems can be categorized into either optical-resolution (OR) PAM, with optical-diffraction-limited lateral resolution, or acoustic-resolution (AR) PAM, with acoustically limited resolution and a deeper maximum imaging depth. In this report, we present a combined OR and AR PAM system with resolutions of 2.2 μm and 40 μm, respectively. Sharing most components between the OR and AR implementations, the system achieves separated illumination for OR and AR imaging by an optical fiber bundle through different channels, and two discrete lasers are used to provide either high-power energy for AR imaging or highrepetition- rate pulses for OR imaging. The design enables automatically co-registered OR and AR photoacoustic imaging in one single system, which extends the usability of current photoacoustic systems and simplifies the imaging procedure.

Photoacoustic imaging uses laser induced ultrasound transients to generate optical absorption maps of the illuminated volume. In this work, we used a custom built photoacoustic imaging system consisting of a 60-channel transducer array, a 50 MHz data acquisition system, and an Nd:YAG pumped OPO laser, to perform simultaneous photoacoustic and fluorescence imaging. A single 780 nm laser pulse generated both ultrasound and fluorescence, enabling reconstruction of images for both modalities with near perfect temporal co-registration. The result highlighted the ability of photoacoustic imaging to supplement fluorescence data when optical scatter reduces fluorescence resolution beyond its useful range.

In order to explore the influence of a finite sensor on the detected photoacoustic pressure, we used the solution of the photoacoustic wave equation for a point-like source to simulate the pressure detected by a flat circular sensor. The sensor was simulated by a mesh of point-like sensors connected in parallel. First, we studied the shape of the photoacoustic pressure when sensor discretization was varied. The artifacts in the predicted pressure decreased as discretization size increased. The above implies that the adequate modeling of pressure depends on the discretization size. Pressure detection at different positions with respect to the sensor location was considered. In this sense we simulated a point-like source located at different positions over an axis in the center of the sensor perpendicular to the sensing surface. We also simulated the behavior of the photoacoustic pressure taking a fixed coordinate system and exploring the variations in the translational and rotational degrees of freedom for the source-sensor system. We found that simulated pressure from the proposed approach for the finite sensor clearly differs from the point-like detection model.

Photoacoustic imaging (PAI) has been proposed as a non-invasive technique for the diagnosis and monitoring of disorders in the neonatal brain. However, PAI of the brain through the intact skull is challenging due to reflection and attenuation of photoacoustic pressure waves by the skull bone. The objective of this work was to develop a phantom for testing the potential limits the skull bone places on PAI of the neonatal brain. Our approach was to make acoustic measurements on materials designed to mimic the neonatal skull bone and construct a semi-realistic phantom. A water tank and two ultrasound transducers were utilized to measure the ultrasound insertion loss (100 kHz to 5MHz) of several materials. Cured mixtures of epoxy and titanium dioxide powder provided the closest acoustic match to neonatal skull bone. Specifically, a 1.4-mm thick sample composed of 50% (by mass) titanium dioxide powder and 50% epoxy was closest to neonatal skull bone in terms of acoustic insertion loss. A hemispherical skull phantom (1.4 mm skull thickness) was made by curing the epoxy/titanium dioxide powder mixture inside a mold. The mold was constructed using 3D prototyping techniques and was based on the hairless head of a realistic infant doll. The head was scanned to generate a 3D model, which in turn was used to build a 3D CAD version of the mold. The mold was CNC machined from two solid blocks of Teflon®. The neonatal skull phantom will enable the study of the propagation of photoacoustic pressure waves under a variety of experimental conditions.

Photoacoustic imaging (PAI) has been proposed as a non-invasive technique for imaging neonatal brain injury. Since PAI combines many of the merits of both optical and ultrasound imaging, images with high contrast, high resolution, and a greater penetration depth can be obtained when compared to more traditional optical methods. However, due to the strong attenuation and reflection of photoacoustic pressure waves at the skull bone, PAI of the brain is much more challenging than traditional methods (e.g. near infrared spectroscopy) for optical interrogation of the neonatal brain. To evaluate the potential limits the skull places on 3D PAI of the neonatal brain, we constructed a neonatal skull phantom (1.4-mm thick) with a mixture of epoxy and titanium dioxide powder that provided acoustic insertion loss (1-5MHz) similar to human infant skull bone. The phantom was molded into a realistic infant skull shape by means of a CNCmachined mold that was based upon a 3D CAD model. To evaluate the effect of the skull bone on PAI, a photoacoustic point source was raster scanned within the phantom brain cavity to capture the imaging operator of the 3D PAI system (128 ultrasound transducers in a hemispherical arrangement) with and without the intervening skull phantom. The resultant imaging operators were compared to determine the effect of the skull layer on the PA signals in terms of amplitude loss and time delay.

We have developed a new photoacoustic endoscopic probe specifically designed for human urogenital imaging. The endoscopic probe uses a parabolic mirror-based mechanical scanning mechanism, providing an angular field of view of 270°. And it has a rigid, dome shaped end section for smooth cavity introduction. Here we introduce the new endoscope’s design and imaging principle, and present experimental results validating its in vivo imaging ability.

To control the overall action of the body, brain consumes a large amount of energy in proportion to its volume. In humans and many other species, the brain gets most of its energy from oxygen-dependent metabolism of glucose. An abnormal metabolic rate of glucose and/or oxygen usually reflects a diseased status of brain, such as cancer or Alzheimer’s disease. We have demonstrated the feasibility of imaging mouse brain metabolism using photoacoustic computed tomography (PACT), a fast, noninvasive and functional imaging modality with optical contrast and acoustic resolution. Brain responses to forepaw stimulations were imaged transdermally and transcranially. 2-NBDG, which diffuses well across the blood-brain-barrier, provided exogenous contrast for photoacoustic imaging of glucose response. Concurrently, hemoglobin provided endogenous contrast for photoacoustic imaging of hemodynamic response. Glucose and hemodynamic responses were quantitatively unmixed by using two-wavelength measurements. We found that glucose uptake and blood perfusion around the somatosensory region of the contralateral hemisphere were both increased by stimulations, indicating elevated neuron activity. The glucose response amplitude was about half that of the hemodynamic response. While the glucose response area was more homogenous and confined within the somatosensory region, the hemodynamic response area showed a clear vascular pattern and spread about twice as wide as that of the glucose response. The PACT of mouse brain metabolism was validated by high-resolution open-scalp OR-PAM and fluorescence imaging. Our results demonstrate that 2-NBDG-enhanced PACT is a promising tool for noninvasive studies of brain metabolism.

Prostate brachytherapy is a form of radiotherapy for treating prostate cancer where the radiation sources are seeds inserted into the prostate. Accurate localization of seeds during prostate brachytherapy is essential to the success of intraoperative treatment planning. The current standard modality used in intraoperative seeds localization is transrectal ultrasound. Transrectal ultrasound, however, suffers in image quality due to several factors such speckle, shadowing, and off-axis seed orientation. Photoacoustic imaging, based on the photoacoustic phenomenon, is an emerging imaging modality. The contrast generating mechanism in photoacoustic imaging is optical absorption that is fundamentally different from conventional B-mode ultrasound which depicts changes in acoustic impedance. A photoacoustic imaging system is developed using a commercial ultrasound system. To improve imaging contrast and depth penetration, absorption enhancing coating is applied to the seeds. In comparison to bare seeds, approximately 18.5 dB increase in signal-to-noise ratio as well as a doubling of imaging depth are achieved. Our results demonstrate that the coating of the seeds can further improve the discernibility of the seeds.

This research investigated anisotropic scattering of ultrasonic and photoacoustic waves from tissues consisting of transverse isotropic structures. Anisotropic scattering refers to the systematic variation in acoustic scattered energy. Take tendon as an example, the maximum occurs when the arrangement of the transducer and fiber orientation is perpendicular and minimum occurs when the arrangement is parallel. Experimental results indicate the apparent integrated backscatter (AIB), which is widely adopted to compute the scattered energy, for photoacoustic as well as ultrasonic waves decayed as the arrangement changed from perpendicular to parallel. The AIB decrement using transducers with center frequency of 3.5 MHz, 5 MHz, and 20 MHz were 10.50 dB, 18.01 dB, and 20.98 dB, respectively. Photoacoustic AIB decrement detected by transducers with center frequency of 3.5 MHz, 5 MHz, and 20 MHz were 7.63 dB, 15.54 dB, and 17.76 dB, respectively. It is shown that higher detection frequency resulted in a larger decrement. A hypothesis is proposed to explain why photoacoustic waves are less affected by the fibrous tissue. In ultrasonic scattering, incident direction for each scatterer were similar due to the relatively planar wavefront, hence the signal amplitudes scattered at the transducer direction are also similar. In photoacoustic scattering, the spherical-like wavefront causes different incident directions for different scatterers, therefore the variation of the signal amplitude collected by the transducer increases, resulting in a lower correlation with the microstructure. In addition, the decrement of backscattered energy decreased for a single scatterer when the incident wave was spherical. Experimental and simulation results verified the hypothesis. The discovery implies that photoacoustic imaging has the potential to detect tissues with transverse isotropic structure that may be overlooked by conventional ultrasound imaging.

We demonstrate a new technique to non-invasively determine the diameter and sound speed of single cells using a combined ultrasonic and photoacoustic technique. Two cell lines, B16-F1 melanoma cells and MCF7 breast cancer cells were examined using this technique. Using a 200 MHz transducer, the ultrasound backscatter from a single cell in suspension was recorded. Immediately following, the cell was irradiated with a 532 nm laser and the resulting photoacoustic wave recorded by the same transducer. The melanoma cells contain optically absorbing melanin particles, which facilitated photoacoustic wave generation. MCF7 cells have negligible optical absorption at 532 nm; the cells were permeabilized and stained with trypan blue prior to measurements. The measured ultrasound and photoacoustic power spectra were compared to theoretical equations with the cell diameter and sound speed as variables (Anderson scattering model for ultrasound, and a thermoelastic expansion model for photoacoustics). The diameter and sound speed were extracted from the models where the spectral shape matched the measured signals. However the photoacoustic spectrum for the melanoma cell did not match theory, which is likely because melanin particles are located around the cytoplasm, and not within the nucleus. Therefore a photoacoustic finite element model of a cell was developed where the central region was not used to generate a photoacoustic wave. The resulting power spectrum was in better agreement with the measured signal than the thermoelastic expansion model. The MCF7 cell diameter obtained using the spectral matching method was 17.5 μm, similar to the optical measurement of 16 μm, while the melanoma cell diameter obtained was 22 μm, similar to the optical measurement of 21 μm. The sound speed measured from the MCF7 and melanoma cell was 1573 and 1560 m/s, respectively, which is within acceptable values that have been published in literature.

Here we attempt to simulate the macroscopic light scattering phenomenon involving digitally time-reversed ultrasound-encoded light, which combines optical phase conjugation with ultrasound encoding, for deep-tissue imaging. By using the focused ultrasound, we can create a virtual source of light frequency shifted due to the acousto-optic effect. Frequency-shifted light emanating from this source is then recorded, and can be used to retrace to the virtual source to form an optical focus deep inside biological tissues. The simulation is done by numerical solutions of Maxwell’s equations, which can accurately account for phase and amplitude of light. The reported simulation enables qualitative and quantitative characterization that may provide important information for enhancement.

Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

Quantification of the optical absorption coefficients of optical absorbers using photoacoustic (PA) pressure waves with broadband frequency was reported. We proposed to use continuous wavelet-transform (CWT) to obtain time-resolved frequency spectra of PA signals and demonstrated the relationship between optical absorption coefficients of optical absorbers and CWT of PA signals. However, the optical absorption coefficients of the optical absorbers were not quantified. Thus, in this research, we quantified optical absorption coefficients of optical absorbers by using the calibration curve which relates the optical absorption coefficients of optical absorbers and CWTs of PA signals. The calibration curve is derived from the simulation. However, due to the frequency response of the acoustic sensor, the simulated PA pressure waves differed from the measured PA signals. Thus, we measured the frequency response of the acoustic sensor. By convolving the frequency response of the acoustic sensor to the simulated pressure waves, we simulated the PA signals which were obtained by measuring the PA pressure wave using the acoustic sensor. The calibration curve derived from the simulated PA signal enabled to quantify optical absorption coefficients of optical absorbers. We verified the method by quantifying optical absorption coefficients of blood vessel phantoms which is tubes filled with diluted inks with optical absorption coefficients from 10 to 40 cm^-1. As results, the simulated PA signals demonstrated close similarity with the measured PA signals, and the optical absorption coefficients of the blood vessel phantoms were quantified with root mean square error of 2.42 cm^-1.

Photoacoustic computed tomography (PACT), also known as optoacoustic tomography or thermoacoustic tomography, is an emerging biomedical imaging technique that combines optical absorption contrast with ultrasound detection principles. Recently, a novel analytic image reconstruction formula has been proposed that operates on a data function expressed in the temporal frequency and spatial domains. The validity the formula has been demonstrated for a two-dimensional (2D) circular measurement geometry. In this study, computer simulation studies are conducted to validate the reconstruction formula for a three-dimensional (3D) spherical measurement geometry. This formula provides new insights into how the spatial frequency components of the sought-after object function can be explicitly determined by the temporal frequency components of the data function measured with a 2D circular or 3D spherical measurement geometry in PACT. Comparing with existing Fourier transform-based reconstruction formulas, the reconstruction formula possesses a simple structure that requires no computation of series expansions or multi-dimensional interpolation in Fourier space.

In this paper, we present the construction of an optical-resolution photoacoustic microscopy (OR-PAM) system and studies done on the characterization of human ovarian tissue with malignant and benign features ex vivo. PAM images of the ovaries showed more detailed blood vessel distributions with much higher resolution compared with conventional photoacoustic images obtained with array transducers. In all, 29 PAM images (20 from normal ovaries and 9 from malignant ovaries) were studied. Eight different features were extracted quantitatively from the PAM images, and a generalized linear model (GLM) was used to classify the ovaries as normal or malignant. By using the GLM, a specificity of 100% and a sensitivity of 100% were obtained for the training set. These preliminary results demonstrate the feasibility of our PAM system in mapping microvasculature networks, as well as characterizing the ovarian tissue, and could be extremely valuable in assisting surgeons for in vivo evaluation of ovarian tissue.

Photoacoustic imaging of cross-sectional slices of extended objects requires ultrasound detectors equipped with an acoustic cylindrical lens rotating around the imaged object. The finite width of the sensor and the small focal depth of lenses with large aperture lead to various imaging artifacts. In this study, these artifacts are on the one hand avoided by the special design of the sensor and on the other hand by a model based, iterative reconstruction algorithm. The integrating property of the cylindrical detector, which exceeds in direction of the cylinder axis the size of the imaged object, avoids the lateral blurring that normally would result from the finite width of a small detector. In addition, an iterative algorithm is presented based on the system matrix that models the signal generation of the device. With this algorithm the imperfect focusing properties of the lens, especially for parts of the object moving out of focus during the rotational scan, are corrected. A direct reconstruction from the measured signals, which in case of the integrating sensor uses the inverse Radon transform, is compared to the reconstruction after iterations, both in a simulation and an experiment. A significant improvement of resolution perpendicular to the section is observed.

Many cancer deaths are related to metastasis to distant organs due to dissemination of circulating tumor cells (CTCs) shed from the primary tumor. For many years, oncologists believed some medical procedures may provoke metastasis; however, no direct evidence has been reported. We have developed a new, noninvasive technology called in vivo photoacoustic (PA) flow cytometry (PAFC), which provides ultrasensitive detection of CTCs. When CTCs with strongly light-absorbing intrinsic melanin pass through a laser beam aimed at a peripheral blood vessel, laser-induced acoustic waves from CTCs were detected using an ultrasound transducer. We focused on melanoma as it is one of the most metastatically aggressive malignancies. The goal of this research was to determine whether melanoma manipulation, like compression, incisional biopsy, or tumor excision, could enhance penetration of cancer cells from the primary tumor into the circulatory system. The ears of nude mice were inoculated with melanoma cells. Blood vessels were monitored for the presence of CTCs using in vivo PAFC. We discovered some medical procedures, like compression of the tumor, biopsy, and surgery may either initiate CTC release in the blood which previously contained no CTCs, or dramatically increased (10-30–fold) CTC counts above the initial level. Our results warn oncologists to use caution during physical examination, and surgery. A preventive anti-CTC therapy during or immediately after surgery, by intravenous drug administration could serve as an option to treat the resulting release of CTCs.

When a blood vessel is injured, the normal physiological response of the body is to form a clot (thrombus) to prevent blood loss. Alternatively, even without injury to the blood vessel, the pathological condition called thromboembolism may lead to the formation of circulating blood clots (CBCs), also called emboli, which can clog blood vessels throughout the body. Veins of the extremities (venous thromboembolism), lungs (pulmonary embolism ), brain (embolic stroke), heart (myocardial infarction), kidneys, and gastrointestinal tract are often affected. Emboli are also common complications of infection, inflammation, cancer, surgery, radiation and coronary artery bypass grafts. Despite the clear medical significance of CBCs, however, little progress has been made in the development of methods for real-time detection and identification of CBCs. To overcome these limitations, we developed a new modification of in vivo photoacoustic (PA) flow cytometry (PAFC) for real-time detection of white, red, and mixed clots through a transient decrease, increase or fluctuation of PA signal amplitude, respectively. In this work, using PAFC and mouse models, we present for the first time direct evidence that some medical procedures, such as conventional or cancer surgery may initiate the formation of CBCs. In conclusion, the PA diagnostic platform can be used in real-time to define risk factors for cardiovascular diseases, assist in the prognosis and potential prevention of stroke by using a well-timed therapy or as a clot count as a marker of therapy efficacy.

In this study, we present a novel photoacoustic contrast agent which is based on bis-carboxylic acid derivative of Indocyanine green (ICG) covalently conjugated to single-wall carbon nanotubes (ICG/SWCNT). Covalently attaching ICG to the functionalized SWCNT provides a more robust system that delivers much more ICG to the tumor site. The detection sensitivity of the new contrast agent in mouse tumor model is demonstrated in vivo by our custom built photoacoustic imaging system. PAT summation signal is defined to show the long-term light absorption of tumor areas in ICG injected mice and ICG/SWCNT injected mice. It is shown that ICG is able to provide 33% enhancement at approximately 20 minutes peak response time referred to pre-injection PAT summation level, while ICG/SWCNT provides 128% enhancement at 80 minutes and even higher enhancement of 196% at the end point of experiments (120 minutes on average). Additionally, the ICG/SWCNT enhancement was mainly observed at the tumor periphery as confirmed by fluorescence images of the tumor samples. This feature is highly valuable in guiding surgeons to assess tumor boundaries and dimensions in vivo and improve surgical resection of tumors for achieving clean tumor margins.

In this paper, we report an ultrafast co-registered ultrasound and photoacoustic imaging system based on FPGA parallel processing. The system features 128-channel parallel acquisition and digitization, along with FPGA-based reconfigurable processing for real-time co-registered imaging of up to 15 frames per second that is only limited by the laser pulse repetition frequency of 15 Hz. We demonstrated the imaging capability of the system by live imaging of a mouse tumor model in vivo, and imaging of human ovaries ex vivo. A compact transvaginal probe that includes the PAT illumination using a fiber-optic assembly was used for this purpose. The system has the potential ability to assist a clinician to perform transvaginal ultrasound scanning and to localize the ovarian mass, while simultaneously mapping the light absorption of the ultrasound detected mass to reveal its vasculature using the co-registered PAT.

We developed the first prototype of dual-modality imager combining optoacoustic tomography (OAT) and laser ultrasound tomography (UST) using computer models followed by experimental validation. The system designed for preclinical biomedical research can concurrently yield images depicting both the absorbed optical energy density and acoustic properties (speed of sound) of an object. In our design of the UST imager, we seek to replace conventional electrical generation of ultrasound waves by laser-induced ultrasound (LU). While earlier studies yielded encouraging results [Manohar, et al., Appl. Phys. Lett, 131911, 2007], they were limited to two-dimensional (2D) geometries. In this work, we conduct computer-simulation studies to investigate different designs for the 3D LU UST imager. The number and location of the laser ultrasound emitters, which are constrained to reside on the cylindrical surface opposite to the arc of detectors, are optimized. In addition to the system parameters, an iterative image reconstruction algorithm was optimized. We demonstrate that high quality volumetric maps of the speed of sound can be reconstructed when only 32 emitters and 128 receiving transducers are employed to record time-of-flight data at 360 tomographic view angles. The implications of the proposed system for small animal and breast-cancer imaging are discussed.

A three-dimensional mouse imaging system combining optoacoustic tomography and laser ultrasound (LOUIS- 3DM) has been developed. It features broadband laser ultrasound emitters positioned opposite an array of transducers. This imaging geometry allows reconstruction of images that either depicts the speed of sound distribution from measured time of flight data, or acoustic attenuation from the measured signal amplitude. We have investigated the performance of two laser ultrasound source designs, both easily adaptable to a commercial imaging system: small diameter source (600 μm) generated off a flat surface, and larger diameter (3 mm) spherical source. Laser energy requirements are modest, well below 1 mJ per pulse for either design. Their performance at normal incidence is comparable both in amplitude and frequency response. However, off-axis generation differs dramatically and the shortcomings of the simple flat emitter design are evident. We show that, in order to achieve optimal performance through proper illumination of the detector array, spherical wave front characteristics are desired.

In this report, we demonstrate the feasibility of using optoacoustic tomography for deducing biodistributions of nanoparticles in animal models. The redistribution of single-walled carbon nanotubes (SWCNTs) was visualized in living mice. Nanoparticle concentrations in harvested organs were measured spectroscopically using the intrinsic optical absorption and fluorescence of SWCNTs. Observed increases in optoacoustic signal brightness in tissues were compared with increases in optical absorptivity coefficients caused by SWCNT accumulation. The methodology presented in this report paves the way for measuring concentrations of optically absorbing agents in small animals using optoacoustic tomography.

Photoacoustic (PA) imaging has been widely used in molecular imaging to detect diseased cells by targeting them with nanoparticle-based contrast agents. However, the sensitivity and specificity are easily degraded because contrast agent signals can be masked by the background. Magnetomotive photoacoustic imaging uses a new type of multifunctional composite particle combining an optically absorptive gold nanorod core and magnetic nanospheres, which can potentially accumulate and concentrate targeted cells while simultaneously enhancing their specific contrast compared to background signals. In this study, HeLa cells molecularly targeted using nanocomposites with folic acid mimicking targeted rare circulating tumor cells (CTCs) were circulated at a 6 ml/min flow rate for trapping and imaging studies. Preliminary results show that the cells accumulate rapidly in the presence of an externally applied magnetic field produced by a dual magnet system. The sensitivity of the current system can reach up to 1 cell/ml in clear water. By manipulating the trapped cells magnetically, the specificity of detecting cells in highly absorptive ink solution can be enhanced with 16.98 dB background suppression by applying motion filtering on PA signals to remove unwanted background signals insensitive to the magnetic field. The results appear promising for future preclinical studies on a small animal model and ultimate clinical detection of rare CTCs in the vasculature.

Iterative image reconstruction algorithms for photoacoustic computed tomography (PACT) have the ability to improve image quality over analytic algorithms due to their ability to mitigate artifacts from incomplete data, incorporate the relevant imaging physics, and model the instrument response. In this work, Kaiser-Bessel functions are employed as the basis functions in an iterative reconstruction algorithm for PACT. Kaiser-Bessel functions, or blobs, can be made arbitrarily smooth (differentiable to arbitrary order), have finite spatial support, and can be made quasi-bandlimited in the spatial Fourier domain. Closed-form solutions exist in the time-domain and the temporal-frequency domain for the pressure signal generated by blobs.

Circulatory shock can lead to death or severe complications, if not promptly diagnosed and effectively treated. Typically, diagnosis and management of circulatory shock are guided by blood pressure and heart rate. However, these variables have poor specificity, sensitivity, and predictive value. Early goal-directed therapy in septic shock patients, using central venous catheterization (CVC), reduced mortality from 46.5% to 30%. However, CVC is invasive and complication-prone. We proposed to use an optoacoustic technique for noninvasive, rapid assessment of peripheral and central venous oxygenation. In this work we used a medical grade optoacoustic system for noninvasive, ultrasound image-guided measurement of central and peripheral venous oxygenation. Venous oxygenation during shock declines more rapidly in the periphery than centrally. Ultrasound imaging of the axillary [peripheral] and internal jugular vein [central] was performed using the Vivid e (GE Healthcare). We built an optoacoustic interface incorporating an optoacoustic transducer and a standard ultrasound imaging probe. Central and peripheral venous oxygenations were measured continuously in healthy volunteers. To simulate shock-induced changes in central and peripheral oxygenation, we induced peripheral vasoconstriction in the upper extremity by using a cooling blanket. Central and peripheral venous oxygenations were measured before (baseline) and after cooling and after rewarming. During the entire experiment, central venous oxygenation was relatively stable, while peripheral venous oxygenation decreased by 5-10% due to cooling and recovered after rewarming. The obtained data indicate that noninvasive, optoacoustic measurements of central and peripheral venous oxygenation may be used for diagnosis and management of circulatory shock with high sensitivity and specificity.

Monitoring of cerebral venous oxygenation is useful to facilitate management of patients with severe or moderate traumatic brain injury (TBI). Prompt recognition of low cerebral venous oxygenation is a key to avoiding secondary brain injury associated with brain hypoxia. In specialized clinical research centers, jugular venous bulb catheters have been used for cerebral venous oxygenation monitoring and have demonstrated that oxygen saturation < 50% (normal range is 55–75%) correlates with poor clinical outcome. We developed an optoacoustic technique for noninvasive monitoring of cerebral venous oxygenation. Recently, we designed and built a novel, medical grade optoacoustic system operating in the near-infrared spectral range for continuous, real-time oxygenation monitoring in the superior sagittal sinus (SSS), a large central cerebral vein. In this work, we designed and built a novel SSS optoacoustic probe and developed a new algorithm for SSS oxygenation measurement. The SSS signals were measured in healthy volunteers during voluntary hyperventilation, which induced changes in SSS oxygenation. Simultaneously, we measured exhaled carbon dioxide concentration (EtCO₂) using capnography. Good temporal correlation between decreases in optoacoustically measured SSS oxygenation and decreases in EtCO₂ was obtained. Decreases in EtCO2 from normal values (35-45 mmHg) to 20-25 mmHg resulted in SSS oxygenation decreases by 3-10%. Intersubject variability of the responses may relate to nonspecific brain activation associated with voluntary hyperventilation. The obtained data demonstrate the capability of the optoacoustic system to detect in real time minor changes in the SSS blood oxygenation.

Photoacoustic imaging (PAI) is a hybrid imaging modality that integrates the strengths from both optical imaging and acoustic imaging while simultaneously overcoming many of their respective weaknesses. In previous work, we reported on a real-time 3D PAI system comprised of a 32-element hemispherical array of transducers. Using the system, we demonstrated the ability to capture photoacoustic data, reconstruct a 3D photoacoustic image, and display select slices of the 3D image every 1.4 s, where each 3D image resulted from a single laser pulse. The present study aimed to exploit the rapid imaging speed of an upgraded 3D PAI system by evaluating its ability to perform dynamic contrast-enhanced imaging. The contrast dynamics can provide rich datasets that contain insight into perfusion, pharmacokinetics and physiology. We captured a series of 3D PA images of a flow phantom before and during injection of piglet and rabbit blood. Principal component analysis was utilized to classify the data according to its spatiotemporal information. The results suggested that this technique can be used to separate a sequence of 3D PA images into a series of images representative of main features according to spatiotemporal flow dynamics.