Optoacoustic imaging has enabled the visualization of optical contrast at high resolutions in deep tissue. Our Multispectral optoacoustic tomography (MSOT) imaging results reveal internal tissue heterogeneity, where the underlying distribution of specific endogenous and exogenous sources of absorption can be resolved in detail. Technical advances in cardiac imaging allow motion-resolved multispectral measurements of the heart, opening the way for studies of cardiovascular disease. We further demonstrate the fast characterization of the pharmacokinetic profiles of lightabsorbing agents. Overall, our MSOT findings indicate new possibilities in high resolution imaging of functional and molecular parameters.

We combined photoacoustic ophthalmoscopy (PAOM) with autofluorescence imaging for simultaneous in vivo imaging of dual molecular contrasts in the retina using a single light source. The dual molecular contrasts come from melanin and lipofuscin in the retinal pigment epithelium (RPE). Melanin and lipofuscin are two types of pigments and are believed to play opposite roles (protective vs. exacerbate) in the RPE in the aging process. We successfully imaged the retina of pigmented and albino rats at different ages. The experimental results showed that multimodal PAOM system can be a potentially powerful tool in the study of age-related degenerative retinal diseases.

Anti-cancer drugs typically exert their pharmacological effect on tumors by inducing apoptosis, or programmed cell death, within the cancer cells, with PCD occurring as soon as 4 hours after treatment. Detection of apoptosis in patients could decisively report a response to treatment days or even weeks before MRI, CAT, and ultrasound indicate morphological changes in the tumor. Here we developed a novel near-infrared dye based imaging probe to directly detect apoptosis with high specificity in cancer cells by utilizing a non-invasive photoacoustic imaging technique. Nude mice bearing head and neck tumors received cisplatin chemotherapy were imaged by PAI after tail vein injection of the contrast agent. In vivo PAI indicated a strong apoptotic response to chemotherapy on the peripheral margins of tumors, whereas untreated controls showed no contrast enhancement by PAI. The apoptotic status of the mouse tumor tissue was verified by immunohistochemical techniques staining for cleaved caspase-3 p11 subunit. The results demonstrated the potential of this imaging probe to guide the evaluation of chemotherapy treatment.

Functional detection in primate brains has particular advantages because of the similarity between non-human primate brain and human brain and the potential for relevance to a wide range of conditions such as stroke and Parkinson's disease. In this research, we used photoacoustic imaging (PAI) technique to detect functional changes in primary motor cortex of awake rhesus monkeys. We observed strong increases in photoacoustic signal amplitude during both passive and active forelimb movement, which indicates an increase in total hemoglobin concentration resulting from activation of primary motor cortex. Further, with PAI approach, we were able to obtain depthresolved functional information from primary motor cortex. The results show that PAI can reliably detect primary motor cortex activation associated with forelimb movement in rhesus macaques with a minimal-invasive approach.

Photoacoustic and thermoacoustic phantom images obtained with a multi-channel breast scanner designed for breast cancer screening are presented here. A tunable laser system (OPOTEK Vibrant 355 I, Calsbad,CA) with a pulse duration of 5 ns was used for photoacoustic irradiation, and a 3.0 GHz microwave source with a pulse width of 0.3-1 μs was used for thermoacoustic tomography. Multiple (>=16) 2.25 MHz single-element unfocused ultrasonic transducers at different depths were scanned simultaneously for a full 360° to obtain a full data set for three-dimensional (3D) tomography. Negative acoustic lenses were attached to these unfocused transducers to increase their acceptance angles. An ultrasound receiving system with 64 parallel receiving channels (Verasonics Inc. Redmond, WA) was used for data acquisition. A filtered backprojection algorithm was used to reconstruct two-dimensional (2D) and 3D images. Different phantoms were imaged to evaluate the performance of the scanner. A lateral resolution of less than 1 mm and an elevational resolution of less than 5 mm were achieved. The phantom studies demonstrate that this scanner can potentially provide high-resolution, dual-modality, three-dimensional images and can potentially be used for human breast cancer screening.

Two-dimensional optoacoustic imaging with a hand-held probe operated in backward mode is being developed for diagnostic imaging of breast cancer to evaluate the feasibility of a dual-modality optoacoustic plus ultrasonic system that maps functional information of anatomical tissue structures with ultrasonic resolution. Tissue is illuminated at 757nm and 1064nm for optical contrast between hypoxic blood of breast carcinomas and normally oxygenated blood in benign masses. The system is optimized and calibrated in phantoms for a pilot clinical study of patients with breast masses suspected for malignancy. Capability of the non-invasive system to improve detection and diagnosis of breast tumors is discussed.

Current imaging modalities are often not able to detect early stages of breast cancer with high imaging contrast. Visualizing malignancy-associated increased hemoglobin concentrations might improve breast cancer diagnosis. Photoacoustic imaging can visualize hemoglobin in tissue with optical contrast and ultrasound resolution, which makes it potentially ideal for breast imaging. The Twente Photoacoustic Mammoscope (PAM) has been designed specifically for this purpose. Based on a successful pilot study in 2007, a large clinical study using PAM has been started in December 2010. PAM uses a pulsed Q-switched Nd:YAG laser at 1064 nm to illuminate a region of interest on the breast. Photoacoustic signals are detected with a 1MHz, unfocused ultrasound detector array. Three dimensional data are reconstructed using an acoustic backprojection algorithm. Those reconstructed images are compared with conventional imaging and histopathology. In the first phase of the study, the goal was to optimize the visualization of malignancies. We performed sixteen technically acceptable measurements on confined breast malignancies. In the reconstructed volumes of all malignancies, a confined high contrast region could be identified at the expected lesion depth. After ten successful measurements, the illumination area was increased and the fluence was substantially decreased. This caused a further significant increase in PAM lesion contrast.

We developed a new optoacoustic microangiography system (OmAS) intended for in-vivo vascular imaging of a human finger. The system employs an arc-shaped acoustic array that is rotated 360 degrees around the finger providing optoacoustic data necessary for tomographic reconstruction of the three-dimensional images of a finger. A near-infrared Q-switched laser is used to generate optoacoustic signals with increased contrast of blood vessels. The laser is coupled through two randomized fiberoptic bundles oriented in orthogonal optoacoustic mode. To demonstrate OmAS capabilities, we present a time-series of optoacoustic images of a human finger taken after the hypothermia stress test. The images show a detailed vascular anatomy of a finger down to the capillary level. A series of quick 30s scans allowed us to visualize the thermoregulatory response within the studied finger as it was manifested via vasomotor activity during the hypothermia recovery. We propose that the developed system can be used for diagnostics of various medical conditions that are manifested in change of the peripheral (finger) blood flow. Examples of the medical conditions that could be diagnosed and staged using the OmAS include the peripheral arterial disease (PAD), thrombosis, frostbite, and traumas.

The development and performance of a cw, TE-cooled DFB quantum cascade laser based sensor for quantitative measurements of ammonia (NH₃) and nitric oxide (NO) concentrations present in exhaled breath will be reported. Human breath contains ~ 500 different chemical species, usually at ultra low concentration levels, which can serve as biomarkers for the identification and monitoring of human diseases or wellness states. By monitoring NH₃ concentration levels in exhaled breath a fast, non-invasive diagnostic method for treatment of patients with liver and kidney disorders, is feasible. The NH3 concentration measurements were performed with a 2f wavelength modulation quartz enhanced photoacoustic spectroscopy (QEPAS) technique, which is suitable for real time breath measurements, due to the fast gas exchange inside a compact QEPAS gas cell. A Hamamatsu air-cooled high heat load (HHL) packaged CW DFB-QCL is operated at 17.5°C, targeting the optimum interference free NH₃ absorption line at 967.35 cm^-1 (λ~10.34 μm), with ~ 20 mW of optical power. The sensor architecture includes a reference cell, filled with a 2000 ppmv NH₃ :N₂ mixture at 130 Torr, which is used for absorption line-locking. A minimum detection limit (1σ) for the line locked NH3 sensor is ~ 6 ppbv (with a 1σ; 1 sec time resolution of the control electronics). This NH₃ sensor was installed in late 2010 and is being clinically tested at St. Luke's Hospital in Bethlehem, PA.

Circulatory shock is lethal, if not promptly diagnosed and effectively treated. Typically, circulatory shock resuscitation is guided by blood pressure, heart rate, and mental status, which have poor predictive value. In patients, in whom early goaldirected therapy was applied using central venous oxygenation measurement, a substantial reduction of mortality was reported (from 46.5% to 30%). However, central venous catheterization is invasive, time-consuming and often results in complications. We proposed to use the optoacoustic technique for noninvasive, rapid assessment of central venous oxygenation. In our previous works we demonstrated that the optoacoustic technique can provide measurement of blood oxygenation in veins and arteries due to high contrast and high resolution. In this work we developed a novel optoacoustic system for noninvasive, automatic, real-time, and continuous measurement of central venous oxygenation. We performed pilot clinical tests of the system in human subjects with different oxygenation in the internal jugular vein and subclavian vein. A novel optoacoustic interface incorporating highly-sensitive optoacoustic probes and standard ultrasound imaging probes were developed and built for the study. Ultrasound imaging systems Vivid i and hand-held Vscan (GE Healthcare) as well as Site-Rite 5 (C.R. Bard) were used in the study. We developed a special algorithm for oxygenation monitoring with minimal influence of overlying tissue. The data demonstrate that the system provides precise measurement of venous oxygenation continuously and in real time. Both current value of the venous oxygenation and trend (in absolute values and for specified time intervals) are displayed in the system. The data indicate that: 1) the optoacoustic system developed by our group is capable of noninvasive measurement of blood oxygenation in specific veins; 2) clinical ultrasound imaging systems can facilitate optoacoustic probing of specific blood vessels; 3) the optoacoustic system provides noninvasive monitoring during rapid changes in blood oxygenation.

We report herein a novel three-dimensional photoacoustic computed tomography (PACT) system for small-animal whole-body imaging. The PACT system, based on a 512-element full-ring ultrasonic transducer array, was cylindrically focused and capable of forming a two-dimensional image in 1.6 seconds. The pulsed laser could either illuminate directly from the top or be reshaped to illuminate the sample from the side. Top illumination was mainly used for mouse brain and mouse embryo imaging. Side illumination provided in vivo anatomical images of an adult mouse. By translating the mouse along the elevational direction, the system provided serial cross-sectional images.

The ability to detect molecular probes in deep tissue, based on optical signatures, has been limited by tissue scattering, which reduces the spatial resolution and complicates quantification. To address this challenge, multispectral optoacoustic tomography (MSOT) has been recently introduced, a hybrid technology that capitalizes on the optoacoustic effect to combine rich optical contrast with the high spatial resolution and real-time operation of ultrasound. Using multiwavelength illumination MSOT can visualize molecular probes based on their distinct optical absorption spectra through several millimeters to centimeters of tissue. Herein we present a whole body multi-spectral optoacoustic tomography system and report on spectral processing techniques for detection of molecular probes in living mice.

In this paper we introduce a novel optical resolution photoacoustic micro-endoscopy system using GRIN-lens focusing. This real-time imaging system takes advantage of an image guide fiber consisting of 100,000 individual single-mode fibers in a 1.4-mm-diameter bundle, and a 532-nm fiber laser with repetition-rates as high as 600 kHz. Fast scanning mirrors were used to scan the entire image guide fiber allowing for the maximum field-of-view. The ability of the proposed system is demonstrated using both phantom and in vivo studies. The system offers <7 μm lateral spatial resolution and several volumetric/C-scans per second frame-rate. The proposed setup can be inserted into the body due to the flexible nature of the image guide and micron-scale footprint of the apparatus.

Using the method of 3D optoacoustic tomography, we studied changes in tissues of the whole body of nude mice as the changes manifested themselves from live to postmortem. The studies provided the necessary baseline for optoacoustic imaging of necrotizing tissue, acute and chronic hypoxia, and reperfusion. They also establish a new optoacoustic model of early postmortem conditions of the whole mouse body. Animals were scanned in a 37°C water bath using a three-dimensional optoacoustic tomography system previously shown to provide high contrast maps of vasculature and organs based on changes in the optical absorbance. The scans were performed right before, 5 minutes after, 2 hours and 1 day after a lethal injection of KCl. The near-infrared laser wavelength of 765 nm was used to evaluate physiological features of postmortem changes. Our data showed that optoacoustic imaging is well suited for visualization of both live and postmortem tissues. The images revealed changes of optical properties in mouse organs and tissues. Specifically, we observed improvements in contrast of the vascular network and organs after the death of the animal. We associated these with reduced optical scattering, loss of motion artifacts, and blood coagulation.

A photoacoustic tomography (PAT) system using a virtual point ultrasonic transducer was developed for transcranial imaging of monkey brains. The virtual point transducer provided a 10 times greater field-of-view (FOV) than finiteaperture unfocused transducers, which enables large primate imaging. The cerebral cortex of a monkey brain was accurately mapped transcranially, through up to two skulls ranging from 4 to 8 mm in thickness. The mass density and speed of sound distributions of the skull were estimated from adjunct X-ray CT image data and utilized with a timereversal algorithm to mitigate artifacts in the reconstructed image due to acoustic aberration. The oxygenation saturation (sO2) in blood phantoms through a monkey skull was also imaged and quantified, with results consistent with measurements by a gas analyzer. The oxygenation saturation (sO2) in blood phantoms through a monkey skull was also imaged and quantified, with results consistent with measurements by a gas analyzer. Our experimental results demonstrate that PAT can overcome the optical and ultrasound attenuation of a relatively thick skull, and the imaging aberration caused by skull can be corrected to a great extent.

Two human tumour cell lines (K562, 293T) were stably transfected to achieve the genetic expression of tyrosinase, which is involved in the production of the pigment eumelanin. The cells were injected subcutaneously into nude mice to form tumour xenografts, which were imaged over a period of up to 26 days using an all-optical photoacoustic imaging system. 3D photoacoustic images of the tumours and the surrounding vasculature were acquired at excitation wavelengths ranging from 600nm to 770nm. The images showed tumour growth and continued tyrosinase expression over the full 26 day duration of the study. These findings were confirmed by histological analysis of excised tumour samples.

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, imbalance of pH regulation may result from or result in serious illness. Even though the regulation system of pH is very robust, tissue pH can be altered in many diseases such as cancer, osteoporosis and diabetes mellitus. Traditional high-resolution optical imaging techniques, such as confocal microscopy, routinely image pH in cells and tissues using pH sensitive fluorescent dyes, which change their fluorescence properties with the surrounding pH. Since strong optical scattering in biological tissue blurs images at greater depths, high-resolution pH imaging is limited to penetration depths of 1mm. Here, we report photoacoustic microscopy (PAM) of commercially available pH-sensitive fluorescent dye in tissue phantoms. Using both opticalresolution photoacoustic microscopy (OR-PAM), and acoustic resolution photoacoustic microscopy (AR-PAM), we explored the possibility of recovering the pH values in tissue phantoms. In this paper, we demonstrate that PAM was capable of recovering pH values up to a depth of 2 mm, greater than possible with other forms of optical microscopy.

Photoacoustic imaging has been widely used in structural and functional imaging. Because of its safety, high resolution, and high imaging depth, it has great potential for a variety of medical studies. Capillaries are the smallest blood vessels and enable the exchange of oxygen and nutrients. Noninvasive flow speed measurement of capillaries in vivo can benefit the study of vascular tone changes and rheological properties of blood cells in capillaries. Recently, there has been a growing interest in photoacoustic velocimetry, such as photoacoustic Doppler and M-mode photoacoustic flow imaging. Methods capable of high-resolution imaging and low-speed flow measurement are suitable to measure blood speeds in capillaries. Previously we proposed photoacoustic correlation spectroscopy (PACS) and shown its feasibility for lowspeed flow measurement. Here, in vivo measurement of blood speeds in capillaries in a chick embryo model by PACS technique is demonstrated. The laser-scanning photoacoustic microscopy system is used for fast imaging acquisition and high-resolution imaging. The measured speed in capillaries is similar to those found in literatures, which confirm the feasibility of the PACS method for blood velocimetry. This technique suggests a fairly simple way to study blood flow speeds in capillaries.

For deep tissue photoacoustic imaging, piezoelectric ultrasound detectors with large element sizes (>1mm) and relatively low centre frequencies (<5MHz) are generally used, as they can provide the required high sensitivity to achieve imaging depths of several centimetres. However, these detectors are generally not optimised in terms of element size and bandwidth. To identify these parameters in order to improve SNR and spatial resolution, two models were employed. The first was a numerical model and was used to investigate the effect of varying the detector element size on the amplitude and SNR of photoacoustic images. The second model was used to optimise the detector bandwidth. For this, the frequency content of simulated photoacoustic signals were studied for a range of depths and acoustic source sizes. The model was based on an analytical solution to the wave equation for a cylindrical source and incorporated the effects of frequency dependent acoustic attenuation. These models provide a new framework for optimising the design of photoacoustic scanners for breast and other deep tissue imaging applications.

A numerical inversion scheme for recovering a map of the absolute conductivity from the absorbed power density map that is conventionally reconstructed in thermacoustic imaging is described. This offers the prospect of obtaining an image that is more closely related to the underlying tissue structure and physiology. The inversion scheme employs a full 3D full wave model of electromagnetic propagation in tissue which is iteratively fitted to the measured absorbed power density map using a simple recursive method. The reconstruction is demonstrated numerically using three examples of absorbers of varying geometries, tissue realistic complex permittivity values and noise. In these examples, the reconstruction is shown to rapidly converge to within good estimates of the true conductivity in less than 20 iterations.

Tyrosinase, a key enzyme in the production of melanin, has shown promise as a reporter of genetic activity. While green fluorescent protein has been used extensively in this capacity, it is limited in its ability to provide information deep in tissue at a reasonable resolution. As melanin is a strong absorber of light, it is possible to image gene expression using tyrosinase with photoacoustic imaging technologies, resulting in excellent resolutions at multiple-centimeter depths. While our previous work has focused on creating and imaging MCF-7 cells with doxycycline-controlled tyrosinase expression, we have now established the viability of these cells in a murine model. Using an array-based photoacoustic imaging system with 5 MHz center frequency, we capture interleaved ultrasound and photoacoustic images of tyrosinase-expressing MCF-7 tumors both in a tissue mimicking phantom, and in vivo. Images of both the tyrosinase-expressing tumor and a control tumor are presented as both coregistered ultrasound-photoacoustic B-scan images and 3-dimensional photoacoustic volumes created by mechanically scanning the transducer. We find that the tyrosinase-expressing tumor is visible with a signal level 12dB greater than that of the control tumor in vivo. Phantom studies with excised tumors show that the tyrosinase-expressing tumor is visible at depths in excess of 2cm, and have suggested that our imaging system is sensitive to a transfection rate of less than 1%.

Photoacoustic (PA) and thermoacoustic (TA) effects are based on the generation of acoustic waves after tissues absorb electromagnetic energy. The amplitude of the acoustic signal is related to the temperature of the absorbing target tissue. A combined photoacoustic and thermoacoustic imaging system built around a modified commercial ultrasound scanner was used to obtain an image of the target's temperature, using reconstructed photoacoustic or thermoacoustic images. To demonstrate these techniques, we used photoacoustic imaging to monitor the temperature changes of methylene blue solution buried at a depth of 1.5 cm in chicken breast tissue from 12 to 42 °C. We also used thermoacoustic imaging to monitor the temperature changes of porcine muscle embedded in 2 cm porcine fat from 14 to 28 °C. The results demonstrate that these techniques can provide noninvasive real-time temperature monitoring of embedded objects and tissue.

As two hallmarks of cancer, angiogenesis and hypermetabolism are closely related to increased blood flow. Volumetric blood flow measurement is important to understanding the tumor microenvironment and developing new means to treat cancer. Current photoacoustic blood flow estimation methods focus on either the axial or transverse component of the flow vector. Here, we propose a method to compute the total flow speed and Doppler angle by combining the axial and transverse flow measurements. Both the components are measured in M-mode. Collating the A-lines side by side yields a 2D matrix. The columns are Hilbert transformed to compare the phases for the computation of the axial flow. The rows are Fourier transformed to quantify the bandwidth for the computation of the transverse flow. From the axial and transverse flow components, the total flow speed and Doppler angle can be derived. The method has been verified by flowing bovine blood in a plastic tube at various speeds from 0 to 7.5 mm/s and at Doppler angles from 30 to 330°. The measurement error for total flow speed was experimentally determined to be less than 0.3 mm/s; for the Doppler angle, it was less than 15°. In addition, the method was tested in vivo on a mouse ear. The advantage of this method is simplicity: No system modification or additional data acquisition is required to use our existing system. We believe that the proposed method has the potential to be used for cancer angiogenesis and hypermetabolism imaging.

The concept of pure optical photoacoustic microscopy(POPAM) was proposed based on optical rastering of a focused excitation beam and optically sensing the photoacoustic signal using a microring resonator fabricated by a nanoimprinting technique. After some refinedment of in the resonator structure and mold fabrication, an ultrahigh Q factor of 3.0×105 was achieved which provided high sensitivity with a noise equivalent detectable pressure(NEDP) value of 29Pa. This NEDP is much lower than the hundreds of Pascals achieved with existing optical resonant structures such as etalons, fiber gratings and dielectric multilayer interference filters available for acoustic measurement. The featured high sensitivity allowed the microring resonator to detect the weak photoacoustic signals from micro- or submicroscale objects. The inherent superbroad bandwidth of the optical microring resonator combined with an optically focused scanning beam provided POPAM of high resolution in the axial as well as both lateral directions while the axial resolution of conventional photoacoustic microscopy (PAM) suffers from the limited bandwidth of PZT detectors. Furthermore, the broadband microring resonator showed similar sensitivity to that of our most sensitive PZT detector. The current POPAM system provides a lateral resolution of 5μm and an axial resolution of 8μm, comparable to that achieved by optical microscopy while presenting the unique contrast of optical absorption and functional information complementing other optical modalities. The 3D structure of microvasculature, including capillary networks, and even individual red blood cells have been discerned successfully in the proof-of-concept experiments on mouse bladders ex vivo and mouse ears in vivo. The potential of approximately GHz bandwidth of the microring resonator also might allow much higher resolution than shown here in microscopy of optical absorption and acoustic propagation properties at depths in unfrozen tissue specimens or thicker tissue sections not now imageable with current optical or acoustic microscopes of comparable resolution.

Small animal models, such as zebrafish, drosophila, C. elegan, is considered to be important models in comparative biology and diseases researches. Traditional imaging methods primarily employ several optical microscopic imaging modalities that rely on fluorescence labeling, which may have potential to affect the natural physiological progress. Thus a label-free imaging method is desired. Photoacoustic (PA) microscopy (PAM) is an emerging biomedical imaging method that combines optical contrast with ultrasonic detection, which is highly sensitive to the optical absorption contrast of living tissues, such as pigments, the vasculature and other optically absorbing organs. In this work, we reported the whole body label-free imaging of zebrafish larvae and drosophila pupa by PAM. Based on intrinsic optical absorption contrast, high resolution images of pigments, microvasculature and several other major organs have been obtained in vivo and non-invasively, and compared with their optical counterparts. We demonstrated that PAM has the potential to be a powerful non-invasive imaging method for studying larvae and pupa of various animal models.

Recently we have reported in vivo near-real-time volumetric optical-resolution photoacoustic microscopy (OR-PAM) using a high pulse-repetition-rate (PRR) nanosecond fiber-laser to realize 2 volumetric image frames per second (fps) within 1mm × 1mm field of view (FOV). Based on our previous OR-PAM system, we are developing a label-free realtime OR-PAM system in reflection mode for higher frame-rates. The system permits imaging of microcirculation hemodynamics, and helps make the technology easier to use for biologists, providing real-time feedback for focusing and positioning. Using a nanosecond-pulsed 532-nm fiber laser combined with fast-scanning mirrors, our proposed system demonstrated its capability of sustained in vivo imaging of horizontal and vertical translation at 0.5 fps for 1mm × 1mm FOV (400 × 400 pixels). Also, real-time in vivo imaging of blood flow at 30 fps for 250μm × 250μm FOV (100 × 100 pixels) was demonstrated. It is anticipated that the real-time nature of the system should prove important in clinical and preclinical adaption, and may prove useful for functional brain imaging studies.

In this report we introduce a novel experimental design for non-invasive scanning optoacoustic microscopy that utilizes an off-axis parabolic mirror. Such reflector provides ideal and lossless conversion of a spherical wavefront into a plane wave and enables diffraction-limited ultrasound focusing. We have designed and build a custom broadband transducer with 0-19 MHz bandwidth and nominal sensitivity of 15 μV/Pa. With 17 dB amplification and noise level of ~ 1.6 mV the estimated sensitivity limit of our experimental setup is 15 Pa. Using the reflector with numerical aperture of 0.5, we have demonstrated lateral resolution limit of ~ 100 micrometers in test phantoms.

We developed multi-contrast photoacoustic microscopy (PAM) for in vivo anatomical, functional, metabolic, and molecular imaging. This technical innovation enables comprehensive understanding of the tumor microenvironment. With multi-contrast PAM, we longitudinally determined tumor vascular anatomy, blood flow, oxygen saturation of hemoglobin, and oxygen extraction fraction.

The acquisition speed of previously reported mechanically-scanned Optical-Resolution Photoacoustic Microscopy (OR-PAM) systems has been limited by both laser pulse repetition rate and mechanical scanning speed. In this paper we introduce a mosaicing scheme wherein a grid of small sub-mm-scale field-of-view (FOV) patches are acquired in 0.5s per patch, and a 3-axis stepper-motor system is used to mechanically move the object to be imaged from patch-to-patch in less than 0.5s. Patch images are aligned and stitched to generate a large FOV image composite. This system retains the SNR-advantages of focused-transducer OR-PAM systems, and is a hybrid approach between optical-scanning and mechanical scanning. With this strategy we reduce the data acquisition time of previously reported large-FOV systems by a factor of around 23. SCID hairless mice are imaged. The wide-FOV, high-speed data acquisition OR-PAM system broadens the potential applications of the imaging modality.

We present a spectrally encoded photoacoustic microscope based on a digital mirror device (DMD). It enables fast spatially resolved spectral measurements of optical absorption. The imaging system can quickly tune the laser illumination spectrum at the laser pulse repetition rate of 2 kHz. To demonstrate multi-wavelength absorption measurements, we imaged optically absorbing solution phantom. Compared with spectral scanning, spectral encoding recovers chromophore absorption spectra with improved accuracy by enhancing photoacoustic amplitude signal-to-noise ratio.

Dependencies of the optoacoustic (OA) transformation efficiency on tissue temperature were obtained for the application in OA temperature monitoring during thermal therapies. Accurate measurement of the OA signal amplitude versus temperature was performed in different ex-vivo tissues in the temperature range 25°C - 80°C. The investigated tissues were selected to represent different structural components: chicken breast (skeletal muscle), porcine lard (fatty tissue) and porcine liver (richly perfused tissue). Backward mode of the OA signal detection and a narrow probe laser beam were used in the experiments to avoid the influence of changes in light scattering with tissue coagulation on the OA signal amplitude. Measurements were performed in heating and cooling regimes. Characteristic behavior of the OA signal amplitude temperature dependences in different temperature ranges were described in terms of changes in different structural components of the tissue samples. Finally, numerical simulation of the OA temperature monitoring with a linear transducers array was performed to demonstrate the possibility of real-time temperature mapping.

The cell differentiation and proliferation of the central nervous system (CNS) are closely related to vascular development. An imaging protocol that integrated optoacoustics (OA) with high-frequency ultrasound (HFU) was developed for in vivo imaging of brain ventricles and vasculature in mouse embryos. A 40-MHz, co-polymer, 5-element annular-array transducer with a geometric focus of 12 mm was modified to accommodate free-beam, coaxial illumination. Three-dimensional (3-D) data sets were acquired by raster scanning the transducer-optics assembly in 50-μm increments. A single intact conceptus from an anesthetized mouse was surgically exposed into PBS-filled Petri-dish. An 800-μm spot illumination from a pulsed, 532-nm, Nd-YAG laser was synchronized with a high-voltage impulse excitation of the central array element to facilitate simultaneous and spatially coregistered OA and HFU data acquisition. The resulting OA and HFU signals from each scan location were recorded on all five array channels and post-processed using a synthetic-focusing algorithm to enhance the depth of field (DOF). Dual-modality images were acquired from mouse embryos at E11.5, E12.5, and E13.5 days of gestation. The extended DOF allowed morphologically accurate visualization of the embryonic head. The brain ventricles were segmented from the HFU data and rendered in 3-D. The OA data provided visualization of the vascular plexus as well as individual blood vessels. Feasibility of spatially co-registered, low-cost dual-modality in vivo imaging of mouse embryos was demonstrated.

The long-term goal of our research is to develop photoacoustic and Doppler ultrasound imaging methods for noninvasive estimation of the oxygen consumption rate (MRO₂) in vivo. Previously, we have demonstrated a combined photoacoustic and high-frequency Doppler ultrasound system and shown the feasibility of flow velocity and oxygen saturation (sO₂) estimation using double-ink flow phantoms. In this work, the results of in vitro sheep blood experiments are presented. Blood oxygen flux has been estimated at different sO2 levels and mean flow speeds, and the uncertainty of the measurement has been quantified. In vivo experiments have been performed on Swiss Webster mice to provide coregistered photoacoustic and Doppler flow images with imaging depths of ~2mm. Doppler bandwidth broadening technique has been used to obtain transverse flow velocity. The diameter of the blood vessel is ~500μm and the mean flow speed is 15cm/s. We are working towards sO₂ estimation in vivo and 3D oxygen consumption imaging of tumors at depths beyond OR-PAM.

Photoacoustic endoscopy (PAE) provides unique functional information with high spatial resolution at super depths. The provision of functional information is predicated on its strong spectroscopic imaging ability, and its deep imaging capability is derived from its ultrasonic detection of diffused photon absorption. To accurately image functional physiological parameters, it is necessary to rapidly alternate laser pulses of sufficient energy and different wavelengths. In this study, we developed peripheral optical systems for PAE based on two identical pulsed-laser systems to achieve the fast laser wavelength switching that is essential for accurate functional imaging. Each laser system was comprised of a tunable dye laser pumped by a solid-state, diode-pumped Nd:YLF laser. Both systems deliver adequate energy at the scanning head of the endoscope for imaging biological tissue in the optically diffusive regime (~0.3-0.6 mJ per pulse with a repetition rate of ~1 kHz). In this paper, we introduce the employed laser systems and design of the light delivery optics, and present results from an ex vivo animal imaging experiment that validates the system's multi-wavelength functional imaging capability.

This paper investigates the use of thin-film optical transmitters to generate focused ultrasound, aiming to develop highamplitude focused ultrasound. Composite films were used as the optoacoustic sources, which consist of carbonnanotubes (CNTs) and elastomeric polymers. As the nano-composites work as excellent optical absorbers and efficient heat converters, thermo-elastic volume deformation within the composites produces strong optoacoustic pressure. These films were formed on concave substrates for optoacoustic generation of the focused ultrasound. A focal waveform was measured using a single-mode fiber-optic hydrophone. A peak positive pressure of ~4 MPa was achieved.

We present a new micro-endoscopy system combining real-time C-scan optical-resolution photoacoustic micro-endoscopy (OR-PAME), and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the OR-PAM sub-system is capable of imaging with a resolution of ~ 7μm. The fluorescence sub-system consists of a diode laser with 445 nm-centered emissions as the light source, an objective lens and a CCD camera. Proflavine, a FDA approved drug for human use, is used as the fluorescent contrast agent by topical application. The fluorescence system does not require any mechanical scanning. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single mode fibers. The absorption of Proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural and functional information given by OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping researchers and clinicians visualize angiogenesis, effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Photoacoustic (PA) imaging has been investigated for intravascular applications. One of the main challenges is that the imaging frame rate is limited by the pulse repetition frequency (PRF), thus making real-time imaging difficult with most high-power solid-state pulse lasers. The goal of this study is to combine omni-directional optical excitation with a ring array transducer for high-frame-rate imaging, so that the image frame rate is the same as the laser PRF. In the preliminary study, we developed a real-time integrated IVUS/IVPA imaging system by modifying an IVUS system in combination with a high-speed Nd:YLF pulsed laser. In addition, an optical fiber with axicon-like distal tip is designed for omni-directional excitation. In this design, a PA image is acquired without rotating the laser light. The imaging frame rate of this integrated imaging system is 19 fps. Both US and PA images are recorded at the same time and co-registered in the fusion image. The US/PA images of tungsten wire, black tube and rabbit's atherosclerotic aorta were acquired with this integrated system to evaluate its imaging performance. The lateral/axial -6 dB resolution of US image is 2.56°/62.4μm. Resolution of PA imaging is 3.76°/91.5μm. The imaging system was also utilized to acquire IVUS/IVPA images of atherosclerotic rabbit's aorta in ex vivo study.

We present a new photoacoustic (PA) cell, which is sealed on the sample side with a 163 μm thick chemical vapor deposition (CVD) diamond window. The investigation of samples containing volatile compounds with an openended PA cell leads to varying conditions in the PA chamber (changing light absorption or relative humidity) and thus causes unstable signals. In contrast the diamond cover ensures stable conditions in the PA chamber and thereby enables sensitive measurements. This is particularly important for the investigation of biological samples with a high water content. Due to the high thermal conductivity of CVD diamond (1800 W/mK) strong PA signals are generated and the broad optical transmission range (250 nm to THz) renders the cell useful for various applications. The performance of the cell is demonstrated by tracking glucose in aqueous keratinocyte solutions with an external-cavity quantum cascade laser (1010-1095 cm^-1). These measurements yield a detection limit of 100 mg/dl (SNR=3). Although glucose measurements within the human physiological range (30-500 mg/dl) are feasible, further improvements are needed for non-invasive glucose monitoring of diabetes patients. First in vivo measurements at the human forearm show an additional PA signal induced by blood pulsation at a frequency around 1 Hz and a steadily increasing relative humidity in the PA chamber due to transepidermal water loss if the cell is neither closed with a diamond window nor ventilated with N₂.

The problem of how to effectively deliver light dynamically to a small volume inside turbid media has been intensively investigated for imaging and therapeutic purposes. Most recently, a new modality termed Time-Reversed Ultrasonically Encoded (TRUE) optical focusing was proposed by integrating the concepts of ultrasound modulation of diffused light with optical phase conjugation. In this work, the diffused photons that travel through the ultrasound focal region are "tagged" with a frequency shift due to the ultrasound modulation. Part of the tagged light is collected in reflection mode and transmitted to a photorefractive crystal, forming there a stationary hologram through interference with a coherent reference optical beam. The hologram is later read by a conjugated optical beam, generating a phase conjugated wavefront of the tagged light. It is conveyed back to the turbid medium in reflection mode, and eventually converges to the ultrasound focal zone. Optical focusing effects from this system are demonstrated experimentally in tissue-mimicking phantoms and ex vivo chicken breast tissue, achieving effective round-trip optical penetration pathlength (extinction coefficient multiplied by round-trip focusing depth) exceeding 160 and 100, respectively. Examples of imaging optical inclusions with this system are also reported.

Photoacoustic tomography (PAT) and ultrasonography (US) of biological tissues usually rely on ultrasonic transducers for the detection of ultrasound. For an optimum sensitivity, transducers require a physical contact with the tissue using a coupling fluid (water or gel). Such a contact is a major drawback in important potential applications such as surgical procedures on human beings and small animal imaging in research laboratories. On the other hand, laser ultrasonics (LU) is a well established optical technique for the non-contact generation and detection of ultrasound in industrial materials. In this paper, the remote optical detection scheme used in industrial LU is adapted to allow the detection of ultrasound in biological tissues while remaining below laser exposure safety limits. Both non-contact PAT (NCPAT) and non-contact US (NCUS) are considered experimentally using a high-power single-frequency detection laser emitting suitably shaped pulses and a confocal Fabry-Perot interferometer in differential configuration. It is shown that an acceptable sensitivity is obtained while remaining below the maximum permissible exposure (MPE) of biological tissues. Results were obtained ex vivo on chicken breast specimens with embedded inclusions simulating blood vessels optical properties. Sub-mm inclusions are readily detected at depths approaching 1 cm. The method is expected to be applicable to living tissues.

The temperature dependence of photoacoustic generation is utilized for monitoring the temperature in flowing blood. A phantom blood vessel is probed with photoacoustic (PA) excitation from a 830nm laser diode whose intensity is sinusoidally modulated at ultrasound frequencies. A second laser diode at the same wavelength is used to photothermally (PT) induce sinusoidal temperature fluctuations in the probed volume. The temperature oscillations lead to modulation sidebands in the PA response. Measurement of the sidebands amplitude as a function of the PT modulation frequency, for different flow rates, reveals a strong dependence of the PT modulation frequency response (MFR) on the flow rate. This is attributed to the thermal properties of the volume under test, and in particular to the heat clearance rate, which is strongly affected by the flow. A simplified lumped model based on the similarity between the system temporal behavior and that of an RC circuit is used to analyze the resulting MFR's. With the addition of an appropriate calibration protocol and by using multispectral PA and/or PT excitation the proposed approach can be used for simultaneous in-vivo measurement of both flow and oxygenation level.

Photoacoustic imaging (PAI) combines high ultrasound resolution with optical contrast. Laser-generated ultrasound is potentially beneficial for cancer detection, blood oxygenation imaging, and molecular imaging. PAI is generally performed using solid state Nd:YAG lasers in combination with optical parametric oscillators. An alternative approach uses laser diodes with higher pulse repetition rates but lower power. Thus, improvement in signal-to-noise ratio (SNR) is a key step towards applying laser diodes in PAI. To receive equivalent image quality using laser diodes as with Nd:YAG lasers, the lower power must be compensated by averaging, which can be enhanced through coded excitation. In principle, perfect binary sequences such as orthogonal Golay codes can be used for this purpose when acquiring data at multiple wavelengths. On the other hand it was shown for a single wavelength that sidelobes can remain invisible even if imperfect sequences are used. Moreover, SNR can be further improved by using an imperfect sequence compared to Golay codes. Here, we show that pseudorandom sequences are a good choice for multispectral photoacoustic coded excitation (MSPACE). Pseudorandom sequences based upon maximal length shift register sequences (m-sequences) are introduced and analyzed for the purpose of use in MSPACE. Their gain in SNR exceeds that of orthogonal Golay codes for finite code lengths. Artefacts are introduced, but may remain invisible depending on SNR and code length.

The method proposed in this work combines the advantages of optical detection (optical and acoustical transparency) with 2D slice imaging, using an optical interferometer combined with an acoustic reflector. The concave reflector has the shape of an elliptical cylinder and concentrates the acoustic wave generated around one focal line in the other one, where an optical beam probes the temporal evolution of acoustic pressure. This yields line projections of the initial acoustic pressure sources at distances corresponding to the time of flight. Image reconstruction from the signals recorded while rotating the sample about an axis perpendicular to the optical detector requires only the application of the inverse Radon transform. The resolution and sensitivity of the detection system were investigated in experiments on phantom samples. Furthermore, the imaging system was tested on a real biological sample.

In this paper we report on remote photoacoustic imaging using an interferometric technique. By utilizing a two-wave mixing interferometer ultrasonic displacements are measured without any physical contact to the sample. This technique allows measurement of the displacements also on rough surfaces. Mixing a plane reference beam with the speckled beam originating from the sample surface is done in a Bi₁₂SiO₂₀ photorefractive crystal. After data acquisition the structure of the specimen is reconstructed using a Fourier-domain synthetic focusing aperture technique. We show three-dimensional imaging on tissue-mimicking phantoms and biological samples. Furthermore, we show remote photoacoustic measurements on a human forearm in-vivo.

The recently developed vibrational photoacoustic (VPA) microscopy allows bond-selective imaging of deep tissues by taking advantage of intrinsic contrast from harmonic vibration of C-H bonds. Due to the spectral similarity of molecules in the overtone vibration region, the compositional information is not available from VPA images acquired by single wavelength excitation. Here we demonstrate that lipids and collagen, two critical markers in many kinds of diseases, can be distinguished by hyperspectral VPA imaging. A phantom consisted of rat tail tendon (collagen) and fat tissue (lipids) was constructed. Wavelengths between 1650 and 1850 nm were scanned to excite the first overtone/combination vibration of C-H bond. B-scan hyperspectral VPA images, in which each pixel contains a spectrum, was analyzed by a Multivariate Curve Resolution - Alternating Least Squares (MCR-ALS) algorism to recover the spatial distribution of two chemical components in the phantom.

We report the development of a novel frequency-domain biomedical photoacoustic (PA) system that utilizes a continuous-wave laser source with a custom intensity modulation pattern for spatially-resolved imaging of biological tissues. The feasibility of using relatively long duration and low optical power laser sources for spatially-resolved PA imaging is presented. We demonstrate that B-mode PA imaging can be performed using an ultrasonic phased array coupled with multi-channel correlation processing and a frequency-domain beamforming algorithm. Application of the frequency-domain PA correlation methodology is shown using tissue-like phantoms with embedded optical contrast, tissue ex-vivo samples and a small animal model in-vivo.

Optical ultrasound detection has become an attractive alternative to piezoelectric ultrasound detection for photoacoustic imaging. The favorable properties of optical detection are high resolution, complete optical and acoustical transparency. Recently, it has been shown that optical phase contrast full field detection in combination with a CCD-camera can be used to record acoustic fields. This allows to obtain two-dimensional photoacoustic projection images in real-time. The present work shows an extension of the technique towards full three-dimensional photoacoustic tomography. The specifications of the detection system, resolution and sensitivity, are 66μm and 3.4kPa. The reconstruction of the initial three dimensional pressure distribution is a two step process. First of all, projection images of the initial pressure distribution are acquired. This is done by back propagating the observed wave pattern in frequency space. In the second step the inverse Radon transform is applied to the obtained projection dataset to reconstruct the initial three dimensional pressure distribution. Simulations and experiments are performed to show the overall applicability of this technique for real-time photoacoustic imaging.

Traumatic brain injury (TBI) and spinal cord injury are a major cause of death for individuals under 50 years of age. In the USA alone, 150,000 patients per year suffer moderate or severe TBI. Moreover, TBI is a major cause of combatrelated death. Monitoring of cerebral venous blood oxygenation is critically important for management of TBI patients because cerebral venous blood oxygenation below 50% results in death or severe neurologic complications. At present, there is no technique for noninvasive, accurate monitoring of this clinically important variable. We proposed to use optoacoustic technique for noninvasive monitoring of cerebral venous blood oxygenation by probing cerebral veins such as the superior sagittal sinus (SSS) and validated it in animal studies. In this work, we developed a novel, medical grade optoacoustic system for continuous, real-time cerebral venous blood oxygenation monitoring and tested it in human subjects at normal conditions and during hyperventilation to simulate changes that may occur in patients with TBI. We designed and built a highly-sensitive optoacoustic probe for SSS signal detection. Continuous measurements were performed in the near infrared spectral range and the SSS oxygenation absolute values were automatically calculated in real time using a special algorithm developed by our group. Continuous measurements performed at normal conditions and during hyperventilation demonstrated that hyperventilation resulted in approximately 12% decrease of cerebral venous blood oxygenation.

Cerebral ischemia after birth and during labor is a major cause of death and severe complications such as cerebral palsy. In the USA alone, cerebral palsy results in permanent disability of 10,000 newborns per year and approximately 500,000 of the total population. Currently, no technology is capable of direct monitoring of cerebral oxygenation in newborns. This study proposes the use of an optoacoustic technique for noninvasive cerebral ischemia monitoring by probing the superior sagittal sinus (SSS), a large central cerebral vein. We developed and built a multi-wavelength, near-infrared optoacoustic system suitable for noninvasive monitoring of cerebral ischemia in newborns with normal weight (NBW), low birth-weight (LBW, 1500 - 2499 g) and very low birth-weight (VLBW, < 1500 g). The system was capable of detecting SSS signals through the open anterior and posterior fontanelles as well as through the skull. We tested the system in NBW, LBW, and VLBW newborns (weight range: from 675 g to 3,000 g) admitted to the neonatal intensive care unit. We performed single and continuous measurements of the SSS blood oxygenation. The data acquisition, processing and analysis software developed by our group provided real-time, absolute SSS blood oxygenation measurements. The SSS blood oxygenation ranged from 60% to 80%. Optoacoustic monitoring of the SSS blood oxygenation provides valuable information because adequate cerebral oxygenation would suggest that no therapy was necessary; conversely, evidence of cerebral ischemia would prompt therapy to increase cerebral blood flow.

Near-field Radio-frequency Thermoacoustic Imaging (NRTI) is an imaging modality that was recently introduced to generate thermoacoustic signals using ultra-short high energy impulses. Because it allows for a higher energy coupling within an ultra-short time, it can achieve higher resolutions and higher signal to noise ratio, compared to traditional thermoacoustic tomography based on radiating sources at single frequencies. As for traditional thermoacoustic imaging the contrast comes from the conductivity and the dielectric properties of the tissues, while the resolution depends on the measured acoustic waves. Since NRTI depends on the efficient generation of high energy short impulses, the ability to control their time width and pulse shape is of high importance. We present here a methodology for generating such impulses based on transmission lines. The ability of such generators to generate impulses in the range of tens of nanoseconds enables high resolution images in the range of tens of microns to hundreds of microns without compromising the amount of the energy coupled. Finally the pulser is used to generate high resolution images of small absorbing insertions, of phantoms with different conductivities and of ex-vivo mouse images. From the phantoms it is possible to see both the capabilities of the system to accurately image small insertions as well as the high quality images generated from imaging phantoms, from ex-vivo mouse images it is possible to see several anatomical characteristics, such as the mouse boundary, the spine and some other characteristics in the mouse abdomens.

Combined intravascular photoacoustic (IVPA) and intravascular ultrasound (IVUS) imaging has been previously established as a viable means for imaging atherosclerotic plaques using both endogenous and exogenous contrast. In this study, IVUS/IVPA imaging of an atherosclerotic rabbit aorta following injection of gold nanorods (AuNR) with peak absorbance within the tissue optical window was performed. Ex-vivo imaging results revealed high photoacoustic signal from localized AuNR. Corresponding histological cross-sections and digital photographs of the artery lumen confirmed the presence of AuNR preferentially located at atherosclerotic regions and in agreement with IVPA signal. Furthermore, an integrated IVUS/IVPA imaging catheter was used to image the AuNR in the presence of luminal blood. The results suggest that AuNR allow for IVPA imaging of exogenously labeled atherosclerotic plaques with a comparatively low background signal and without the need for arterial flushing.

Trapping and manipulation of micro-scale objects mimicking metastatic cancer cells in a flow field have been demonstrated with magnetomotive photoacoustic (mmPA) imaging. Coupled contrast agents combining gold nanorods (15 nm × 50 nm; absorption peak around 730 nm) with 15 nm diameter magnetic nanospheres were targeted to 10 μm polystyrene beads recirculating in a 1.6 mm diameter tube mimicking a human peripheral vessel. Targeted objects were then trapped by an external magnetic field produced by a dual magnet system consisting of two disc magnets separated by 6 cm to form a polarizing field (0.04 Tesla in the tube region) to magnetize the magnetic contrast agents, and a custom designed cone magnet array with a high magnetic field gradient (about 0.044 Tesla/mm in the tube region) producing a strong trapping force to magnetized contrast agents. Results show that polystyrene beads linked to nanocomposites can be trapped at flow rates up to 12 ml/min. It is shown that unwanted background in a photoacoustic image can be significantly suppressed by changing the position of the cone magnet array with respect to the tube, thus creating coherent movement of the trapped objects. This study makes mmPA imaging very promising for differential visualization of metastatic cells trafficking in the vasculature.

Normally, urine flows down from kidneys to bladders. Vesicoureteral reflux (VUR) is the abnormal flow of urine from bladders back to kidneys. VUR commonly follows urinary tract infection and leads to renal infection. Fluoroscopic voiding cystourethrography and direct radionuclide voiding cystography have been clinical gold standards for VUR imaging, but these methods are ionizing. Here, we demonstrate the feasibility of a novel and nonionizing process for VUR mapping in vivo, called photoacoustic cystography (PAC). Using a photoacoustic (PA) imaging system, we have successfully imaged a rat bladder filled with clinically being used methylene blue dye. An image contrast of ~8 was achieved. Further, spectroscopic PAC confirmed the accumulation of methylene blue in the bladder. Using a laser pulse energy of less than 1 mJ/cm², bladder was clearly visible in the PA image. Our results suggest that this technology would be a useful clinical tool, allowing clinicians to identify bladder noninvasively in vivo.

Cytochrome c is a heme protein normally bound to mitochondria and is important for mitochondrial electron transport and apoptosis initiation. Since cytochrome c is nonfluorescent, it is always labeled with fluorescent molecules for imaging, which, however, may affect normal cellular functions. Here, label-free photoacoustic microscopy (PAM) of mitochondrial cytochrome c was realized for the first time by utilizing the optical absorption around the Soret peak. PAM was demonstrated to be sensitive enough to image mitochondrial cytochrome c at 422 nm wavelength. Mitochondrial cytochrome c in the cytoplasm of fixed fibroblasts was clearly imaged by PAM as confirmed by fluorescent labeling. By showing mitochondrial cytochrome c in various cells, we demonstrated the feasibility of PAM for label-free histology of mouse ear sections. Therefore, PAM can sensitively image cytochrome c in unstained cells at 422 nm wavelength and has great potential for functional imaging of cytochrome c in live cells or in vivo.

Ultraviolet photoacoustic microscopy (UVPAM) can image cell nuclei in vivo with high contrast and resolution noninvasively without staining. Here, we used UV light at wavelengths of 210-310 nm for excitation of DNA and RNA to produce photoacoustic waves. We applied the UVPAM to in vivo imaging of cell nuclei in mouse skin, and obtained UVPAM images of the unstained cell nuclei at wavelengths of 245-282 nm as ultrasound gel was used for acoustic coupling. The largest ratio of contrast to noise was found for the images of cell nuclei at a 250 nm wavelength.

Filtered backprojection (FBP) algorithms are commonly employed for image reconstruction in optoacoustic tomography (OAT). A limitation of FBP algorithms is that they require the measured acoustic data to be densely sampled, which necessitates expensive ultrasound arrays that possess a large number of elements or increased data-acquisition times if mechnical scanning is employed. Additionally, FBP algorithms are based on idealized imaging models that do not accurately model the response of the transducers and fail to exploit the statistical characteristics of noisy measurement data to minimize noise levels in the reconstructed images. Iterative image reconstruction algorithms can circumvent these difficulties. However, to date, iterative reconstruction algorithms have not been successfully applied to three-dimensional (3D) OAT. In this work we investigate the use of an iterative image reconstruction method in 3D OAT. The large computational burden of 3D iterative image reconstruction is circumvented by implementing the reconstrution algorithm with graphics processing units (GPUs). The ability of the reconstruction algorithm to mitigate artifacts due to incomplete data is demonstrated.

In photoacoustic imaging, upon short laser pulse irradiation, absorbers generate N-shaped pulses which can be detected by ultrasound transducers. Radio frequency signals from different spatial locations are then reconstructed taking into account the ultrasound transducer angular response. Usually, the directivity is part of the "a priori" characterization of the transducer and it is assumed to be constant in the reconstruction algorithm. This approach is valid in both transmission and reflection ultrasound imaging, where any echo resembles the transducer frequency response. Center frequency and bandwidth of any echo are almost the same, and the ultrasound transducer collect signals with the same "fixed" acceptance angle. In photoacoustics, instead, absorbers generate echoes whose time duration is proportional to the absorber size. Large absorbers generate low frequency echoes, whereas small absorber echoes are centered at higher frequencies. Thus for different absorber sizes, different pulse frequencies are obtained and different directivities need to be applied. For this purpose once a radio-frequency signal is aquired, it is pre-processed with a sliding window: every segment is Fourier transformed to extract the central frequency. Then, a proper directivity can be estimated for each segment. Finally signals can be reconstructed via a backprojection algorithm, according to the system's geometry. Echoes are backprojected over spheres with the angular extension being adapted to the frequency content of the photoacoustic sources. Simulation and experimental validation of this approach are discussed showing promising results in terms of image contrast and resolution.

We report on an investigation of the role of shear waves in transcranial PAT brain imaging. Using a recently developed PAT image reconstruction method for use with layered media, we quantify the extent to which accounting for shear waves in the reconstruction method can improve image quality. The effects of shear waves propagating in the solid layer on the ability to estimate Fourier components of the object are investigated as a function of the thickness of the layer supporting shear waves as well as the incidence angle of the field in the planewave representation. These results clarify the role of shear waves in transcranial PAT image formation and indicate that further research is warranted to develop reconstruction algorithms that account for shear waves.

As for any other imaging technique spatial resolution and sensitivity are important features for a photoacoustic imaging device. It is already well known that spatial resolution depends on the size and the bandwidth of the detectors. Therefore for photoacoustic image reconstruction usually small point-like and broadband detectors are assumed, which measure the pressure as a function of time on a detection surface around the sample. But in reality point-like detectors are not ideal at all: because of the small detector volume the thermodynamic fluctuations (= noise) get high and the signal amplitude is low, which results in a bad signal-to-noise ratio (SNR). For a bigger detector volume the fluctuations are less and the signal amplitude is better, which gives a better SNR. But on the other hand the photoacoustic pressure signal is averaged over the whole detector volume, which results in blurring and a reduced spatial resolution if reconstruction algorithms for point-like detectors are used. To characterize this trade-off between spatial resolution and sensitivity for a varying detector volume in a quantitative way the pressure is described by a random variable having the measured pressure as a mean value and noise as random fluctuations around that mean value ("stochastic process"). For a PVDF detector the optimum for the detector size is given.

In optoacoustic tomography (OAT), also known as photoacoustic tomography, a variety of analytic reconstruction algorithms, such as filtered backprojection (FBP) algorithms, have been developed. Analytic algorithms are typically computationally more efficient than iterative image reconstruction algorithms but possess disadvantages that include the inabilty to accurately compensate for the response of the measurement system and stochastic noise. While these shortcomings can be circumvented by use of iterative image reconstruction methods, threedimensional (3D) iterative reconstruction is computationally burdensome. In this work, we present a novel datarestoration method that seeks to recover an accurate estimate of the pressure data with reduced noise levels from knowledge of the experimentally acquired transducer output data. From knowledge of the "restored" pressure data, a computationally efficient analytic algorithm can be applied for image reconstruction. Accordingly, this approach combines the advantages of an iterative reconstruction algorithm with the computational efficiency of an analytic algorithm. Curvelet-based data-space restoration is demonstrated by use of computer-simulation studies.

In optical scattering media such as biological tissue, light propagation is randomized by multiple scattering. Beyond one transport mean free path, where photon propagation is in the diffusive regime, direct light focusing becomes infeasible. The resulting loss of light localization poses serious challenge to optical imaging in thick scattering media. Ultrasound modulated optical tomography (UOT) combines high optical contrast and good ultrasonic resolution, and is therefore an ideal imaging modality for soft biological tissue. A variety of detection techniques have been developed in UOT in an effort to discriminate the ultrasonically encoded diffused light as the imaging signal. We developed a photorefractive crystal based detection system, which has the ability to image both the optical and acoustic properties of biological tissues. With the improved photorefractive crystal based detection, tissue-mimicking phantom samples as thick as 9.4 cm can be imaged. We further exploit the virtual source concept in UOT and combine it with optical time reversal to achieve diffusive light focusing into scattering media. Experimental implementation of this new technology is presented.

Near infrared spectroscopy is a widely adopted optical sensing technique to measure tissue oxygenation non-invasively in human tissues such as the brain and muscle. However, in many situations, the region of interest is beneath a superficial layer, e.g., muscle overlaid by a superficial layer of skin and fat, which can affect the accuracy of the optical measurement. By applying focused ultrasound in the region of interest, acousto-optic (AO) sensing techniques can potentially provide a measurement less susceptible to physiological changes in the superficial layer. In this work, a digital correlator based AO system has been used to perform a series of phantom experiments to assess the sensitivities of the AO and optical measurements (both in reflection modes) to absorption changes (μa = 0.0235, 0.05, 0.1, 0.2, and 0.3 cm^-1) in three different locations, including one deep location (23 mm away from the surface) and two superficial locations (8 mm away from the surface). Our results show that the AO measurements have a higher sensitivity factor to the absorption change in the deeper location (177 % cm) than the optical measurements (16 % cm). For the two more superficial locations, the AO measurements have lower sensitivity factors (-5 % cm and 56 % cm) than the optical measurements (194 % cm and 101 % cm). This study has shown the potential of the AO technique for physiological monitoring, which requires a technique to be sensitive to oxygenation changes in the deeper layer while minimizing any contamination from the superficial layer.

Ultrasound imaging has benefited from non-linear approaches to improve image resolution and reduce the effects of side-lobes. A system for performing second harmonic ultrasound modulated optical tomography is demonstrated which incorporates both pulsed optical illumination and acoustic excitation. A pulse acoustic inversion scheme is employed which allows the second harmonic ultrasound modulated optical signal to be obtained while still maintaining a short pulse length of the acoustic excitation. For the experiments carried out the method demonstrates a reduction in the effective line spread function from 4mm for the fundamental to 2.4mm for the second harmonic. The first use of pulsed ultrasound modulated optical tomography in imaging fluorescent targets is also discussed. Simple experiments show that by changing the length of the acoustic pulse the image contrast can be optimized. The modulation depth of the detected signal is greatest when the length of the object along the acoustic axis is an odd number of half wavelengths and is weakest when the object is an integer multiple of an acoustic wavelength. Preliminary ultrasound modulated imaging results are also presented where the target generates light within the medium without the use of an external light source. Although signal to noise ratio is likely to be a major challenge, this result highlights a potentially useful application of ultrasound modulated optical tomography in bio- or chemi-luminescence imaging.

Adequate capillary blood flow is a critical parameter for tissue vitality. We present a novel non-invasive method for measuring blood flow based on the acousto-optic effect, using ultrasound modulated diffused light. The benefits of the presented method are: deep tissue sampling (> 1cm), continuous real time measurement, simplicity of apparatus and ease of operation. We demonstrate the ability of the method to measure flow of scattering fluid using a calibrated flow phantom model. Fluid flow was generated by a calibrated syringe pump and the phantom's sampled volume contained millimeter size flow channels. Results demonstrate linear dependence of flow as measured by the presented technique (CFI) to actual flow values with R²=0.91 in the range of 0 to 2 ml/min, and a linear correlation to simultaneous readings of a laser Doppler probe from the same phantom. This data demonstrates that CFI readings provide a non-invasive platform form measuring tissue microcirculatory blood flow.

We demonstrate the feasibility of quantitative optical absorption imaging by using Monte Carlo simulation of combined photoacoustic tomography (PAT) and ultrasound-modulated optical tomography (UOT). Our simulation results show that the optical fluence on initial photoacoustic (PA) pressure waves can be accurately compensated for recovering exact optical absorption coefficients by using a fluence map acquired from UOT signals. Further, when the optical heterogeneities of a sample were varied, the recovered optical absorption coefficients from a target remained constant while PA amplitudes fluctuated. In practice, PAT and UOT systems can be potentially combined because both imaging systems can easily share light illumination and ultrasound application patterns.

We report on experimental demonstration of focused photoacoustic (PA) imaging for simultaneous recovery of the acoustic and optical properties of absorber in homogeneous media. The PA signals are reconstructed from tissue-like phantom experiments using Hilbert transform (HT) algorithm coupled with a focused PA imaging system. The results demonstrate that the HT-based PA signal occurs at the edge of heterogeneous sample. The average acoustic velocity could be obtained by the size dividing the traveling time. In addition, the absorption coefficient of absorber could be reconstructed by the intensity of the HT-based PA signal at the edge of sample based on the theoretical analysis.

Perfluorocarbon droplets containing optical absorbing nanoparticles have been developed for use as theranostic agents (for both imaging and therapy) and as dual-mode contrast agents. Droplets can be used as photoacoustic contrast agents, vaporized via optical irradiation, then the resulting bubbles can be used as ultrasound imaging and therapeutic agents. The photoacoustic signals from micron-sized droplets containing silica coated gold nanospheres were measured using ultra-high frequencies (100-1000 MHz). The spectra of droplets embedded in a gelatin phantom were compared to a theoretical model which calculates the pressure wave from a spherical homogenous liquid undergoing thermoelastic expansion resulting from laser absorption. The location of the spectral features of the theoretical model and experimental spectra were in agreement after accounting for increases in the droplet sound speed with frequency. The agreement between experiment and model indicate that droplets (which have negligible optical absorption in the visible and infrared spectra by themselves) emitted pressure waves related to the droplet composition and size, and was independent of the physical characteristics of the optical absorbing nanoparticles. The diameter of individual droplets was calculated using three independent methods: the time domain photoacoustic signal, the time domain pulse echo ultrasound signal, and a fit to the photoacoustic model, then compared to the diameter as measured by optical microscopy. It was found the photoacoustic and ultrasound methods calculated diameters an average of 2.6% of each other, and 8.8% lower than that measured using optical microscopy. The discrepancy between the calculated diameters and the optical measurements may be due to the difficulty in resolving the droplet edges after being embedded in the translucent gelatin medium.

The laminar myocardial sheet architecture and its dynamic change play a key role in myocardial wall thickening. Histology, confocal optical microscopy (COM), and diffusion tensor MRI (DTI) have been used to unveil the structures and functions of the myocardial sheets. However, histology and COM require fixation, sectioning, and staining processes, which dehydrate and deform the sheet architecture. Although DTI can delineate sheet architecture nondestructively in viable hearts, it cannot provide cellular-level resolution. Here we show that photoacoustic microscopy (PAM), with high resolution (~1 μm) and label-free detection, is appropriate for imaging 3D myocardial architecture. Perfused half-split mouse hearts were also imaged by PAM in vitro without fixation, dehydration, nor staining. The laminar myocardial sheet architecture was clearly visualized within a 0.15 mm depth range. Two populations of oppositely signed sheet angles were observed. Therefore, PAM promises to access dynamic changes of myocardial architectures in ex vivo perfused-viable hearts.

Direct optical measurements in scattering media offer poor resolution due to the high scattering. Ultrasound is scattered orders of magnitude less in tissue compared with light and therefore offers good resolution. Photoacoustics and acoustooptics are both relatively new hybrid techniques that enable measurements of optical properties in scattering media by combining ultrasound and light. Quantified measurements of the fluence and absorption coefficient however are desired and can not be performed by these separate techniques. A new approach to achieve this goal is to combine both hybrid techniques. By combining photoacoustic and acousto-optic measurements there is sufficient information to calculate the absorption coefficient and fluence at the ultrasound focus used for the acousto-optics. We require knowledge on the interaction of light and sound inside tissue, so the size of the so called tagging volume can be determined. This tagging volume is defined by the size and shape of the ultrasound focus used in the acousto-optic measurements. A stochastic model for acousto-optics is under development that used existing knowledge on the in the interaction between light and sound. By separating light transport and the interactions of light and sound and writing this interaction as a probability density function it is possible to find the effective geometrical properties of the tagging volume. At the moment multiple interaction mechanisms of sound and light are added to this model. In the future this model will be validated in phantoms and biological tissue.

We demonstrate the ability of a novel device employing ultrasound modulation of near infrared light (referred as "Ultrasound tagged light" or UTL) to perform non-invasive monitoring of blood flow in the microvascular level in tissue. Monitoring microcirculatory blood flow is critical in clinical situations affecting flow to different organs, such as the brain or the limbs. . However, currently there are no non-invasive devices that measure microcirculatory blood flow in deep tissue continuously. Our prototype device (Ornim Medical, Israel) was used to monitor tissue blood flow on anesthetized swine during controlled manipulations of increased and decreased blood flow. Measurements were done on the calf muscle and forehead of the animal and compared with Laser Doppler (LD). ROC analysis of the sensitivity and specificity for detecting an increase in blood flow on the calf muscle, demonstrated AUC = 0.951 for 23 systemic manipulations of cardiac output by Epinephrine injection, which is comparable to AUC = 0.943 using laser Doppler. Some examples of cerebral blood flow monitoring are presented, along with their individual ROC curves. UTL flowmetry is shown to be effective in detecting changes in cerebral and muscle blood flow in swine, and has merit in clinical applications.

We successfully encapsulated ICG in an ultrasound-triggerable perfluorocarbon double emulsion that prevents ICG from binding with plasma proteins. Photoacoustic spectral measurements on point target as well as 2-D photoacoustic images of blood vessels revealed that the photoacoustic spectrum changes significantly in blood when the ICG-loaded emulsion undergoes acoustic droplet vaporization (ADV), which is the conversion of liquid droplets into gas bubbles using ultrasound. Other than providing a new photoacoustic contrast agent, the ICG encapsulated double emulsion, when imaged with photoacoustic tomography, could facilitate spatial and quantitative monitoring of ultrasound initiated drug delivery.

Photoacoustic tomography (PAT) suppresses speckles by prominent boundary buildups. We theoretically study the dependence of PAT speckles on the boundary roughness, which is quantified by the root-mean-squared (RMS) value and the correlation length of the height. The speckle visibility and the correlation coefficient between the reconstructed and actual boundaries are quantified as a function of the boundary roughness. The statistics of PAT speckles is studied experimentally.

Spectral analysis of photoacoustic (PA) molecular imaging (PMI) of ferritin expressed in human melanoma cells (SK-24) was performed in vitro. Ferritin is a ubiquitously expressed protein which stores iron that can be detected by PA imaging, allowing ferritin to act as a reporter gene. To over-express ferritin, SK-24 cells were co-transfected with plasmid expressing Heavy chain ferritin (H-FT) and plasmid expressing enhanced green fluorescent protein (pEGFP-C1) using Lipofectamine^TM 2000. Non-transfected SK-24 cells served as a negative control. Fluorescent imaging of EGFP confirmed transfection and transgene expression in co-transfected cells. To detect iron accumulation in SK-24 cells, a focused high frequency ultrasonic transducer (60 MHz, f/1.5), synchronized to a pulsed laser (<20mJ/cm²), was used to scan the PA signal from 680 nm to 950 nm (in 10 nm increments) from the surface of the 6-well culturing plate. PA signal intensity from H-FT transfected SK-24 cells was not different from that of non-transfected SK-24 cells at wavelengths less than 770 nm, but was over 4 dB higher than non-transfected SK-24 cells at 850 ~ 950 nm. Fluorescent microscopy indicates significant accumulation of ferritin in H-FT transfected SK-24 cells, with little ferritin expression in non-transfected SK-24 cells. The PA spectral analysis clearly differentiates transfected SK-24 cells from nontransfected SK-24 cells with significantly increased iron signal at 850 ~ 950 nm, and these increased signals were associated with transfection of H-FT plasmid. As such, the feasibility of ferritin as a reporter gene for PMI has been demonstrated in vitro. The use of ferritin as a reporter gene represents a new concept for PA imaging, and may provide various opportunities for molecular imaging and basic science research.

An image reconstruction formula is presented for photoacoustic computed tomography (PCT) that is valid for a layered medium in which some of the layers may be solids and detection is performed on a planar measurement surface. It is assumed that the optical absorber is embedded in a single fluid layer and any elastic solid layers present are separated by one or more fluid layers. Computer-simulation studies are used to validate the proposed reconstruction formula.

In this paper we present a method to visualize the pressure field of an ultrasound beam in a single shot of the CCD and to image the shear wave propagation based on acousto-optic laser speckle contrast analysis. The contrast images show features in the near field, far field and central region of the ultrasound beam and the pressure profile fits with that measured with a hydrophone. The shear wave propagation was acquired by changing the imaging delay time after the ultrasound burst. This method can be used to study the shear wave properties of common tissue phantoms to guide experiments on tissue.

structural and functional imaging. Image reconstruction of PAI requires the solution of an inverse source problem, where the source represents the optical energy absorption distribution in the object. PAI in spherical or circular geometry gives good image resolution yet is slow in signal acquisition and image formation. Reducing the number of detection angles can ameliorate such issues. Besides, it is almost impossible to cover the entire surface of tissue. This will restrict it in the medical application with incomplete projection data. To resolve such limiting factors, in this thesis, a preconditioned conjugate gradient method is applied to the normal equations (PCCGNR method) for reconstructing the absorption distribution. Under the common assumption, a zero-mean Gaussian noise is added to the projection signals and a computer simulated has been used for the evaluation. This algorithm works well in rectification of the measurement and converges quickly onto an accurate estimate of the distribution of absolute absorption. It not only runs much faster than the FBP algorithm, but also shows stronger robustness in that it provides better image quality with detection data. We observed that diagonal preconditioners offer some improvement in convergence rate for image reconstruction, and reconstructed image preconditioning with ω = 0 (diagonal scaling) is closer to the true image than with ω> 0 . In addition, a physical experiment that will be done with our experiment equipment system further demonstrates the potential of the proposed algorithm in practical applications.

Co-registered Ultrasound and Photoacoustic images provide complimentary structure and functional information for cancer diagnosis and assessment of therapy response. In SPIE Photonics West 2011, we reported a system that acquires from 64 channels and displays up to 1 frame per second (fps) ultrasound pulse-echo images, 5 fps photoacoustic images, and 0.5 fps co-registered images. In this year, we report an upgraded system which acquires from 128 channels and displays up to 15 fps co-registered ultrasound and photoacoustic images limited by our laser pulse repetition rate. The system architecture is novel and it provides real-time co-registration of images, the ability of acquiring the channel RF data for both modalities, and the flexibility of adjusting every parameter involved in the imaging process for both modalities. The digital signal processor board is upgraded to an FPGA-based PCIe board that collects the data from the acquisition modules and transfers them to the PC memory at 2.5GT/s rate through an x8 DDR PCIe bus running at 100MHz clock frequency. The modules FPGA code is also upgraded to form a beam line in 90 microseconds and to communicate through ultrafast differential tracks with the PCIe board. Furthermore, the printed circuit board (PCB) design of the system was adjusted to provide a maximum of 80dB signal-to-noise ratio at 60dB gain, which is comparable to some commercial ultrasound machines. The real-time system allows capturing co-registered US/PAT images free of motion artifacts and also provides ultrafast dynamic information when a contrast agent is used. The system is built for clinical use to assist the diagnosis of ovarian cancer. However, the hardware is still under testing and evaluation stage, experimental and clinical results will be reported later.

Optical resolution photoacoustic microscopy (OR-PAM) has been shown as a promising tool for label-free microvascular and single-cell imaging in clinical and bioscientific applications. However, most OR-PAM systems are realized by using a bulky laser for photoacoustic excitation. The large volume and high price of the laser may restrain the popularity of OR-PAM. In this study, we develop a low-cost and compact OR-PAM system based on a commercially available DVD pickup head. We showed that the DVD pickup head have the required laser energy and focusing optics for OR-PAM. The firmware of a DVD burner was modified to enable its laser diode to provide a 13-ns laser pulse with 1.3-nJ energy at 650 nm. Two excitation wavelengths at 650 and 780 nm were available. The laser beam was focused onto the target after passing through a 0.6-mm thick DVD transparent polycarbonate coating, and then aligned to be confocal with a 50-MHz focused ultrasonic transducer in forward mode. To keep the target on focus, a scan involving auto-tracking procedure was performed. The lateral resolution was verified via cross-sectional imaging of a 6-μm carbon fiber. The measured -6 dB width of the carbon fiber was 6.66 μm which was in agreement with optical diffraction limit. The proposed OR-PAM has potential as an economically viable and compact blood screening tool available outside of large laboratories due to its low cost and portability. Furthermore, a better spatial resolution could be provided by using a blue ray DVD pickup head.

Properties of excitation laser are the important parameters that affect the photoacoustic image quality. As for the pulse width, it is closely related to signal strength and image resolution, which reported as a result of an experiment using a laser diode that can control the pulse width easily1. However, though a solid-state laser is promising for a medical application due to its high pulse energy creating high photo acoustic signal, its influence on waveform or the image quality has not been discussed in detail because the pulse width is hardly changeable in a solid-state laser. We use two kinds of solid-state lasers, i.e., Q-switched Nd:YAG and Ti-Sapphire Laser, in this study and generate different pulse width between 4.5 and 45 ns by changing wavelength and excitation energy. These laser pulses are entered into a silicon tube composed of carbon-particle suspension as absorber whose wavelength dependence for absorption is small. We detect the generated laser-induced photoacoustic waves by hydrophone. The photoacoustic temporal waveform shows sharper as the pulse width is shorter, which also indicates high frequency signal components increase. The width of the first peak on the temporal waveform is corresponding to the pulse width. Additionally, as a result of the photoacoustic imaging experiment preformed with 192-channel PZT linear array probe to image a thin wire, the modulation transfer function shows that the narrower the pulse width, the slightly better the image resolution.

In photoacoustic imaging, an adaptive beamforming method with coherence factor (ABF-CF) was previously introduced for improving spatial resolution and signal-to-noise ratio (SNR) over a conventional delay-and-sum beamforming method (DAS). However, the ABF-CF method is not suitable for being used in practical diagnosis since it is overly-sensitive for off-axis interferences and noises. In this paper, a new adaptive beamforming method with spatially-smoothed coherence factor (ABF-SSCF) is presented for ultrasound and photoacoustic combined imaging to enhance the contrast and spatial resolution while preserving the target information by applying a spatial-smoothing technique into CF coefficients from multiple sub-arrays within an array probe. To evaluate the ABF-SSCF method, computer simulation and ex vivo experiments were conducted. For the computer simulation, 64-channel radio-frequency (RF) data with one channel amplitude-varying off-axis interference was generated. Also, The ex vivo experiments were conducted where 128-channel pre-beamformed RF data were captured from a microcalcification-contained breast core specimen with a commercial ultrasound system equipped with a research package by using a 7-MHz linear array probe (SonixTouch, Ultrasonix Corp., BC, Canada) and an Nd:Yag laser excitation system (Surelite III-10 and Surelite OPO Plus, Continuum Inc., Santa Clara, CA, USA). From the simulation and ex vivo experiments, the proposed ABF-SSCF method provides better contrast and spatial resolutions comparable than the DAS method. Also, compared to the ABFCF method, image information is clearly presented without being degraded by off-axis interferences. These results indicate that the proposed ABF-SSCF method can simultaneously enhance the image quality and efficacy of the ABF method for ultrasound and photoacoustic combined imaging.

Near-infrared spectroscopy (NIRS) can provide an estimate of the mean oxygen saturation in tissue. This technique is limited by optical scattering, which reduces the spatial resolution of the measurement, and by absorption, which makes the measurement insensitive to oxygenation changes in larger deep blood vessels relative to that in the superficial tissue. Acousto-optic (AO) techniques which combine focused ultrasound (US) with diffuse light have been shown to improve the spatial resolution as a result of US-modulation of the light signal, however this technique still suffers from low signal-to-noise when detecting a signal from regions of high optical absorption. Combining an US contrast agent with this hybrid technique has been proposed to amplify an AO signal. Microbubbles are a clinical contrast agent used in diagnostic US for their ability to resonate in a sound field: in this work we also make use of their optical scattering properties (modelled using Mie theory). A perturbation Monte Carlo (pMC) model of light transport in a highly absorbing blood vessel containing microbubbles surrounded by tissue is used to calculate the AO signal detected on the top surface of the tissue. An algorithm based on the modified Beer-Lambert law is derived which expresses intravenous oxygen saturation in terms of an AO signal. This is used to determine the oxygen saturation in the blood vessel from a dual wavelength microbubble-contrast AO measurement. Applying this algorithm to the simulation data shows that the venous oxygen saturation is accurately recovered, and this measurement is robust to changes in the oxygenation of the superficial tissue layer.

Multi-Spectral Optoacoustic Tomography (MSOT) offers real time imaging that simultaneously exploits high ultrasound resolutions and strong optical contrast. It allows visualizing absorbers in tissue by using their distinct spectral absorption profiles. This work presents a non-invasive in vivo study of kinetics involved in the clearance of carboxylated dye in mouse kidneys. The distinctio

We investigate laser-induced ultrasound generated in a plane semi-transparent layered polymer structure. The scope is to study relations between generated ultrasound, as e.g. amplitude, and centre frequency and bandwidth of its frequency spectrum, and properties of the polymer layers, like thickness and absorption. This knowledge can then be used when designing polymer film based, semi-transparent ultrasonic devices specifically for photoacoustic applications. The experimental study is set-up as a factorial experiment with a completely randomised design. In the experiments, the light source is a pulsed Nd:YAG laser. As absorber, a semi-transparent, non-conductive polymer film in a plane layered structure of one or more layers on a glass substrate is used. The frequency spectra of the generated ultrasound spans 2 to 20 MHz, which is recorded by a broadband PVDF ultrasonic transducer. The results show that an increased thickness of the polymer layer structure relate to a lower center frequency and a lower bandwidth, and that an increased optical absorption and a decreased layer structure thickness is related to a higher ultrasound amplitude.

In photo-acoustic (PA) imaging, valuable medical applications based on optical absorption spectrum such as contrast agent imaging and blood oxygen saturation measurement have been investigated. In these applications, there is an essential requirement to determine optical absorption coefficients accurately. In present, PA signal intensities have been commonly used to determine optical absorption coefficients. This method achieves practical accuracy by combining with radiative transfer analysis. However, time consumption of radiative transfer analysis and effects of signal generation efficiencies were problems of this method. In this research, we propose a new method to determine optical absorption coefficients using continuous wavelet transform (CWT). We used CWT to estimate instantaneous frequencies of PA signals which reflects optical absorption distribution. We validated the effectiveness of CWT in determination of optical absorption coefficients through an experiment. In the experiment, planar shaped samples were illuminated to generate PA signal. The PA signal was measured by our fabricated PA probe in which an optical fiber and a ring shaped P(VDFTrFE) ultrasound sensor were coaxially aligned. Optical properties of samples were adjusted by changing the concentration of dye solution. Tunable Ti:Sapphire laser (690 - 1000 nm) was used as illumination source. As a result, we confirmed strong correlation between optical absorption coefficients of samples and the instantaneous frequency of PA signal obtained by CWT. Advantages of this method were less interference of light transfer and signal generation efficiency.

The specificity of the hemodynamic response function (HRF) is determined spatially by the vascular architecture and temporally by the evolution of hemodynamic changes. Here, we used functional photoacoustic microscopy (fPAM) to investigate the spatiotemporal evolution of the HRFs of hemoglobin concentration (HbT), cerebral blood volume (CBV) and hemoglobin oxygen saturation (SO₂) in single cerebral vessels to rat left-forepaw stimulation. The HRF changes in specific cerebral vessels responding to different stimulation intensities and durations were bilaterally imaged with 36 × 65-μm spatial resolution. Various electrical stimulations were applied with stimulation intensities at 1, 2, 6 and 10-mA combined with 5-s and 15-s stimulation durations, respectively. Our main findings were as follows: 1) the functional HbT and SO₂ increased sub-linearly with increasing stimulus intensities and 2) the results suggested that the CBV changes are more linearly correlated with arterioles than HbT and SO₂ within a limited dynamic range of stimulation intensities and duration. The findings in this study indicate that the regulation of hemodynamic changes in single cerebral vessels can be reliable studied by the fPAM technique without the use of contrast agents.

Breast calcification is one of the most important indicators for early breast cancer detection. In this study, based on a medical ultrasound array imaging platform, we attempt to develop a real-time and high penetration photoacoustic (PA) array imaging system for visualization of breast calcifications. Phantom studies were used to verify the imaging capability and penetration depth of the developed PA array system for calcification imaging. Intralipid gelatin phantoms with different-sized hydroxyapatite (HA) particles - major chemical composition of the breast calcification associated with malignant breast cancers - embedded were imaged. Laser at 750 nm was used for photoacoustic excitation and a custom-made 5-MHz photoacoustic array transducer with linear light guides was applied for photoacoustic signal detection. Experimental results demonstrated that this system is capable of calcification imaging of 0.3-0.5 mm HA particles. For the 0.5-mm HA particles, the imaging contrast was about 34 dB and the achievable penetration was 20 mm where the axial, lateral, and elevational resolution of this PA array imaging system is 0.39 mm, 0.38 mm, and 1.25 mm, respectively. The highest frame rate was 10 frames/sec limited by the laser pulse rate. Overall, our results demonstrate that it is promising for PA imaging as a real-time diagnosis and biopsy guidance tool of breast micro-calcifications outside mass lesion. Future work will focus on optimization of the photoacoustic transducer to further improve the penetration depth and development of photoacoustic and ultrasound dual-modal imaging to enhance the calcification imaging capability.

The photoacoustic (PA) signal attenuation was affected by many factors in an imaging system. In this presentation, the factors lead to the signal attenuation and their characters were discussed based on tissue optics, acoustic transportation and detection in a long-focal-zone PA imaging system. A method to recover the detected PA signals was presented and employed to image a thyroid sample in vitro. The experimental results demonstrated that the method could be used to improve the imaging depth and quality in the PA system.

For photoacoustic imaging detectors which provide high spatial resolution while being highly sensitive are essential. Integrating line detectors made of single mode polymer fibers achieve these requirements. In this paper several approaches and preliminary experiments for single mode polymer fiber line detectors are presented. Operation point stabilization by utilizing a fiber-based phase shifter is shown as well as results using different fiber couplers in the setup.

Detection of acoustic waves is the cornerstone of photoacoustic tomography (PAT). Detection has conventionally been performed mechanically using ultrasonic transducers, or optically by interferometric techniques. We propose an interferometric detection scheme using low coherence interferometry (LCI) and discuss the challenges, advantages and limitations of applying this technique to photoacoustics.

Photoacoustic signals originating from weak sources can be hard to discriminate from higher intensity signals resulting from the photoacoustic background. In order to reveal these weaker signals we propose a method where the expected background signal is subtracted from the actual signals. In this method an ultrasound image provides the geometry for the pressure distribution used in a photoacoustic wave field simulation. The simulated photoacoustic signals are subtracted from the actual recordings and the residual is used for image reconstruction. This method was successfully validated experimentally with a vessel phantom containing three optical absorption irregularities within the vessel wall.

Genetically encoded probes powerfully and non-invasively target specific tissues, cells, and subcellular locations. iRFP, a novel near-infrared fluorescent protein with low quantum yield whose absorption and fluorescence maxima are located at wavelengths longer than the Q-band of hemoglobin absorption, is ideal for PAT. Here, we report on an in vitro comparison of iRFP with other far-red fluorescent proteins, and its use in imaging a mouse tumor xenograft model. In an in vivo experiment, we stably transfected iRFP into MTLn3 adenocarcinoma cells and injected them into the mammary fat pad of female SCID/NCr mice, then imaged the resulting tumors two and three weeks post injection. The contrast increase from the protein expression was high enough to clearly separate the tumor region from the rest of the animal.

We have investigated different types of optical diffusers for the image quality assessment of photoacoustic tomography (PAT). PAT has been adapted in many biomedical research efforts over the past decade, however, studies on image quality of PAT have not been performed as much as that for photoacoustic microscopy. We developed a simple imaging phantom using strings of red plastic embedded in gelatinous base. Using a 532 nm Nd:YAG laser and focused/unfocused transducers, we reconstructed PAT images of the phantom with various types of optical diffusers placed on top of phantoms. Our initial results showed that the uniformity of the diffuser did not affect the PAT image quality, while the degree of light scattering contributed relatively more to the image quality. Image quality of biological samples will be presented and discussed.

A custom-made first prototype of a linear array ultrasound transducer for breast imaging is presented. Large active area transducer elements (5 mm × 5 mm) with 1 MHz resonance frequency are chosen to obtain a relatively high sensitivity. Acoustic lenses are used to enlarge the narrow acceptance angle of such transducer elements. The minimum detectable pressure, frequency bandwidth and electrical impedance of the transducer elements are characterized. The results show the transducer has a minimum detectable pressure of 0.8 Pa, which is superior than the transducers used in the Twente Photoacoustic Mammoscope system previously developed in our group. The bandwidth of the transducer is relative small, however it can be improved when using optimized matching layer thickness in future. We also observed a strong lateral resonance at 330 kHz, which may cause problems in various aspects for a photoacoustic imaging system. We discuss the future improvement and plans for the transducer optimizations.

Red blood cells (RBCs) aggregate in the presence of increased plasma fibrinogen and low shear forces during blood flow. RBC aggregation has been observed in deep vein thrombosis, sepsis and diabetes. We propose using photoacoustics (PA) as a non-invasive imaging modality to detect RBC aggregation. The theoretical and experimental feasibility of PA for detecting and characterizing aggregation was assessed. A simulation study was performed to generate PA signals from non-aggregated and aggregated RBCs using a frequency domain approach and to study the PA signals' dependence on hematocrit and aggregate size. The effect of the finite bandwidth nature of transducers on the PA power spectra was also investigated. Experimental confirmation of theoretical results was conducted using porcine RBC samples exposed to 1064 nm optical wavelength using the Imagio Small Animal PA imaging system (Seno Medical Instruments, Inc., San Antonio, TX). Aggregation was induced with Dextran-70 (Sigma-Aldrich, St. Louis, MO) and the effect of hematocrit and aggregation level was investigated. The theoretical and experimental PA signal amplitude increased linearly with increasing hematocrit. The theoretical dominant frequency content of PA signals shifted towards lower frequencies (<30 MHz) and 9 dB enhancements in spectral power were observed as the size of aggregates increased compared to non-aggregating RBCs. Calibration of the PA spectra with the transducer response obtained from a 200 nm gold film was performed to remove system dependencies. Analysis of the spectral parameters from the calibrated spectra suggested that PA can assess the degree of aggregation at multiple hematocrit and aggregation levels.

Photoacoustic imaging is a hybrid imaging modality capable of producing images based on optical contrast, but with depth penetration and resolution similar to ultrasound imaging. In this work, a staring, sparse approach to 3D photoacoustic imaging was used to image a number of objects. The photoacoustic system described in this paper improved upon a previous generation that contained 30 commercial transducers, by incorporating 60 custom-built transducers into a compact hemispherical array. Imaging was performed by acquiring an experimental estimate of the imaging operator and solving a linear system model to provide an estimate of the object. The imaging operator contained 18,000 voxels, each at 0.5-mm isotropic resolution. The dimensions of the imaging operator were 30 mm × 30 mm × 2.5 mm. In the first experiment, a black wire was arranged in a triangular shape and imaged in a 0.3% Intralipid^TM solution. The second experiment utilized a rotating human hair, where the hair was imaged at different angular positions. Both objects were successfully captured with reasonable accuracy, though image artifacts were present in both sets of images. The experimental results demonstrated that objects of substantial geometrical complexity could be reconstructed using measurements from only 60 transducers with prior knowledge of the imaging operator.

Photoacoustic Doppler Flowmetry has several potential advantages over its purely acoustical counterpart. The key ones are better inherent contrast and potential molecular information. It is therefore highly desired to continue to develop this modality into a viable complementary tool alongside with Doppler Ultrasound flowmetry. Working towards this goal we have constructed a Photoacoustic Doppler setup based on a combined pair of laser diodes at 830nm and a 10MHz focused acoustical transducer. Using tone-burst intensity modulation, depth-resolved Doppler spectrograms of a phantom vessel containing flowing suspension of carbon particles, were obtained. In order to investigate the conditions required for successful photoacoustic Doppler measurement in blood a k-space photoacoustic simulation was performed. It tested the photoacoustic response which is obtained for moving random spatial distributions of red blood cells and the effect of several parameters, such as particles density, ultrasonic frequency and optical spot size.

Photoacoustic microscopy (PAM) has been shown to be a valuable tool for quantifying hemoglobin oxygenation within single vessels. Recently, optical-resolution PAM was developed to achieve higher resolution by reducing the laser beam diameter, which increased the light intensity. As intensity increases, saturation of the optical absorption and multiphoton/ multi-step absorption can occur, which, together with the temperature dependence of thermal expansion, result in a non-linear dependence of the photoacoustic signal on the excitation pulse fluence. For hemoglobin, the major absorber in tissue for photoacoustic imaging, these non-linear phenomena have strong wavelength dependence. To enable quantitative photoacoustic measurements at different wavelengths in the presence of nonlinearity, a careful wide range analysis of the intensity-dependent absorption is required. Here, we built a photoacoustic spectrometer, using a tunable nanosecond optical parametric oscillator that operates between 410 nm and 2400 nm as our light source. To reduce uncertainty in our measurements due to inhomogeneous spatial distribution of the optical fluence, we used a flat-top beam illumination and a flat transducer which was mounted in reflection mode, effectively reducing quantitative measurements to a one dimensional problem. Intensity-dependent non-linear spectra of the photoacoustic signals of oxyand deoxy-hemoglobin were obtained. These measurements have the potential to contribute significantly to quantitative functional PAM.

We discuss a method for using the pseudoinverse of the system matrix to perform photoacoustic image reconstruction. In our method, the regularization levels are set and the pseudoinverse matrices are calculated just once for all possible objects, so the reconstruction step consists only of a matrix-vector multiplication, which is very fast. We expect this method to work well in photoacoustic imaging because the dominant noise mechanism is usually transducer thermal noise, which is object independent. We find that this reconstruction method offers improvements in ideal observer signal-to-noise ratio, resolution, and the length of streak artifacts compared to standard filtered backprojection.

Radiofrequency ablation (RFA) procedures are used to destroy abnormal electrical pathways in the heart that can cause cardiac arrhythmias. Current methods relying on fluoroscopy, echocardiography and electrical conduction mapping are unable to accurately assess ablation lesion size. In an effort to better visualize RFA lesions, photoacoustic (PA) and ultrasonic (US) imaging were utilized to obtain co-registered images of ablated porcine cardiac tissue. The left ventricular free wall of fresh (i.e., never frozen) porcine hearts was harvested within 24 hours of the animals' sacrifice. A THERMOCOOL^R Ablation System (Biosense Webster, Inc.) operating at 40 W for 30-60 s was used to induce lesions through the endocardial and epicardial walls of the cardiac samples. Following lesion creation, the ablated tissue samples were placed in 25 °C saline to allow for multi-wavelength PA imaging. Samples were imaged with a Vevo^R 2100 ultrasound system (VisualSonics, Inc.) using a modified 20-MHz array that could provide laser irradiation to the sample from a pulsed tunable laser (Newport Corp.) to allow for co-registered photoacoustic-ultrasound (PAUS) imaging. PA imaging was conducted from 750-1064 nm, with a surface fluence of approximately 15 mJ/cm² maintained during imaging. In this preliminary study with PA imaging, the ablated region could be well visualized on the surface of the sample, with contrasts of 6-10 dB achieved at 750 nm. Although imaging penetration depth is a concern, PA imaging shows promise in being able to reliably visualize RF ablation lesions.

We have developed a 2.5-mm outer diameter photoacoustic endoscopic mini-probe to use in the instrument channel (typically 2.8 or 3.7 mm in diameter) of standard video endoscopes. To achieve adequate signal sensitivity, we fabricated a focused ultrasonic transducer using a highly-sensitive PMN-PT piezoelectric material. We quantified the PMN-PT transducer's operating parameters and validated the mini-probe's endoscopic imaging capability through an ex vivo imaging experiment on a rat colon.

As a new class of sentinel lymph node (SLN) tracers for photoacoustic (PA) imaging, Au nanocages offer the advantages of noninvasiveness, strong optical absorption in the near-infrared region (for deep penetration), and accumulation in higher concentrations than the initial injected solution. By monitoring the amplitude changes of PA signals in an animal model, we quantified the accumulations of nanocages in SLNs over time. Based on this method, we quantitatively evaluated the kinetics of gold nanocages in SLN in terms of concentration, size, and surface modification. We could detect the SLN at an Au nanocage injection concentration of 50 pM and a dose of 100 μL in vivo. This concentration is about 40 times less than the previously reported value. We also investigated the influence of nanocages' size (50 nm and 30 nm in edge length), and the effects of surface modification (with positive, or neutral, or negative surface charges). The results are helpful to develop this AuNC-based PA imaging system for noninvasive lymph node mapping, providing valuable information about metastatic cancer staging.

Pressure changes caused by an ultrasound pattern inside tissue will modify its density and hence its refractive index. We present an integrated computational imaging approach to minimize multiple-scattered reflected light from tissue. It is based on optimum modulation of the refractive index of tissue using an ultrasound pattern. We examine issues related the design of such pattern using COMSOL Multiphysics. An optimum ultrasound pattern could be used to design and implement an integrated- computational optical coherence tomography (IC-OCT) system with extended depth of imaging.

The penetration depth of ballistic optical imaging technologies is limited by light scattering. To study the effect of scattering on optical-resolution photoacoustic microscopy (OR-PAM), we divided the signals in OR-PAM into two classes: one is from the target volume defined by the optical resolution cell (Class I); the other is from the rest of the acoustic resolution cell (Class II). We developed a way to simulate the point spread function (PSF) of our OR-PAM system considering both optical illumination and acoustic detection, then used the PSF to calculate the contributions of each class of signal to the total signal at different focal depths. Our simulation results showed that: 1) The Class II signal decays much more slowly than the Class I signal; 2) The full width at half maximum (FWHM) of the PSF for the focal depth of 0.9 transport mean free path (TMFP) is not broadened much (~10%) compared with that for a clear medium; 3) Image contrast is degraded with increasing depth when there is a uniform absorption background.

We have developed dichroism optical-resolution photoacoustic microscopy, capable of imaging polarization-dependent optical absorption (i.e., dichroism) with excellent specificity. This technical innovation enriches molecular photoacoustic contrasts and holds particular potential for detecting amyloid-associated neurodegenerative and cardiovascular diseases.

We have recently developed Transient Absorption Ultrasonic Microscopy (TAUM) as an ultrahigh-resolution photoacoustic microscopy technique. The amplitude of the multiphoton pump-probe interaction is dependent on the interpulse delay between the pump and probe pulses. Measuring the interpulse delay dependent TAUM amplitude maps out the ground state recovery time of the chromophore. The ground state recovery time is a molecular signature that may be used to differentiate multiple chromophores, analogous to fluorescence lifetime. We have used TAUM to measure the ground state recovery time of Rhodamine 6G to be 3.65 ns, which matches well with known literature values. Whole blood is also investigated, with measured ground state recovery times of 3.74 ns for oxygenated blood and 7.9 ns for deoxygenated blood. The distinct difference in lifetimes for the oxidized and reduced forms suggests the feasibility of subcellular SO2 images maps in future iterations of TAUM.

Massive small bowel resection (SBR) results in villus angiogenesis and intestinal adaptation. The exact mechanism that causes intestinal villus angiogenesis remains unknown. We hypothesize that hemodynamic changes within the remnant bowel after SBR will trigger intestinal angiogenesis. To validate this, we used photoacoustic microscopy (PAM) to image the microvascular system of the intestine in C57B6 mice and to measure blood flow and oxygen saturation (sO₂) of a supplying artery and vein. Baseline measurements were made 6 cm proximal to the ileal-cecal junction (ICJ) prior to resection. A 50% proximal bowel resection was then performed, and measurements were again recorded at the same location immediately, 1, 3 and 7 days following resection. The results show that arterial and venous sO₂ were similar prior to SBR. Immediately following SBR, the arterial and venous sO2 decreased by 14.3 ± 2.7% and 32.7 ± 6.6%, respectively, while the arterial and venous flow speed decreased by 62.9 ± 17.3% and 60.0 ± 20.1%, respectively. Such significant decreases in sO₂ and blood flow indicate a hypoxic state after SBR. Within one week after SBR, both sO₂ and blood flow speed had gradually recovered. By 7 days after SBR, arterial and venous sO₂ had increased to 101.0 ± 2.9% and 82.7 ± 7.3% of the baseline values, respectively, while arterial and venous flow speed had increased to 106.0 ± 21.4% and 150.0 ± 29.6% of the baseline values, respectively. Such increases in sO₂ and blood flow may result from angiogenesis following SBR.

We have previously demonstrated that tyrosinase is a reporter gene for photoacoustic imaging since tyrosinase is the rate-limiting step in the synthesis of melanin, a pigment capable of producing strong photoacoustic signals. We previously created a cell line capable of inducible tyrosinase expression (important due to toxicity of melanin) by stably transfecting tyrosinase in MCF-7 Tet-On^R cell line (Clontech) which expresses a doxycycline-controlled transactivator. Unfortunately, Clontech provides few Tet-On Advanced cell lines making it difficult to have inducible tyrosinase expression in cell lines not provided by Clontech. In order to simplify the creation of cell lines with inducible expression of tyrosinase, we created a single plasmid that encodes both the transactivator as well as tyrosinase. PCR was used to amplify both the transactivator and tyrosinase from the Tet-On^R Advanced and pTRE-Tight-TYR plasmids, respectively. Both PCR products were cloned into the pEGFP-N1 plasmid and the newly created plasmid was transfected into ZR-75-1, MCF-7, and MIA PaCa-1 cells using lipofectamine. After several days, brown melanin was only observed in cells incubated with doxycycline, suggesting that the newly created single plasmid allowed inducible tyrosinase expression in many different cells lines.

Ultrasound array systems with conventional delay-and-sum beamformers are often adopted for high frame rate photoacoustic imaging. In this report, we propose a coherence factor (CF) based imaging approach to further improve the image contrast of such array systems by suppressing sidelobes of the acoustic diffraction pattern. Specifically, minimum variance based coherence factor (CFMVDR) is used. Furthermore, because CF-based weighting is susceptible to variations in signal-to-noise-ratio (SNR), we also adopt a Wiener filter approach to alleviate this problem so that the method can perform well under all SNR conditions. This is of particular interest as the SNR in photoacoustic imaging is typically low. To test this method, a human hair and a graphite phantom were used as test subjects. The imaging system consisted of a 523nm pulsed laser, and a 128-channel linear array (bandwidth from 5 to 10MHz) for photoacoustic signal detection. It is demonstrated that the beam widths (i.e., lateral resolution) can be effectively improved and the noise background is suppressed by 20 dB. The contrast improvement is also evident.

Laser thermal therapy involves heating tissue using light to temperatures between 55 °C and 95 °C for several minutes resulting in coagulation and cell death. This treatment method has been under investigation for use as a minimally invasive method for treating solid tumors and cancer cells. Heating tissues results in highly variable outcomes and challenges; for example, ensuring complete coagulation of the target tissue while avoiding damage to surrounding healthy tissues. Overcoming such challenges requires precise and real-time monitoring. Optoacoustic imaging has been proposed as a real-time, noninvasive method for monitoring laser thermal. Ex-vivo porcine tenderloin samples were heated using a 1000 μm core optical fiber coupled to an 810 nm diode laser at a constant power of 7 W for 10 minutes. Lesions (6-7 mm diameter) were scanned using a prototype reverse-mode optoacoustic system consisting of a pulsed laser which operates at 1064 nm coupled to a bifurcated fibre bundle, and an 8 element annular array wideband ultrasound transducer with a central frequency ~5 MHz. Scanning was done across native and coagulated tissue with an energy of 6.5 mJ at a 1064 nm wavelength. Three lesions of similar size, shape and coagulation state were chosen for analysis. Thermal coagulation effects were analyzed using optoacoustic signal amplitude and spectral analysis of the optoacoustic RF data. Results show that the signal amplitude and the intercept and midband fit of the power spectrum exhibit interesting differences between native and coagulated tissue states.

An Interferometric Polymer Optical Waveguide Sensor (IPOWS) for optoacoustic signal detection has been fabricated by UV-imprinting method. The sensor has been characterized in sensitivity, dynamic range and frequency bandwidth. The noise equivalent pressure (NEP) of the sensor is around 100 Pa for a bandwidth range of 20 MHz. We have compared experimentally the performance of the IPOWS with a piezoelectric ultra wideband sensor and other optical fiber sensors based on single-mode silica and polymer optical fibers. All sensors are designed for the detection of optoacoustic wave sources with a frequency bandwidth that exceed 10MHz.

Gold nanorods (GNR) with a peak absorption wavelength of 760 nm were prepared using a seed-mediated method. A novel protocol has been developed to replace hexadecyltrimethylammonium bromide (CTAB) on the surface of GNR with 16-mercaptohexadecanoic acid (MHDA) and metoxy-poly(ethylene glycol)-thiol (PEG), and the monoclonal antibodies: HER2 or CD33. The physical chemistry property of the conjugates was monitored through optical and zetapotential measurements to confirm surface chemistry. The plasmon resonance is kept in the near infrared area, and changes from strong positive charge for GNR-CTAB to slightly negative for GNR-PEG-mAb conjugates are observed. The conjugates were investigated for different cells lines: breast cancer cells and human leukemia lines in vivo applications. These results demonstrate successful tumor accumulation of our modified PEG-MHDA conjugates of GNR for HER2/neu in both overexpressed breast tumors in nude mice, and for thermolysis of human leukemia cells in vitro. The conjugates are non-toxic and can be used in pre-clinical applications, as well as molecular and optoacoustic imaging, and quantitative sensing of biological substrates.

We have applied optical-resolution photoacoustic microscopy (OR-PAM) for longitudinal monitoring of cerebral metabolism through the intact skull of mice before, during, and up to 72 hours after a 1-hour transient middle cerebral artery occlusion (tMCAO). The high spatial resolution of OR-PAM enabled us to develop vessel segmentation techniques for segment-wise analysis of cerebrovascular responses.

A new design of light illumination scheme for deep tissue photoacoustic (PA) imaging, a light catcher, is proposed and evaluated by in silico simulation. Finite element (FE)-based numerical simulation model was developed for photoacoustic (PA) imaging in soft tissues. In this in silico simulation using a commercially available FE simulation package (COMSOL MultiphysicsTM, COMSOL Inc., USA), a short-pulsed laser point source (pulse length of 5 ns) was placed in water on the tissue surface. Overall, four sets of simulation models were integrated together to describe the physical principles of PA imaging. Light energy transmission through background tissues from the laser source to the target tissue or contrast agent was described by diffusion equation. The absorption of light energy and its conversion to heat by target tissue or contrast agent was modeled using bio-heat equation. The heat then causes the stress and strain change, and the resulting displacement of the target surface produces acoustic pressure. The created wide-band acoustic pressure will propagate through background tissues to the ultrasound detector, which is governed by acoustic wave equation. Both optical and acoustical parameters in soft tissues such as scattering, absorption, and attenuation are incorporated in tissue models. PA imaging performance with different design parameters of the laser source and energy delivery scheme was investigated. The laser light illumination into the deep tissues can be significantly improved by up to 134.8% increase of fluence rate by introducing a designed compact light catcher with highly reflecting inner surface surrounding the light source. The optimized parameters through this simulation will guide the design of PA system for deep tissue imaging, and help to form the base protocols of experimental evaluations in vitro and in vivo.

High-energy and short-duration outputs from lasers are desirable to improve the photoacoustic image quality when imaging deeply-seated lesions. In many clinical applications, optical fibers are used to couple the high-energy laser pulse to tissue. These high peak intensity pulses can damage an optical fiber input face if the damage threshold is exceeded. It is necessary to reduce the peak intensity to minimize the fiber damage and to delivery sufficient light for imaging. In this paper, a laser-pulse-stretching technique is introduced to reduce the peak intensity of laser pulses. To demonstrate the technique, an initial 17ns pulse was stretched to 37ns by a ring-cavity laser-pulse-stretching system, and the laser peak power reduced to 42%. The stretched pulse increased the fiber damage threshold by 1.5-fold. Three ultrasound transducers centered at 1.3MHz, 3.5MHz, 6MHz frequencies were simulated and the results showed that the photoacoustic signal of 0.5mm-diameter target obtained with 37ns pulse was about 98%, 91% and 80% respectively using the same energy as with the 17ns pulse. Simulations were validated using a broadband hydrophone. Quantitative comparisons of photoacoustic images obtained with three corresponding ultrasound transducers showed that the image quality was not affected by stretching the pulse.

Traditional Photoacoustic tomography provides the distribution of absorbed optical energy densities which are the products of the optical absorption coefficients and fluences. However, the absorption coefficient is the only functional parameter that is related to disease diagnosis, such as cancer. In this paper, we report the experimental investigation of an improved fitting procedure which can quantitatively characterize optical absorption coefficients of multiple targets. The original fitting procedure was proposed by us and used for a single target. The fitting procedure included a complete photoacoustic forward model, which incorporated an analytical model of light transport and a model of acoustic propagation. Using the target information from the PAT images and the background information from diffuse optical measurements (DOM), the fitting method minimizes the photoacoustic measurements and forward model data and recovers the target absorption coefficient quantitatively. The fitting errors in the absorption coefficients can reach 20% to 100% if the original fitting procedure is directly used on multiple targets. In our improved fitting method, the ratio between the photoacoustic intensities is introduced and served as extra input to the fitting procedure. As a result, the total number of unknown parameters is reduced and fitting accuracy is improved. The hybrid system used in the experiment combines a 64-channel photoacoustic system with a frequency-domain diffused optical system. The experiment was performed in the reflection geometry suitable for breast imaging. Phantom experiments include the combination of high contrast and low contrast targets with absorption coefficients ranging from 0.07 to 0.28 cm^-1 and with different spatial separations. The phantoms were inserted into a chicken breast tissue. The fitting errors of multiple targets were reduced to less than 20% for both high and low contrast targets. These results illustrate the potential application of this quantitative DOM-assisted photoacoustic fitting procedure to image and diagnose breast cancer having multiple and complex tumor distribution.

Photoacoustic (PA) tomography (PAT) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Multi-wavelength PAT can noninvasively monitor hemoglobin oxygen saturation (sO2) with high sensitivity and fine spatial resolution. However, accurate quantification in PAT requires knowledge of the optical fluence distribution, acoustic wave attenuation, and detection system bandwidth. We propose a method to circumvent this requirement using acoustic spectra of PA signals acquired at two optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560 and 575 nm were quantified with errors of ><5%.