This PDF file contains the front matter associated with SPIE Proceedings Volume 8943 including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

Ex vivo imaging of fresh prostate specimens was performed to test the hypothesis that the thermoacoustic (TA) contrast mechanism generated with very high frequency electromagnetic (EM) irradiation is sensitive to prostate cancer. Ex vivo imaging was performed immediately after radical prostatectomy, performed as part of normal care. Irradiation pulsewidth was 700 ns and duty cycle was extremely low. Typical specific absorption rate (SAR) throughout the prostate was 70-90 kW/kg during pulsing, but time-averaged SAR was below 2 W/kg. TA pressure pulses generated by rapid heating due to EM energy deposition were detected using single element transducers. 15g/L glycine powder mixed into DI water served as acoustic couplant, which was chilled to prevent autolysis. Spatial encoding was performed by scanning in tomographic “step-and-shoot” mode, with 3 mm translation between slices and 1.8-degree rotation between tomographic views. Histology slides for 3 cases scanned with 2.25 MHz transducers were marked for comparison to TA reconstructions. These three cases showed little, moderate, and severe involvement in the histology levels surrounding the verumontanum. TA signal strength decreased with percent cancerous involvement. When VHF is used for tissue heating, the TA contrast mechanism is driven by ionic content and we observed suppressed TA signal from diseased prostate tissue in the peripheral zone. For the 45 regions of interest analyzed, a reconstruction value of 0.4 mV provides 100% sensitivity but only 29% specificity.

In this paper, human ovarian tissues with malignant and benign features were imaged ex vivo by using an opticalresolution photoacoustic microscopy (OR-PAM) system. Several features were quantitatively extracted from PAM images to describe photoacoustic signal distributions and fluctuations. 106 PAM images from 18 human ovaries were classified by applying those extracted features to a logistic prediction model. 57 images from 9 ovaries were used as a training set to train the logistic model, and 49 images from another 9 ovaries were used to test our prediction model. We assumed that if one image from one malignant ovary was classified as malignant, it is sufficient to classify this ovary as malignant. For the training set, we achieved 100% sensitivity and 83.3% specificity; for testing set, we achieved 100% sensitivity and 66.7% specificity. These preliminary results demonstrate that PAM could be extremely valuable in assisting and guiding surgeons for in vivo evaluation of ovarian tissue.

Endonasal surgeries to remove pituitary tumors incur the deadly risk of carotid artery injury due to limitations with real-time visualization of blood vessels surrounded by bone. We propose to use photoacoustic imaging to overcome current limitations. Blood vessels and surrounding bone would be illuminated by an optical fiber attached to the endonasal drill, while a transducer placed on the pterional region outside of the skull acquires images. To investigate feasibility, a plastisol phantom embedded with a spherical metal target was submerged in a water tank. The target was aligned with a 1-mm optical fiber coupled to a 1064nm Nd:YAG laser. An Ultrasonix L14-5W/60 linear transducer, placed approximately 1 cm above the phantom, acquired photoacoustic and ultrasound images of the target in the presence and absence of 2- and 4-mm-thick human adult cadaveric skull specimens. Though visualized at 18 mm depth when no bone was present, the target was not detectable in ultrasound images when the 4-mm thick skull specimen was placed between the transducer and phantom. In contrast, the target was visible in photoacoustic images at depths of 17-18 mm with and without the skull specimen. To mimic a clinical scenario where cranial bone in the nasal cavity reduces optical transmission prior to drill penetration, the 2-mm-thick specimen was placed between the phantom and optical fiber, while the 4-mm specimen remained between the phantom and transducer. In this case, the target was present at depths of 15-17 mm for energies ranging 9-18 mJ. With conventional delay-and-sum beamforming, the photoacoustic signal-tonoise ratios measured 15-18 dB and the contrast measured 5-13 dB. A short-lag spatial coherence beamformer was applied to increase signal contrast by 11-27 dB with similar values for SNR at most laser energies. Results are generally promising for photoacoustic-guided endonasal surgeries.

Intravascular ultrasound (IVUS) plays a vital role in assessing the severity of atherosclerosis and has greatly enriched our knowledge on atherosclerotic plaques. However, it mainly reveals the structural information of plaques. In contrast, spectroscopic and molecular photoacoustic imaging can potentially improve plaque composition identification, inflammation detection, and ultimately the stratification of plaque vulnerability and risk. In this work, we developed an integrated intravascular ultrasound and optical-resolution photoacoustic microscopy (IVUS-PAM) system with a single catheter as small as 1 mm in diameter, comparable to that of existing clinical IVUS catheters. In addition, by using a GRIN lens to focus the excitation laser pulse, the system provides an optical-diffraction limited photoacoustic lateral resolution as fine as 19.6 micrometers, ~10-fold finer than that of conventional intravascular photoacoustic imaging and existing IVUS technology. The system employs a custom-made miniaturized single-element ultrasonic transducer with a dimension of ~0.5 mm, a centre frequency of ~40 MHz, and a fractional bandwidth of ~60%. The IVUS-PAM can simultaneously acquire co-registered IVUS images with an axial resolution of ~40 micrometers and a lateral resolution of ~200 micrometers. In the future, IVUS-PAM may open up new opportunities for improved high-resolution vulnerable plaque imaging and image-guided stent deployment.

Measuring intracellular temperature is critical to understanding many cellular functions but still remains challenging. Here we present a technique – fluorescence-assisted photoacoustic thermometry (FAPT) – for intracellular temperature mapping applications. To demonstrate FAPT, we monitored the intracellular temperature distribution of HeLa cells with sub-degree (0.7 °C) temperature resolution and sub-micron (0.23 μm) spatial resolution at a sampling rate of 1 kHz. Compared to traditional fluorescence-based methods, FAPT features the unique capability of transforming a regular fluorescence probe into a concentration- and excitation-independent temperature sensor, bringing a large collection of commercially available generic fluorescent probes into the realm of intracellular temperature sensing.

It is beneficial to study tumor angiogenesis and microenvironments by imaging the microvasculature and cells at the same time. Photoacoustic microscopy (PAM) is capable of sensitive three-dimensional mapping of microvasculature, while fluorescence microscopy may be applied to assessment of tissue pathology. In this work, a fiber-optic based PAM and confocal fluorescence microscopy (CFM) dual-modality imaging system was designed and built, serving as a prototype of a miniaturized dual-modality imaging probe for endoscopic applications. As for the design, we employed miniature components, including a microelectromechanical systems (MEMS) scanner, a miniature objective lens, and a small size optical microring resonator as an acoustic detector. The system resolutions were calibrated as 8.8 μm in the lateral directions for both PAM and CFM, and 19 μm and 53 μm in the axial direction for PAM and CFM, respectively. Images of the animal bladders ex vivo were demonstrated to show the ability of the system in imaging not only microvasculature but also cellular structure.

We present novel concave Fabry Perot (FP) sensor arrays for photoacoustic imaging which were fabricated using a high-precision inkjet printing approach to produce the cavity and employed physical vapor deposition to form the dielectric mirrors. Our concave FP cavity design provides excellent beam confinement within the cavity enabling high finesse and thus high sensitivity to be achieved. Two such concave sensors are evaluated in terms of their sensitivity and acoustic bandwidth. A 66 μm thick concave sensor is shown to provide a noise equivalent pressure (NEP) of 85 Pa and an acoustic bandwidth of 16 MHz, and can potentially be used as a sensitive broadband sensor for superficial imaging. A 250 μm thick sensor with an NEP of 12 Pa and acoustic bandwidth of 4 MHz was also developed for deep tissue imaging applications.

A method to detect and differentiate circulating melanoma tumor cells (CTCs) from blood cells using ultrasound and photoacoustic signals with frequencies over 100 MHz is presented. At these frequencies, the acoustic wavelength is similar to the dimensions of a cell, which results in unique features in the signal; periodically varying minima and maxima occur throughout the power spectrum. The spacing between minima depends on the ratio of the size to sound speed of the cell. Using a 532 nm pulsed laser and a 375 MHz center frequency wide-bandwidth transducer, the ultrasound and photoacoustic signals were measured from single cells. A total of 80 cells were measured, 20 melanoma cells, 20 white blood cells (WBCs) and 40 red blood cells (RBCs). The photoacoustic spectral spacing Δf between minima was 95 ± 15 MHz for melanoma cells and greater than 230 MHz for RBCs. No photoacoustic signal was detected from WBCs. The ultrasonic spectral spacing between minima was 46 ± 9 MHz for melanoma cells and 98 ± 11 for WBCs. Both photoacoustic and ultrasound signals were detected from melanoma cells, while only ultrasound signals were detected from WBCs. RBCs showed distinct photoacoustic spectral variations in comparison to any other type of cell. Using the spectral spacing and signal amplitudes, each cell type could be grouped together to aid in cell identification. This method could be used for label-free counting and classifying cells in a sample.

Tissue oxygen plays a critical role in maintaining tissue viability and in various diseases, including response to therapy. Images of oxygen distribution provide the history of tissue hypoxia and evidence of oxygen availability in the circulatory system. Currently available methods of direct measuring or imaging tissue oxygen all have significant limitations. Previously, we have reported a non-invasive in vivo imaging modality based on photoacoustic lifetime. The technique maps the excited triplet state of oxygen-sensitive dye, thus reflects the spatial and temporal distribution of tissue oxygen. We have applied PALI on tumor hypoxia in small animals, and the hypoxic region imaged by PALI is consistent with the site of the tumor imaged by ultrasound. Here, we present two studies of applying PALI to monitor changes of tissue oxygen by modulations. The first study involves an acute ischemia model using a thin thread tied around the hind limb of a normal mouse to reduce the blood flow. PALI images were acquired before, during, and after the restriction. The drop of muscle pO₂ and recovery from hypoxia due to reperfusion were observed by PALI tracking the same region. The second study modulates tissue oxygen by controlling the percentage of oxygen the mouse inhales. We demonstrate that PALI is able to reflect the change of oxygen level with respect to both hyperbaric and hypobaric conditions. We expect this technique to be very attractive for a range of clinical applications in which tissue oxygen mapping would improve therapy decision making and treatment planning.

Photoacoustic computed tomography (PACT) with a linear transducer array suffers from limited detection view. To increase the detection aperture, it is possible to circularly scan the linear transducer array around the object at the expense of imaging speed. Here we propose an alternative method to double or triple the detection view angle without sacrificing the imaging speed. By using a planar acoustic reflector which creates a virtual linear transducer array, the detection view angle is doubled. Similarly, by using two planar acoustic reflectors placed at 120 degrees to each other, we can form two virtual linear transducer arrays, and the detection view angle is tripled. This paper comparatively studies the two cases. We found that the planar acoustic reflectors greatly increase the detection aperture and thus significantly enhance the image quality of linear-array-base PACT systems.

In photoacoustic imaging, nanoparticle contrast agents offer strong signal intensity and long-term stability, but are limited by poor biodistribution and clearance profiles. Conversely, small molecules offer renal clearance, but relatively low photoacoustic signal. Here we describe a cellulose-based nanoparticle with photoacoustic signal superior to gold nanorods, but that undergoes enzymatic cleavage into constituent glucose molecules for renal clearance. Cellulose nanoparticles (CNPs) were synthesized through acidic cleavage of cellulose linters and purified with centrifugation. TEM indicated that the nanoparticles were 132 ± 46 nm; the polydispersity index was 0.138. Ex vivo characterization showed a photoacoustic limit of detection of 0.02 mg/mL CNPs, and the photoacoustic signal of CNPs was 1.5- to 3.0-fold higher than gold nanorods (also at 700 nm resonance) on a particle-to-particle basis. Cell toxicity assays suggested that overnight doses below 0.31 mg/mL CNPs produced no significant (p>0.05) impact on cell metabolism. Intravenous doses up to 0.24 mg were tolerated well in nude mice. Subcutaneous and orthotopic tumor xenografts of the OV2008 ovarian cancer cell line were then created in nude mice. Data was collected with a Nexus128 scanner from Endra LifeSciences. Spectral data used a LAZR system from Visualsonics both at 700 nm excitation. We injected CNPs (0.024 mg, 0.048 mg, and 0.80 mg) via tail vein and showed that the tumor photoacoustic signal reached maximum increase between 10 and 20 minutes. All injected concentrations were statistically (p<0.05) elevated relative to the control group with n=3 mice in each group, and dose and signal had a linear relationship at R²>0.96 suggesting quantitative signal. CNP biodegradation was demonstrated ex vivo with a glucose assay. CNPs in the presence of cellulase were reduced to free glucose in under than four hours. The glucose concentration before addition of cellulase was not detectable, but increased to 92.1 μg/mL in four hours. CNPs in the absence of cellulase did not produce glucose. Small fragments of nanoparticle in the treated cohort were observed with electron microscopy. There are few photoacoustic contrast agents that offer both high signal intensity and obvious clearance/biodegradation profiles. To the best of our knowledge, this is the first example of a sugar-based photoacoustic contrast agent with important implications for clinical translation of this emerging molecular imaging modality.

In this work we introduce an improved prototype of three-dimensional imaging system that combines optoacoustic tomography (OAT) and laser ultrasound tomography (LUT) to obtain coregistered maps of tissue optical absorption and speed of sound (SoS). The OAT scan is performed by a 360 degree rotation of a mouse with respect to an arc-shaped array of ultrasonic transducers. A Q-switched laser system is used to establish optoacoustic illumination pattern appropriate for deep tissue imaging with a tunable (730-840 nm) output wavelengths operated at 10 Hz pulse repetition rate. A 532 nm wavelength output, being mostly absorbed within a narrow superficial layer of skin, is used to outline the visualized biological object. Broadband laser ultrasound emitters are arranged in another arc pattern and are positioned opposite and orthogonal to the array of transducers. This imaging geometry allows reconstruction of volumes that depict SoS distributions from the measured time of flight data. The reconstructed LUT images can subsequently be employed by an optoacoustic reconstruction algorithm to compensate for acoustic wavefield aberration and thereby improve accuracy of the reconstructed images of the absorbed optical energy. The coregistered OAT-LUT imaging is validated in a phantom and live mouse using a single-slice system prototype.

Characterizing disease progression and identifying possible therapeutic interventions in stroke is greatly aided by the use of longitudinal function imaging studies. In this study, we investigate the applicability of real-time multispectral optoacoustic tomography (MSOT) as a tool for non-invasive monitoring of the progression of stroke in the whole brain. The middle cerebral artery occlusion (MCAO) method was used to induce stroke. Mice were imaged under isoflurane anesthesia preoperatively and at several time points during and after the 60-minute occlusion. The animals were sacrificed after 24 hours and their excised brains frozen at -80°C for sectioning. The cryosection were stained using H&E staining to identify the ischemic lesion. Major vessels are readily identifiable in the whole mouse head in the in vivo optoacoustic scans. During ischemia, a reduction in cerebral blood volume is detectable in the cortex. Post ischemia, spectral unmixing of the optoacoustic signals shows an asymmetry of the deoxygenated hemoglobin in the hemisphere affected by MCAO. This hypoxic area was mainly located around the boundary of the ischemic lesion and was therefore identified as the ischemic penumbra. Non-invasive functional MSOT imaging is able to visualize the hypoxic penumbra in brains affected by stroke. Stopping the spread of the infarct area and revitalizing the penumbra is central in stroke research, this new imaging technique may therefore prove to be a valuable tool in the monitoring and developing new treatments.

We present a novel approach to photoacoustic imaging of prostate brachytherapy seeds utilizing an existing urinary catheter for transurethral light delivery. Two canine prostates were surgically implanted with brachyther- apy seeds under transrectal ultrasound guidance. One prostate was excised shortly after euthanasia and fixed in gelatin. The second prostate was imaged in the native tissue environment shortly after euthanasia. A urinary catheter was inserted in the urethra of each prostate. A 1-mm core diameter optical fiber coupled to a 1064 nm Nd:YAG laser was inserted into the urinary catheter. Light from the fiber was either directed mostly parallel to the fiber axis (i.e. end-fire fire) or mostly 90° to the fiber axis (i.e. side-fire fiber). An Ultrasonix SonixTouch scanner, transrectal ultrasound probe with curvilinear (BPC8-4) and linear (BPL9-5) arrays, and DAQ unit were utilized for synchronized laser light emission and photoacoustic signal acquisition. The implanted brachytherapy seeds were visualized at radial distances of 6-16 mm from the catheter. Multiple brachytherapy seeds were si- multaneously visualized with each array of the transrectal probe using both delay-and-sum (DAS) and short-lag spatial coherence (SLSC) beamforming. This work is the first to demonstrate the feasibility of photoacoustic imaging of prostate brachytherapy seeds using a transurethral light delivery method.

Novel ultrasound transducers were created with a composite of carbon nanotubes (CNTs) and polydimethylsiloxane (PDMS) that was dip coated onto the end faces of optical fibers. The CNTs were functionalized with oleylamine to allow for their dissolution in xylene, a solvent of PDMS. Ultrasound pulses were generated by illuminating the composite coating with pulsed laser light. At distances of 2 to 16 mm from the end faces, ultrasound pressures ranged from 0.81 to 0.07 MPa and from 0.27 to 0.03 MPa with 105 and 200 μm core fibers, respectively. Using an optical fiber hydrophone positioned adjacent to the coated 200 µm core optical fiber, ultrasound reflectance measurements were obtained from the outer surface of a sheep heart ventricle. The results of this study suggest that ultrasound transducers that comprise optical fibers with CNT-PDMS composite coatings may be suitable for miniature medical imaging probes.

Optoacoustic (OA) imaging will experience broadest clinical application if implemented in epi-style with the irradiation optics and the acoustic probe integrated in a single probe. This will allow most flexible imaging of the human body in a combined system together with echo ultrasound (US). In such a multimodal combination, the OA signal could provide functional information within the anatomical context shown in the US image, similar to what is already done with colour flow imaging. Up to date, successful deep epi-OA imaging was difficult to achieve, owing to clutter and acoustic aberrations. Clutter signals arise from strong optical absorption in the region of tissue irradiation and strongly reduce contrast and imaging depth. Acoustic aberrations are caused by the inhomogeneous speed of sound and degrade the spatial resolution of deep tissue structures, further reducing contrast and thus imaging depth. In past years we have developed displacement-compensated averaging (DCA) for clutter reduction based on the clutter decorrelation that occurs when palpating the tissue using the ultrasound probe. We have now implemented real-time DCA on a research ultrasound system to evaluate its clutter reduction performance in freehand scanning of human volunteers. Our results confirm that DCA significantly improves image contrast and imaging depth, making clutter reduction a basic requirement for a clinically successful combination of epi-OA and US imaging. In addition we propose a novel technique which allows automatic full aberration correction of OA images, based on measuring the effect of aberration spatially resolved using echo US. Phantom results demonstrate that this technique allows spatially invariant diffraction-limited resolution in presence of a strong aberrator.

A new experimental approach for measurements of temperature dependence of the Grüneisen parameter in optically absorbing solutions is proposed. Two-dimensional optoacoustic (OA) imaging is used to improve accuracy of signal amplitude measurements and spatial localization of the studied samples. We estimated OA response of optically absorbing solutions measuring median intensity of OA images within the region of interest (ROI) as a function of temperature. We showed that when normalized to its value at a particular temperature, OA image intensity becomes an accurate metric reflecting temperature changes of Grüneisen parameter regardless of local optical fluence and absorbance, assuming those remain constant with temperature. Using the proposed method we studied temperature dependence of aqueous solutions of nickel and cupric sulfate in the range from 4 to 40°C. Obtained results were compared with temperature dependence for the Grüneisen parameter of DI-water, which we measured by using carbon ink colloid. We also found that Grüneisen-temperature relationship for nickel sulfate exhibits linear trend with respect to the concentration, and is independent of coupling medium and laser excitation wavelength.In the future, the developed methodology could be adopted for important applications of in vivo optoacoustic temperature monitoring.

We report on a hand-held imaging probe for real-time optoacoustic visualization of deep tissues in three dimensions. The proposed solution incorporates a two-dimensional array of ultrasonic sensors densely distributed on a spherical surface, whereas illumination is performed coaxially through a cylindrical cavity in the array. Visualization of three-dimensional tomographic data at a frame rate of 10 images per second is enabled by parallel recording of 256 time-resolved signals for each individual laser pulse along with a highly efficient GPUbased real-time reconstruction. A liquid coupling medium (water), enclosed in a transparent membrane, is used to guarantee transmission of the optoacoustically generated waves to the ultrasonic detectors. Excitation at multiple wavelengths further allows imaging spectrally distinctive tissue chromophores such as oxygenated and deoxygenated haemoglobin. The performance is showcased by video-rate tracking of deep tissue vasculature and three-dimensional measurements of blood oxygenenation in a healthy human volunteer. The flexibility provided by the hand-held hardware design, combined with the real-time operation, makes the developed platform highly usable for both small animal research and clinical imaging in multiple indications, including cancer, inflammation, skin and cardiovascular diseases, diagnostics of lymphatic system and breast

Accurate and efficient identification of nerves is of great importance during many ultrasound-guided clinical procedures, including nerve blocks and prostate biopsies. It can be challenging to visualise nerves with conventional ultrasound imaging, however. One of the challenges is that nerves can have very similar appearances to nearby structures such as tendons. Several recent studies have highlighted the potential of near-infrared optical spectroscopy for differentiating nerves and adjacent tissues, as this modality can be sensitive to optical absorption of lipids that are present in intra- and extra-neural adipose tissue and in the myelin sheaths. These studies were limited to point measurements, however. In this pilot study, a custom photoacoustic system with a clinical ultrasound imaging probe was used to acquire multi-spectral photoacoustic images of nerves and tendons from swine ex vivo, across the wavelength range of 1100 to 1300 nm. Photoacoustic images were processed and overlaid in colour onto co-registered conventional ultrasound images that were acquired with the same imaging probe. A pronounced optical absorption peak centred at 1210 nm was observed in the photoacoustic signals obtained from nerves, and it was absent in those obtained from tendons. This absorption peak, which is consistent with the presence of lipids, provides a novel image contrast mechanism to significantly enhance the visualization of nerves. In particular, image contrast for nerves was up to 5.5 times greater with photoacoustic imaging (0.82 ± 0.15) than with conventional ultrasound imaging (0.148 ± 0.002), with a maximum contrast of 0.95 ± 0.02 obtained in photoacoustic mode. This pilot study demonstrates the potential of photoacoustic imaging to improve clinical outcomes in ultrasound-guided interventions in regional anaesthesia and interventional oncology.

Circulatory shock is a dangerous medical condition, in which blood flow cannot provide the necessary amount of oxygen to organs and tissues. Currently, its diagnosis and therapy decisions are based on hemodynamic parameters (heart rate, blood pressure, blood gases) and mental status of a patient, which all have low specificity. Measurement of mixed or central venous blood oxygenation via catheters is more reliable, but highly invasive and associated with complications. Our previous studies in healthy volunteers demonstrated that optoacoustic systems provide non-invasive measurement of blood oxygenation in specific vessels, including central veins. Here we report our first results of a clinical study in coronary artery bypass graft (CABG) surgery patients. We used a medical-grade OPO-based optoacoustic system developed in our laboratory to measure in real time blood oxygenation in the internal jugular vein (IJV) of these patients. A clinical ultrasound imaging system (GE Vivid e) was used for IJV localization. Catheters were placed in the IJV as part of routine care and blood samples taken via the catheters were processed with a CO-oximeter. The optoacoustic oxygenation data were compared to the CO-oximeter readings. Good correlation between the noninvasive and invasive measurements was obtained. The results of these studies suggest that the optoacoustic system can provide accurate, noninvasive measurements of central venous oxygenation that can be used for patients with circulatory shock.

Due to the high scattering coefficient of tissue over the wavelength range used for photoacoustic (PA) imaging, most studies employ bulky, low repetition rate lasers to provide sufficient pulse energies at depth to image within the body. The size and cost of these lasers has impeded integration of photoacoustics into conventional, routinely-used ultrasound (US) scanners. Here, we present an approach leveraging the capabilities of modern, high repetition rate fiber lasers to produce a clinically translatable system providing integrated US/PA images at frame rates > 30 Hz. The system uses a portable, low-cost, low pulse-energy (1 mJ/pulse), high repetition rate (1 kHz), 1064 nm laser and is designed for integrated US/PA imaging of the peripheral vasculature or any relevant diseased region, such as a tumor. Using a rotating galvo-mirror system, the incident laser beam is quickly scanned over the imaging area. Multiple PA images covering the scan area are integrated to form a single PA image. Additionally, ultrasound firings are integrated into the scan sequence to provide an US image reconstructed over the same frame period. We acquired PA images of a 1.5-mmdiameter cylindrical absorber (absorption coefficient 5 cm^-1) embedded in a tissue-mimicking gelatin phantom at 6-cm depth. A 2 cm × 1 cm (depth × lateral) area was reconstructed. We obtained a signal-to-noise ratio of more than 30 dB, comparable to conventional PA methods using high energy, low repetition rate lasers. The current system produces an integrated US/PA frame at a 32 Hz rate, and 100 Hz frame rates are possible with our present approach.

The scanning mechanism is a major technical focus in optical-resolution photoacoustic microscopy. Flexible scanning access with fast scanning speed is desired to monitor biological and physiological dynamics with high temporal resolution. We developed random-access optical-resolution photoacoustic microscopy (RA-OR-PAM) using a digital micromirror device (DMD). Each micromirror on the DMD can be independently controlled, allowing imaging of regions of interest with arbitrary user-selected shapes without extraneous information. A global structural image is first acquired, and the regions of interest are selected. The laser beam then scans these regions exclusively, resulting in a faster frame rate than in a conventional raster scan. This system can rapidly scan arbitrarily shaped regions of interest with a lateral resolution of 3.6 μm within a 40×40 μm² imaging area, a size comparable to the focal spot size of a 50 MHz ultrasound transducer. We demonstrated the random-access ability of RA-OR-PAM by imaging a monolayer of red blood cells. This system was then used to monitor blood flow in vivo within user-selected capillaries in a mouse ear. By imaging only the capillary of interest, the frame rate was increased by up to 13.3 times.

For three-dimensional imaging of optical absorbance, the existing technology of photoacoustic microscopy (PAM) has quite poor axial resolution, the tens of microns to hundreds of microns. This is despite the fact that PAM has recently achieved lateral resolutions on the order of a micron or submicron, comparable to that of optical microscopy. In this paper, a pure optical photoacoustic microscopy (POPAM) with optical rastering of a focused excitation beam and optically sensing of the photoacoustic signal using a microring resonator was developed with the super broad bandwidth of the system more than 350MHz. With unprecedented broad bandwidth of POPAM, 3.8μm axial resolution was achieved without deconvolution processing. Sectioning imaging ability along axial direction presenting 3D morphologic features was shown based on imaging printed phantom. The impact of this approach will be similar to how confocal optical microscopy revolutionized the conventional optical microscopy by enabling the axial sectioning capability. Tissue imaging comparing POPAM and conventional PAM based on needle hydrophone demonstrated that though such broad bandwidth compromised the sensitivity of POPAM 4.35 times than that of conventional PAM, the noise equivalent detectable pressure (NEDP) was estimated as 74Pa, still able to get the tissue imaging.

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 attempt to develop a compact, portable, and low cost OR-PAM based on a consumer Blu-ray (405 nm) DVD pickup head for label-free micro-vascular imaging and red-blood-cell related blood examination. According to the high optical absorption of the hemoglobin at 405 nm, the proposed OR-PAM has potential to be an alternative for the conventional optical microscopy in the examinations of hematological morphology for blood routine. We showed that the Blu-ray DVD pickup head owns the required laser energy and focusing optics for OR-PAM. The firmware of a Blu-ray DVD drive was modified to allow its pickup head to generate nano-second laser pulses with a tunable pulse repetition rate of >30 kHz and a tunable pulse width ranging from 10 to 30 ns. The laser beam was focused onto the target after passing through a transparent cover slide, 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 measured maximum achievable lateral resolution was 1 μm which was mainly limited by the minimum step size of the used motorized stage. A blood smear was imaged without any staining. The red blood cells were well resolved and the biconcave structure could be clearly visualized. In addition, to verify the in vivo imaging capability of the proposed OR-PAM, the micro-vasculature of a mouse ear was imaged without any contrast agent. The results showed that it performed better than a 200x digital optical microscope in terms of image contrast and vascular morphology. In summaries, the proposed OR-PAM has been demonstrated as a promising tool for label-free blood imaging in both small animal studies and blood examinations, and potentially can be a compact and low-cost OR-PAM platform.

The axial resolution of photoacoustic microscopy (PAM) can be enhanced by reducing the speed of sound within the imaging region of interest. This principle was demonstrated on a previously-reported PAM system, which utilized a 125 MHz ultrasonic transducer for signal detection and the Wiener deconvolution for signal processing. With sound slowed by silicone oil immersion, we have achieved a finest axial resolution of 5.8 μm for PAM, as validated by phantom experiments. The axial resolution was also enhanced in vivo when mouse ears injected with silicone oil were imaged. After injection of silicone oil, the blood vessels were resolved more clearly. When tissue-compatible low-speed liquids become available, this approach may find applications in PAM as well as in other imaging modalities, such as photoacoustic computed tomography and ultrasound imaging.

Controllable light delivery to the region of interest is essential to biomedical optical imaging methods like photoacoustic microscopy. It is, however, challenging beyond superficial depths in biological tissue (~1 mm beneath human skin) due to the strong scattering of light that scrambles the photon propagation paths. Recently, optical wavefront shaping has been proposed to modulate the incident light wavefront to compensate for the scattering-induced phase distortions, and consequentially, convey light optimally to a desired location behind or inside turbid media. To reach an optimum wavefront, a searching algorithm is usually required to optimize a feedback signal. In this work, we present our latest explorations, which use photoacoustic signals as the feedback to remotely and non-invasively guide the wavefront shaping process. Our method does not require direct optical access to the target region or the invasive embedding of fluorescence probes inside turbid media. Experimentally, we have demonstrated that diffuse light can be converged to the ultrasound focus by maximizing the amplitude of photoacoustic emissions from the intended absorbing site. Moreover, we show that wavefront-shaped light focusing can enhance existing optical imaging modalities like photoacoustic microscopy, in regard to signal-to-noise ratio, imaging depth, and potentially, resolution.

To achieve localized light delivery beyond turbid layers, TRUE optical focusing has been previously implemented by both analog and digital devices. The digital scheme offers a higher energy gain than the analog version. In many biological applications, the reflection-mode configuration, which uses backscattered light from the sample, is more suitable than the transmission-mode configuration. Although reflection-mode analog TRUE focusing has been demonstrated, its digital implementation has not been explored. Here, we report a reflection-mode digital TRUE focusing to concentrate light through a turbid layer. Further, by simply moving the ultrasound focus, we show the system's dynamic focusing capability.

The interaction of ultrasound and light in biological tissues results in a small amount of the scattered light being shifted relative to the carrier frequency (typically 1 part in 10⁸). We have developed an inherently efficient and low noise quantum memory based technique to selectively absorb these ‘ultrasound tagged’ photons in a pair of atomic frequency combs, and recover them delayed in time as a photon echo. In this manner we have demonstrated record ultrasoundmodulated sideband-to-carrier discrimination (49dB). Further, we confirm that the technique is compatible with highly scattering samples, and present initial acoustic pulse tracking measurements. This strongly suggests the suitability of the technique for biological tissue imaging.

Optoacoutic mesoscopy aims to bridge the gap between optoacoustic microscopy and optoacoustic tomography. We have developed a setup for optoacoustic mesoscopy where we use a high frequency, high numerical aperture spherically focused ultrasound transducer, with a wide bandwidth of 25-125 MHz. The excitation is performed using a diode laser capable of >500 μJ/pulse, 1.8ns pulse width, 1.4 kHz pulse repetition rate, at 515 nm. The system is capable to penetrate more than 5 mm with a resolution of 7 μm axially and 30 μm transversally. Using high-speed stages and scanning the transducer in a quasi-continuous mode, a field of view of 2×2 mm₂ is scanned in less than 2 minutes. The system is suitable for imaging biological samples that have a diameter of 1-5 mm; zebrafish, drosophila melanogaster, and thin biological samples such as the mouse ear and mouse extremities. We have used our mesoscopic setup to generate 3- dimensional images of genetically modified drosophila fly, and drosophila pupae expressing GFP from the wings, high resolution images were generated in both cases, in the fly we can see the wings, the legs, the eyes, and the shape of the body. In the pupae the outline of the pupae, the spiracles at both ends and a strong signal corresponding to the location of the future wings are observed.

To provide more information for classification and assessment of biological tissues, photoacoustic spectrum analysis (PASA) moves beyond the quantification of the intensities of the photoacoustic (PA) signals by the use of the frequency-domain power distribution, namely power spectrum, of broadband PA signals. The method of PASA quantifies the linear-fit to the power spectrum of the PA signals from a biological tissue with 3 parameters, including intercept, midband-fit and slope. Intercept and midband-fit reflect the total optical absorption of the tissues whereas slope reflects the heterogeneity of the tissue structure. Taking advantage of the optical absorption contrasts contributed by lipid and blood at 1200 and 532 nm, respectively and the heterogeneous tissue microstructure in fatty liver due to the lipid infiltration, we investigate the capability of PASA in identifying histological changes of fatty livers in mouse model. 6 and 9 pairs of normal and fatty liver tissues from rat models were examined by ex vivo experiment with a conventional rotational PA measurement system. One pair of rat models with normal and fatty livers was examined non-invasively and in situ with our recently developed ultrasound and PA parallel imaging system. The results support our hypotheses that the spectrum analysis of PA signals can provide quantitative measures of the differences between the normal and fatty liver tissues and that part of the PA power spectrum can suffice for characterization of microstructures in biological tissues. Experimental results also indicate that the vibrational absorption peak of lipid at 1200nm could facilitate fatty liver diagnosis.

An assessment has been made of various experimental factors affecting the accuracy of flow velocities measured using a pulsed time correlation photoacoustic Doppler technique. In this method, Doppler time shifts are quantified via crosscorrelation of pairs of photoacoustic waveforms generated in moving absorbers using pairs of laser light pulses, and the photoacoustic waves are detected using an ultrasound transducer. The acoustic resolution mode is employed by using the transducer focal width, rather than the large illuminated volume, to define the lateral spatial resolution. This enables penetration depths of several millimetres or centimetres, unlike methods using the optical resolution mode, which limits the maximum penetration depth to approximately 1 mm. In the acoustic resolution mode, it is difficult to detect time shifts in highly concentrated suspensions of flowing absorbers, such as red blood cell suspensions and whole blood, and this challenge supposedly arises because of the lack of spatial heterogeneity. However, by assessing the effect of different absorption coefficients and tube diameters, we offer an alternative explanation relating to light attenuation and parabolic flow. We also demonstrate a new signal processing method that surmounts the previous problem of measurement under-reading. This method is a form of signal range gating and enables mapping of the flow velocity profile across the tube as well as measurement of the average flow velocity. We show that, using our signal processing scheme, it is possible to measure the flow of whole blood using a relatively low frequency detector. This important finding paves the way for application of the technique to measurements of blood flow several centimetres deep in living tissue.

We introduce an innovative detection approach for photoacoustic tomography. The pressure induced surface displacement is obtained in 2D by Electronic Speckle Pattern Interferometry (ESPI) in a repetitive measurement with a variable time delay between excitation- and detection pulses. The detection approach works without any physical contact to the object surface and is very versatile in terms of an adjustable object surface area and an adjustable temporal sampling rate. Furthermore, the approach is potentially very fast by the use of a high speed camera and a high repetition laser excitation and detection. In a proof of concept measurement, transparent silicone cubes with black silicone sphere absorbers are measured. In order to validate the acquired displacement data, the pressure is measured using a lipstick needle hydrophone and correlated to the measured displacement.

The metabolic rate and oxygen consumption of the brain is reflected in jugular venous oxygen saturation. In many clinical conditions, such as head trauma, stroke, and low cardiac output states, the brain is at risk for hypoxic-ischemic injury. The current gold standard for monitoring brain oxygenation is invasive and requires jugular vein catheterization under fluoroscopic guidance; and therefore it is rarely used. Photo-acoustic tomography in combination with ultrasound can be used to estimate oxygen saturation of the internal jugular vein in real-time. This noninvasive method will enable earlier detection and prevention of impending hypoxic brain injury. A wavelength-tunable dye laser pumped by a Nd:YAG laser delivers light through an optical fiber bundle, and a modified commercial ultrasound imaging system (Philips iU22) detects both the pulse-echo ultrasound (US) and photoacoustic (PA) signals. A custom-built multichannel data acquisition system renders co-registered ultrasound and photoacoustic images at 5 frames per second. After the jugular vein was localized in healthy volunteers, dualwavelength PA images were used to calculate the blood hemoglobin oxygen saturation from the internal jugular vein in vivo. The preliminary results raise confidence that this emerging technology can be used clinically as an accurate, noninvasive indicator of cerebral oxygenation.

Lack of sensory feedback during laser surgery prevents surgeons from keeping track of the exact lesion profile and cutting depth. As a result, duration and complexity of the treatments are significantly increased. In this study we propose a new method for enabling three-dimensional tracking of the exact lesion profile, based on detection of shock waves emanating from the ablated tissue and subsequent reconstruction of the incision location using time-of-flight data obtained from multiple acoustic detectors. Ablation was performed in fresh bovine tissue samples using a Q-switched Nd-YAG laser, delivering 8 ns duration 150mJ pulses at a wavelength of 1064nm and repetition rate of 5Hz. The beam was focused by a 50mm lens on the tissue surface, which resulted in a deep cut of up to 9mm depth. The generated shock waves were detected using a spherical matrix ultrasonic array. The exact cutting profile was subsequently rendered by reconstructing the origin of shockwaves detected during the entire procedure. Different combinations of the detector positions were considered with respect to the resulting reconstruction quality. It was observed that, by utilizing at least 12 detection elements, the lesion profile could be characterized with high accuracy in all three dimensions, which was confirmed by histological evaluations. The proposed method holds promise for delivering highly precise and accurate real-time feedback during laser surgeries.

Imaging is crucial for stem cell therapy to monitor the location(s), numbers, and state of the implanted cells. Real-time imaging in particular can ensure proper cell delivery for best engraftment. However, established imaging tools such as MRI are limited by their temporal resolution for guidance during delivery. In contrast, photoacoustic imaging is ideally suited for real time, image-guided therapy. Here, we use silica-coated gold nanorods as photoacoustic contrast agents and deploy them to image and quantitate mesenchymal stem cells during implant into the muscle tissue of live mice. Silica-coated gold nanorods (SiGNRs) were created with standard methods and loaded into mesenchymal stem cells (MSCs) without transfection agents. There was no significant (p<0.05) toxicity or changes to cell proliferation after incubating MSCs with 0.05 nM SiGNRs for 3 hours. A panel of cytokines should only minor upregulation of inflammatory markers including interleukin-6. We used electron microscopy to illustrate vacuole-bound SiGNRs inside the cells. This cell staining increased photoacoustic signal 175% relative to MSCs without contrast agent—the silica coat itself increased signal 55% relative to uncoated GNRs. Using inductively coupled plasma spectroscopy, we found that there were 100,000 SiGNRs per MSC. This value was 5-fold higher than a MSC population stained with GNRs in the absence of silica coat. After labeling, cells were washed and injected into murine muscle tissue to simulate a muscular dystrophy patient. Mice (N=5) treated with these SiGNRlabeled MSCs exhibited no adverse events and implants up to 5 mm deep were easily visualized. The in vivo detection limit was 90,000 cells in a 100 uL bolus in mouse thigh muscle. Here, the B-mode signal is useful for orienting the treatment area and visualizing the delivery catheter while the photoacoustic mode offers cell-specific content. The photoacoustic signal was validated with histology a long-term fluorescent tracking dye after MSC transplant.

Neovascularity also known as angiogenesis is an early feature of inflammatory arthritis disease. Therefore, identifying the development of neovascularity is one way to potentially detect and characterize arthritis. Laser-based photoacoustic imaging (PAI) is an emerging biomedical imaging modality which may aid in detection of both early and continued development of neovascularity. In this work, we investigated the feasibility of PAI to measure angiogenesis, for the purpose of evaluating and monitoring inflammatory arthritis after treatment. The imaging results on an arthritis rat model demonstrate that 1) there is noticeable enhancement in image intensity in the arthritic ankle joints when compared to the normal joints, and 2) there is noticeable decrease in image intensity in the arthritic ankle joints after treatment when compared to the untreated arthritic joints. In order to validate the findings from PAI, we performed positron emission tomography (PET) and histology on the same joints. The diameters of the ankle joints, as a clinical score of the arthritis, were also measured at each time point.

Vascular therapy in oncology exploits the differences between normal blood vessels and abnormal tumour neoangiogenesis to selectively target cancer. For optimal treatment efficacy, and translation of novel compounds, the response of the tumour vasculature needs to be assessed. Photoacoustic tomography (PAT) is capable of this as it provides highly spatially resolved 3D images of vascular networks in biological tissue to cm depths. In preclinical models of cancer this is sufficient to encompass entire subcutaneous tumours, and can therefore be used to evaluate pharmacological intervention directed at the vasculature. In this study the vascular disrupting agent OXi4503 was used to treat subcutaneous tumour mouse models of two human colorectal carcinoma tumour types (SW1222, LS174T) at a range of concentrations (40mg/kg, 10mg/kg, 1mg/kg and sham dose control). The characteristic destruction of tumour vasculature caused by OXi4503 was observed by PAT and confirmed ex vivo via histology. Differences observed between the two tumour types assessed demonstrate the importance of tumour microenvironment and pathophysiology on response to therapy. Differential response to different doses of OXi4503 was observed, with outward tumour growth only seen once entire tumour viability had been re-established; this demonstrates the potential of PAT to act as a biomarker of response for the translation of novel anti-vascular compounds and also within the clinic. This study shows clearly that PAT can accurately assess the time course of drug action and relapse of pharmacodynamic effect in preclinical models of cancer and the important translational prospects for vascular targeted tumour therapies.

We propose time and frequency domain methods for homogenous flow measurement based on the photoacoustic Doppler effect. Excited by spatially modulated laser pulses, the flowing medium induces a Doppler frequency shift in the received photoacoustic signals. The frequency shift is proportional to the component of the flow speed projected onto the acoustic beam axis. These methods do not rely on particle heterogeneity in the medium. A red-ink phantom flowing in a tube immersed in water was used to validate the methods in both frequency and time domains.

We present two-photon absorption-induced photoacoustic measurements. Since two-photon photoacoustics is a nonlinear effect numerous physical effects might lead to similar observations. In the presented paper we focus on the effect of the temperature dependent Grüneisen coefficient of water. We discuss the expected theoretical behavior and compare measurements with theoretical predictions. Finally, we demonstrate multimodal photoacoustic and luminescence microscopy via two-photon absorption, and show that it is possible to image objects with localized dyes. Excitation is performed using a femtosecond laser with a wavelength of 800nm. The photoacoustic signals, the luminescence intensity and its spectrum were detected simultaneously.

Currently, laser fluence calibration is typically required for quantitative measurement of particle concentration in photoacoustic microscopy. In this paper, we present another quantitative approach to measure absolute absorber concentrations by photoacoustic correlation spectroscopy. The proposed method is based on the fact that the Brownian motion induces particle count fluctuation in the detection volume. We first derived a theoretical model for photoacoustic signals and then applied our method to quantitative measurement of different concentrations of various particles. The experimental results agreed well with the predictions from the theoretical model, suggesting that our method can be used for absolute particle concentrations measurement.

Photoacoustic imaging is a novel imaging technology for visualizing optically-absorbing structures in vivo with simultaneous high contrast and high spatial resolution. However, differentiation of photoacoustic signals is non-trivial hence specificity can be poor. We propose multimodality photoacoustic and Raman imaging system using surfaceenhanced Raman scattering nanoparticles, previously demonstrated to have extremely high specificity and multiplexing capabilities. Moreover, we propose to target cancer cells with both magnetic and surface-enhanced Raman scattering nanoparticles, which can further improve our system to be used for magnetic enrichment and detection of circulating tumor cells.

We have developed an intraoperative surgical photoacoustic microscopy (IS-PAM) system by integrating an optical resolution photoacoustic microscopy (OR-PAM) and conventional surgical microscope. Based on the common optical path in the OR-PAM and microscope system, we can acquire the PAM and microscope images at the same time. Furthermore, by utilizing a mini-sized beam projector, 2D PAM images are back-projected onto the microscope view plane as augmented reality. Thus, both the conventional microscopic and 2D cross-sectional PAM images are displayed on the plane through an eyepiece lens of the microscope. In our method, additional image display tool is not required to show the PAM image. Therefore, it potentially offers significant convenience to surgeons without movement of their sights during surgeries. In order to demonstrate the performance of our IS-PAM system, first, we successfully monitored needle intervention in phantoms. Moreover, we successfully guided needle insertion into mice skins in vivo by visualizing surrounding blood vessels from the PAM images and the magnified skin surfaces from the conventional microscopic images simultaneously.

We have demonstrated a novel microbubbles methylene blue solution, called to “MB²” solution for a dual modality contrast. We have photoacoustically and ultrasonically imaged and quantified aqueous solutions of MB² by varying the concentration of either microbubbles or methylene blue to investigate the dual modal imaging capability. Interestingly, as the microbubbles concentration increased with the constant methylene blue concentration, photoacoustic (PA) signal was greatly attenuated in the MB² solution. Conversely, when methylene blue concentration increased with the fixed microbubbles concentration, no interference was observed in ultrasound (US) signals. To further confirm our findings, we switched the PA and ultrasound (US) signals using conventional ultrasound. We compared the PA and US signals of the MB² solution before and after sonication. The PA amplitude increased 2.5 times. Conversely, the US signals were initially strong, but decreased 2.5 times following sonication. Moreover, we used a clinically modified PA/US imaging system to disrupt the microbubbles in MB² and recover the PA signals.

A nanoemulsion contrast agent with a perfluorohexane core and optically absorptive gold nanospheres (GNSs) assembled on the surface, is presented to improve the specificity of photoacoustic (PA) molecular imaging in differentiating targeted cells or aberrant regions from heterogeneous background signals. Compared to distributed GNSs, clustered GNSs at the emulsion oil-water interface produce a red-shifted and broadened absorption spectrum, exhibiting fairly high absorption in the near-infrared region commonly used for deep tissue imaging. Above a certain laser irradiation fluence threshold, a phase transition creating a microbubble in the emulsion core leads to more than 10 times stronger PA signals compared with conventional thermal-expansion-induced PA signals. These signals are also strongly non-linear, as verified by a differential scheme using recorded PA images at different laser fluences. Assuming a linear relation between laser fluence and the PA signal amplitude, differential processing results in nearly perfect suppression of linear sources, but retains a significant residue for the non-linear nanoemulsion with more than 35 dB enhancement. This result demonstrates that contrast specificity can be improved using the nanoemulsion as a targeting agent in PA molecular imaging by suppressing all background signals related to a linear PA response. Furthermore, combined with a system providing simultaneous laser/ultrasound excitation, cavitation-generated bubbles have the potential to be a highly specific contrast agent for ultrasound molecular imaging and harmonic imaging, as well as a targeted means for noninvasive ultrasound-based therapies.

Photoacoustic tomography (PAT) has become a powerful tool for biomedical imaging, particularly pre-clinical small animal imaging. Several different measurement systems have been demonstrated, in particular, optically addressed Fabry-Perot interferometer (FPI) sensors have been shown to provide exquisite images when a planar geometry is suitable. However, in its current incarnation the measurements must be made at each point sequentially, so these devices therefore suffer from slow data acquisition time. An alternative to this point-by-point interrogation scheme, is to interrogate the whole sensor with a series of independent patterns, so each measurement is the spatial integral of the product of the pattern and the acoustic field (as in the single-pixel Rice camera). Such an interrogation scheme allows compressed sensing to be used. This enables the number of measurements to be reduced significantly, leading to much faster data acquisition. An experimental implementation will be described, which employs a wide NIR tunable laser beam to interrogate the FPI sensor. The reflected beam is patterned by a digital micro-mirror device, and then focused to a single photodiode. To demonstrate the idea of patterned and compressed sensing for ultrasound detection, a scrambled Hadamard operator is used in the experiments. Photoacoustic imaging experiments of phantoms shows good reconstructed results with 20% compression.

Photoacoustic (PA) tissue characterization relies on the analysis of ultrasound (US) signals generated through the PA effect. The probability distributions of PA signal amplitude as well as the frequency content of the PA signals are typically not considered. We present a phantom study where we introduce the combined use of US/PA signal envelope statistics along with analysis of the frequency content of the US/PA signals for the purposes of monitoring physical changes in the absorbers. The phantoms were constructed using black polystyrene beads (radius 1.77 μm and 7.36 μm). Tissue microenvironment was emulated by homogenously mixing 10 beads/imaging-transducer-resolution-volume in order to accommodate large numbers of sub-resolved beads. The phantoms were imaged with the Vevo LAZR US/PA integrated imaging system (Fujifilm-VisualSonics) using a 40 MHz linear array probe and 680 nm illumination. US/PA signals from the same region of interest were analyzed by mapping the signal amplitudes distributions from 5 phantom locations, fitting the data to the Generalized Gamma (GG) distribution and extracting the fit parameters while computing the normalized power spectra to retrieve the spectral slope (SS) and midband fit (MBF) spectral parameters. The GG scale parameter increased by 500x for US images and 12x for PA equivalents as the size of the beads increased by ~4.5x. The SS decreased by 0.8x for US and 0.4x for PA. These changes can be attributed variations in size and spatial organization of the beads suggesting that combined US/PA statistical and spectral analysis can potentially monitor normal and abnormal tissue physiological changes.

Image reconstruction in optoacoustics usually employs algorithms that assume ultrasonic transducers to be confined to points. This assumption deviates strongly from realistic detectors and is the cause of severe artifacts in the reconstructions. We propose two model-based image reconstruction algorithms that account for the shape of cylindrically focused ultrasonic transducers in three-dimensional optoacoustic tomography. The algorithms have been proved favorable in simulations and experiments.

Photoacoustic computed tomography (PACT) is a hybrid technique that combines optical excitation and ultrasonic detection to provide high resolution images in deep tissues. In the image reconstruction, a constant speed of sound (SOS) is normally assumed. This assumption, however, is often not strictly satisfied in deep tissue imaging, due to acoustic heterogeneities within the object and between the object and coupling medium. If these heterogeneities are not accounted for, they will cause distortions and artifacts in the reconstructed images. In this paper, we incorporated ultrasonic computed tomography (USCT), which measures the SOS distribution within the object, into our full-ring array PACT system. Without the need for ultrasonic transmitting electronics, USCT was performed using the same laser beam as for PACT measurement. By scanning the laser beam on the array surface, we can sequentially fire different elements. As a first demonstration of the system, we studied the effect of acoustic heterogeneities on photoacoustic vascular imaging. We verified that constant SOS is a reasonable approximation when the SOS variation is small. When the variation is large, distortion will be observed in the periphery of the object, especially in the tangential direction.

In order to monitor dynamic physiological events in near-real time, a variety of photoacoustic computed tomography (PACT) systems have been developed that can rapidly acquire data. Previously reported studies of dynamic PACT have employed conventional static methods to reconstruct a temporally ordered sequence of images on a frame-by-frame basis. Frame-by-frame image reconstruction (FBFIR) methods fail to exploit correlations between data frames and are known to be statistically and computationally suboptimal. In this study, a low-rank matrix estimation-based spatio-temporal image reconstruction (LRME-STIR) method is investigated for dynamic PACT applications. The LRME-STIR method is based on the observation that, in many PACT applications, the number of frames is much greater than the rank of the ideal noiseless data matrix. Using computer-simulated photoacoustic data, the performance of the LRME-STIR method is compared with that of conventional FBFIR method. The results demonstrate that LRME-STIR method is not only computationally more efficient but also produces more accurate dynamic PACT images than a conventional FBFIR method.

Quantitative ultrasound (QUS) estimates derived from power spectra of pulse-echo signals are sensitive to mi- crostructure and potentially can differentiate among tissues. However, QUS estimates do not provide molecular specificity. We investigated the feasibility of obtaining quantitative photoacoustic (QPA) estimates for sensi- tivity to microstructure and chromophores for tissue classification. QPA methods were tested using gel-based phantoms containing uniformly dispersed, black polyethylene spheres (1E5 particles/ml) with nominal mean diameters of 23.5, 29.5, 42.0, and 58.0 μm. A pulsed, 532-nm laser excited the photoacoustic (PA) response. A single-element, 34-MHz transducer with a 12-mm focal length was raster scanned over the phantom to acquire 3D PA data. Normalized power spectra were generated from the PA signals within 2079, moving (50% overlap), 1-mm-cube regions-of-interest (ROIs) to provide three QPA estimates: spectral slope (SS), spectral intercept (SI), and effective absorber size (EAS). SS and SI were computed using a linear-regression approximation to the normalized spectrum in the -6-dB band. EAS was computed by fitting the normalized spectrum in the -20-dB band to the multi-sphere analytical solution. All estimates were correlated with the size of particles dispersed in the phantoms. SS decreased while SI increased with an increase in particle size. EAS was correlated with nominal particle diameter, but particles aggregation and the finite bandwidth of the PAI system resulted in outliers. SS, SI, and EAS for the 23.5-μm-phantom were -0.14±-0.04 dB/MHz, 4.8±1.3 dB, and 25.4±6.3 μm, respectively; the corresponding values for the 58.0-μm phantom were -0.47±-0.03 dB/MHz, 15.6±0.9 dB, and 82.7±0.9 μm.

Detector arrays enable parallel detection for faster photoacoustic imaging than by moving a single detector, but the detector spacing for arrays cannot be smaller than the size of an array element. Spatial over-sampling is scanning with a step-size smaller than the size of the detector element and is possible only for a moving single detector. For a detector with finite sized surface the measured acoustic signal is a spatial average of the pressure field over the detector surface. If the reconstruction is performed assuming point-like detection over-sampling brings no advantage as e.g. for spherical or cylindrical detection surfaces the blurring caused by a finite detector size is proportional to the distance from the rotation center and is equal to the detector size at the detection surface.

Iterative reconstruction algorithms or inverting directly the imaging matrix can take the finite size of real detectors directly into account, but the numerical effort is significantly higher compared to direct algorithms assuming point-like detection. Another reconstruction with less numerical effort is to use a direct algorithm assuming point-like detectors and run a deconvolution algorithm for deblurring afterwards. For such reconstruction methods spatial over-sampling makes sense because it reduces the blurring significantly.

The effect of step size on the reconstructed image is systematically examined using simulated and experimental data. Experimental data are obtained on a plastisol cylinder with thin holes filled with an absorbing liquid. Data acquisition is done by utilization of a piezoelectric detector (PVDF stripe) which is rotated around the plastisol cylinder.

Resting-state functional connectivity (RSFC) imaging is an emerging neuroimaging approach that aims to identify spontaneous cerebral hemodynamic fluctuations and their associated functional connections. Clinical studies have demonstrated that RSFC is altered in brain disorders such as stroke, Alzheimer’s, autism, and epilepsy. However, conventional neuroimaging modalities cannot easily be applied to mice, the most widely used model species for human brain disease studies. For instance, functional magnetic resonance imaging (fMRI) of mice requires a very high magnetic field to obtain a sufficient signal-to-noise ratio and spatial resolution. Functional connectivity mapping with optical intrinsic signal imaging (fcOIS) is an alternative method. Due to the diffusion of light in tissue, the spatial resolution of fcOIS is limited, and experiments have been performed using an exposed skull preparation. In this study, we show for the first time, the use of photoacoustic computed tomography (PACT) to noninvasively image resting-state functional connectivity in the mouse brain, with a large field of view and a high spatial resolution. Bilateral correlations were observed in eight regions, as well as several subregions. These findings agreed well with the Paxinos mouse brain atlas. This study showed that PACT is a promising, non-invasive modality for small-animal functional brain imaging.

Ultrasound endoscopy has been proven effective in identifying and staging relatively advanced tumors in esophageal or colon wall lining. When combined with optoacoustic imaging modality, the endoscopy examination may prove beneficial in detecting also early stage tumors as well as more accurate staging of advanced tumors based on functional - anatomical maps. Here we present a prototype of a dual-modality optoacoustic – laser ultrasound (OA-LUS) endoscopy system with enhanced imaging capabilities. The system consists of a rotating 90° off-axis parabolic reflector which is acoustically coupled to a flat 8-element transducer array. A parabolic mirror serves dual purpose of directing light towards a sample and reflecting incoming optically generated ultrasound signals towards a detector. LUS modality is enabled by placing an optically absorbing and acoustically transparent polymeric membrane in the path of laser light to generate broadband and non-reverberating transient ultrasound waves propagating towards the sample. Focused system detects ultrasound signals and reconstructs the image similar to optoacoustic mode. Presence of a delay between optically generated and reflected acoustic signals allows concurrent image acquisition in OA and LUS modalities.

Optoacoustic microscopy (OAM) is an emerging technology combining the beneficial features of optical contrast and ultrasound resolution, to form a hybrid imaging technique capable of multi-scale, high-contrast and high-resolution imaging through optically scattering biological tissues. In the past 15 years, two system modifications have been developed for optoacoustic / photoacoustic microscopy: acoustic-resolution AR-OAM and optical-resolution OR-OAM. Typically, acoustic resolution systems can image deeper tissues structures, however, with resolution at least an order of magnitude worse than the systems of optical-resolution. It would be attractive for variety of biomedical applications to attain high (submicron) resolution at a depth exceeding the present limit of the optical resolution optoacoustic microscopy. Here we introduce a novel, all-optical method for OAM, in which not only thermal energy deposition, but also optoacoustic signal detection is achieved optically. In our design the probe laser beam was used as an ultrawide-band ultrasonic transducer. In this method the acoustic pressure wave amplitude is proportional to the angle of deflection of the probing CW laser beam incident on a balanced dual photodiode. Such laser beam deflection (LBD) method overcomes the limitations of conventional piezoelectric ultrasound transducers and optical interferometers. LBD method allows one to use high numerical aperture objectives for better focusing, avoid distortions associated with the system elements that separate optical and acoustic paths, and provides better sensitivity than any optical interferometer. It also provides a non-contact method that is insensitive to optical and acoustic artifacts typical of backward mode of optoacoustic imaging. The LBD sensitivity depends on a large number of system parameters such as probe beam power, spot size, interaction length, optical refraction index of the coupling medium, laser wavelength, photodiode sensitivity, proximity to the optoacoustic source, and thus, can be optimized. The basic setup of OR-LBD-OAM shows high sensitivity competitive with commercial ultrasonic transducers. We report first images of biological cells and tissues obtained using this technique.

The endogenous contrast in thermoacoustic imaging is due to the water and ionic content in tissue. This results in poor tissue speci city between high water content tissues. As a result, exogenous contrast agents have been employed to improve tissue speci city and also increase the SNR. An investigation into the sources of contrast produced by several exogenous contrast agents is described. These include three gadolinium based MRI contrast agents, iron oxide particles, single wall carbon nanotubes, saline and sucrose solutions. Both the dielectric and magnetic properties of contrast agents at 3GHz have been measured using microwave resonant cavities. The DC conductivity of the contrast agents were also measured. It is shown that the measured increase in dielectric contrast, relative to water, is due to dipole rotational loss of polar non electrolytes, ionic loss of electrolytes or a combination of both. It is shown that for the same dielectric contrast, electrolytes make better thermoacoustic contrast agents than non-electrolytes, for thermoacoustic imaging.

In current photoacoustic tomography (PAT), l-D or 2-D ultrasound arrays and multi-channel data acquisition (DAQ) electronics are used to detect the photoacoustic signals simultaneously for “real-time” image construction. However, as the number of transducer elements and DAQ channels increase, the construction and operation of the ultrasound receiving system will become complex and costly. This situation can be addressed by using parallel acoustic delay lines (PADLs) to create true time delays in multiple PA signal channels. The time-delayed PA signals will reach the ultrasound transducer at different times and therefore can be received by one single-element transducer without mixing with each other. In this paper, we report the development of the first miniaturized PADL probe suitable for handheld operations. Fusedsilica optical fibers with low acoustic attenuation were used to construct the 16 PADLs with specific time delays. The handheld probe structure was fabricated using precision laser-micromachining process to provide robust mechanical support and accurate alignment of the PADLs with minimal acoustic distortion and inter-channel coupling. The 16 optical-fiber PADLs were arranged to form one input port and two output ports. Photoacoustic imaging of a black-ink target embedded in an optically-scattering phantom was successfully conducted using the handheld PADL probe with two single-element transducers and two DAQ channels (equal to a channel reduction ratio of 8:1). Our results show that the PADL technique and the handheld probe could provide a promising solution for real-time PAT with significantly reduced complexity and cost of the ultrasound receiver system.

Conventional photoacoustic microscopy (PAM) employs light pulses to produce a photoacoustic (PA) effect and detects the resulting acoustic waves using an ultrasound transducer acoustically coupled to the target. The resolution of conventional PAM is limited by the sensitivity and bandwidth of the ultrasound transducer. We investigated a versatile, all-optical PAM (AOPAM) system for characterizing in vivo as well as ex vivo biological specimens. The system employs non-contact interferometric detection of PA signals that overcomes limitations of conventional PAM. A 532-nm pump laser with a pulse duration of 5 ns excites the PA effect in tissue. Resulting acoustic waves produce surface displacements that are sensed using a 532-nm continuous-wave (CW) probe laser in a Michelson interferometer with a 1- GHz bandwidth. The pump and probe beams are coaxially focused using a 50X objective giving a diffraction-limited spot size of 0.48 μm. The phase-encoded probe beam is demodulated using homodyne methods. The detected timedomain signal is time reversed using k-space wave-propagation methods to produce a spatial distribution of PA sources in the target tissue. A minimum surface-displacement sensitivity of 0.19 pm was measured. PA-induced surface displacements are very small; therefore, they impose stringent detection requirements and determine the feasibility of implementing an all-optical PAM in biomedical applications. 3D PA images of ex vivo porcine retina specimens were generated successfully. We believe the AOPAM system potentially is well suited for assessing retinal diseases and other near-surface biomedical applications such as sectionless histology and evaluation of skin burns and pressure or friction ulcers.

We propose a multilayer film structure to generate high frequency and narrowband ultrasound. It consists of three light-absorbing layers and two light-transmittance layers. The amplitude is tunable by adjusting the optical absorption coefficient of light-absorbing layers. The delay can be adjusted by changing thicknesses of light-transmittance layers. In one example, the generated high frequency narrowband ultrasound signal has a center frequency of 18.4MHz and 32.6% fractional bandwidth using the proposed multilayer structure. Compared with this result, the single layer structure produces a center frequency of 20.2MHz and 125.7% fractional bandwidth. In addition, a single laser pulse was employed to generate US on the multilayer film as an US source and PA signals of the high optical absorption region of the phantom at the same time. Because the spectral characteristics of the ultrasound signals generated by the multi-layer film are tunable, it can be designed such that the US echo and PA echo are spectrally separable, thus enabling simultaneous US/PA imaging using only a single laser pulse. Feasibility of this proposed method was demonstrated by imaging of a cyst-like phantom.

We developed an all-optical 3D photoacoustic imaging probe consisting of an optical fiber probe for ultrasound detection and a bundle of hollow optical fibers for excitation of photoacoustic waves. The fiber probe for ultrasound is based on a single-mode optical fiber with a thin polymer film attached to the output end surface that works as a Fabry Perot etalon. The input end of the hollow fiber bundle is aligned so that each fiber in the bundle is sequentially excited. A thin and flexible probe can be obtained because the probe system does not have a scanning mechanism at the distal end.

Most concurrent photoacoustic tomography systems are based on traditional ultrasound measurement regime, which requires the contact or acoustic coupling material between the biological tissue and the ultrasound transducer. This study investigates the feasibility of non-contact measurement of photacoustic signals generated inside biomedical tissues by observing the vibrations at the surface of the tissues with a commercial laser Doppler vibrometer. The vibrometer with 0- 2MHz measurement bandwidth and 5 MHz sampling frequency was integrated to a conventional rotational PAT data acquisition system. The data acquisition of the vibrometer was synchronized to the laser illumination from an Nd:YAG laser with output at 532nm. The laser energy was tuned to 17.5mJ per square centimeter. The PA signals were acquired at 120 angular locations uniformly distributed around the scanned objects. The frequency response of the measurement system was first calibrated. 2-inch-diamater cylindrical phantoms containing small rubber plates and biological tissues were afterwards imaged. The phantoms were made from 5% intralipid solution in 10% porcine gelatin to simulate the light scattering in biological tissue and to backscatter the measurement laser from the vibrometer. Time-domain backprojection method was used for the image reconstruction. Experiments with real-tissue phantoms show that with laser illumination of 17.5 mJ/cm² at 532 nm, the non-contact photoacoustic (PA) imaging system with 15dB detection bandwidth of 2.5 MHz can resolve spherical optical inclusions with dimension of 500μm and multi-layered structure with optical contrast in strongly scattering medium. The experiment results prompt the potential implementation of the non-contact PAT to achieve “photoacoustic camera”.

Photoacoustic imaging has been used to determine the spatial distribution of fluorophores, such as exogenous dyes and genetically expressed proteins, from images acquired in phantoms and in vivo. Most methods involve the acquisition of multiwavelength images and rely on differences in the absorption spectra of the tissue chromophores to estimate the spatial distribution and abundance of the latter using spectral decomposition techniques, such as model based inversion schemes. However, the inversion of 3-D images can be computationally expensive. Experimental approaches to localising contrast agents may therefore be useful, especially if quantification is not essential. This work aims to develop a method for determining the spatial distribution of a near-infrared fluorescent cell marker from images acquired using dual wavelength excitation. The excitation wavelengths coincided with the absorption and emission spectrum of the fluorophore. The contrast mechanism relies on reducing the excited state lifetime of the fluorophore by inducing stimulated emission. This changes the amount of energy thermalized by the fluorophore, and hence the photoacoustic signal amplitude. Since this is not observed in endogenous chromophores, the background may be removed by subtracting two images acquired with and without pulse delay between the pump and probe pulses. To characterise the fluorophore, the signal amplitude is measured in a cuvette as a function of pulse delay, concentration, and fluence. The spatial distribution of the fluorophore is determined from images acquired in realistic tissue phantoms. This method may be suitable for in vivo applications, such as imaging of exogenous or genetically expressed fluorescent cell markers.

A decade of research has pushed photoacoustic computed tomography to the forefront of molecular-level imaging, notes SPIE Fellow Lihong Wang (Washington University, St. Louis) in his plenary talk, "Photoacoustic Tomography: Ultrasonically Beating Optical Diffusion and Diffraction." Modern optical microscopy has resolution and diffraction limitations. But noninvasive functional photoacoustic computed tomography has overcome this limit, offering deep penetration with optical contrast and ultrasonic resolution of 1 cm depth or more -- up to 7 cm of penetration in some cases, such as evaluating sentinel lymph nodes for breast cancer staging. This opens up applications in whole body imaging, brain function, oxygen saturation, label-free cell analysis, and noninvasive cancer biopsies.

A high-throughput ultrasound/photoacoustic probe for delivering high contrast and signal-to-noise ratio images was designed, constructed, and tested. The probe consists of a transvaginal ultrasound array integrated with four 1mm-core optical fibers and a sheath. The sheath encases transducer and is lined with highly reflecting aluminum for high intensity light output and uniformity while at the same time remaining below the maximum permissible exposure (MPE) recommended by the American National Standards Institute (ANSI). The probe design was optimized by simulating the light fluence distribution in Zemax. The performance of the probe was evaluated by experimental measurements of the fluence and real-time imaging of polyethylene-tubing filled with blood. These results suggest that our probe has great potential for in vivo imaging and characterization of ovarian cancer.

Circulatory shock may be fatal unless promptly recognized and treated. The most commonly used indicators of shock (hypotension and tachycardia) lack sensitivity and specificity. In the initial stages of shock, the body compensates by reducing blood flow to the peripheral (skin, muscle, etc.) circulation in order to preserve vital organ (brain, heart, liver) perfusion. Characteristically, this can be observed by a greater reduction in peripheral venous oxygenation (for instance, the axillary vein) compared to central venous oxygenation (the internal jugular vein). While invasive measurements of oxygenation are accurate, they lack practicality and are not without complications. We have developed a novel optoacoustic system that noninvasively determines oxygenation in specific veins. In order to test this application, we used lower body negative pressure (LBNP) system, which simulates hemorrhage by exerting a variable amount of suction on the lower body, thereby reducing the volume of blood available for central circulation. Restoration of normal blood flow occurs promptly upon cessation of LBNP. Using two optoacoustic probes, guided by ultrasound imaging, we simultaneously monitored oxygenation in the axillary and internal jugular veins (IJV). LBNP began at -20 mmHg, thereafter was reduced in a step-wise fashion (up to 30 min). The optoacoustically measured axillary oxygenation decreased with LBNP, whereas IJV oxygenation remained relatively constant. These results indicate that our optoacoustic system may provide safe and rapid measurement of peripheral and central venous oxygenation and diagnosis of shock with high specificity and sensitivity.

In this work, we investigate a novel reconstruction method for laser-induced ultrasound computed tomography (USCT) breast imaging that circumvents limitations of existing methods that rely on ray-tracing. There is currently great interest in developing hybrid imaging systems that combine optoacoustic tomography (OAT) and USCT. There are two primary motivations for this: (1) the speed-of-sound (SOS) distribution reconstructed by USCT can provide complementary diagnostic information; and (2) the reconstructed SOS distribution can be incorporated in the OAT reconstruction algorithm to improve OAT image quality. However, image reconstruction in USCT remains challenging. The majority of existing approaches for USCT breast imaging involve ray-tracing to establish the imaging operator. This process is cumbersome and can lead to inaccuracies in the reconstructed SOS images in the presence of multiple ray-paths and/or shadow zones. To circumvent these problems, we implemented a partial differential equation-based Eulerian approach to USCT that was proposed in the mathematics literature but never investigated for medical imaging applications. This method operates by directly inverting the Eikonal equation without ray-tracing. A numerical implementation of this method was developed and compared to existing reconstruction methods for USCT breast imaging. We demonstrated the ability of the new method to reconstruct SOS maps from TOF data obtained by a hybrid OAT/USCT imager built by our team.

Focusing light inside highly scattering media beyond the ballistic regime is a challenging task in biomedical optical imaging, manipulation, and therapy. This challenge can be overcome by time reversing ultrasonically encoded (TRUE) diffuse light to the ultrasonic focus inside a turbid medium. In TRUE optical focusing, a photorefractive crystal or polymer is used as the phase conjugate mirror for optical time reversal. Accordingly, a relatively long ultrasound burst, whose duration matches the response time of the photorefractive material, is used to encode the diffuse light. With this long ultrasound burst, the resolution of the TRUE focus along the acoustic axis is poor. In this work, we used two transducers, emitting two intersecting ultrasound beams at 3.4 MHz and 3.6 MHz respectively, to modulate the diffuse light within their intersection volume at the beat frequency. We show that light encoded at the beat frequency can be time-reversed and converge to the intersection volume. Experimentally, TRUE focusing with an acoustic axial resolution of ~1.1 mm was demonstrated inside turbid media, agreeing with the theoretical estimation.

Time-reversed ultrasonically encoded (TRUE) optical focusing is an emerging technique that focuses light deep into scattering media by phase-conjugating ultrasonically encoded diffuse light. In previous work, the speed of TRUE focusing was limited to no faster than 1 Hz by the response time of the photorefractive phase conjugate mirror, or the data acquisition and streaming speed of the digital camera; photorefractive-crystal-based TRUE focusing was also limited to the visible spectral range. These time-consuming schemes prevent this technique from being applied in vivo, since living biological tissue has a speckle decorrelation time on the order of a millisecond. In this work, using a Tedoped Sn₂P₂S₆ photorefractive crystal at a near-infrared wavelength of 793 nm, we achieved TRUE focusing inside dynamic scattering media having a speckle decorrelation time as short as 7.7 ms. As the achieved speed approaches the tissue decorrelation rate, this work is an important step forward toward in vivo applications of TRUE focusing in deep tissue imaging, photodynamic therapy, and optical manipulation.

Both the spatial resolution and maximum penetration depth of optical-resolution photoacoustic microscopy (ORPAM) deteriorate sharply with depth due to strong light scattering in tissue. To reduce tissue scattering, we propose to use glycerol as an optical clearing agent in OR-PAM. Our results show that the imaging performance of OR-PAM can be greatly enhanced by optical clearing both in vitro and in vivo.

To improve the penetration depth in photoacoustic microscopy while preserving high spatial resolution, we have proposed two-photon absorption-induced photoacoustic microscopy (TP-PAM). However, in tissue imaging, unwanted one-photon photoacoustic signals impair the image constructed from the two-photon photoacoustic signals, because the cross-section of two-photon absorption is smaller than that of one-photon absorption. To overcome this drawback, it is important to enhance (or extract) only the photoacoustic signals generated by two-photon absorption. In this study, to improve the detection selectivity and efficiency of two-photon photoacoustic signals, we investigated the dependence of TP-PAM signal intensity and image quality on the detection frequency range and excitation pulse duration in detail. The comparison among photoacoustic signals generated by optical pulses with various pulse durations (femtosecond to sub-nanosecond) enabled us to find that, the shorter the pulse duration is, the higher the generation efficiency of two-photon photoacoustic signals is. We also applied the confocal configuration between optical (excitation) and acoustic (detection) foci to TP-PAM. The optimization of the pulse duration, frequency filtering and confocal configuration improves the selectivity and efficiency of the TP-PAM signal. Such improvements can reduce the photon number required to obtain TP-PAM images and thus make the imaging speed faster and avoid tissue damage.

Ultrasound-induced inertial cavitation is a mechanical process used for site-localized therapies such as non-invasive surgery. Initiating cavitation in tissue requires very high intensity focused ultrasound (HIFU) and low-frequencies. Hence, some applications like thrombolysis require targeted contrast agents to reduce peak intensities and the potential for secondary effects. A new type of theranostic nanoemulsion has been developed as a combined ultrasound (US)/photoacoustic(PA) agent for molecular imaging and therapy. It includes a nanoscale emulsion core encapsulated with a layer of gold nanospheres at the water/ oil interface. Its optical absorption exhibits a spectrum broadened up to 1100 nm, opening the possibility that 1064 nm light can excite cavitation nuclei. If optically-excited nuclei are produced at the same time that a low-frequency US wave is at peak negative pressure, then highly localized therapies based on acoustic cavitation may be enabled at very low US pressures. We have demonstrated this concept using a low-cost, low energy, portable 1064 nm fiber laser in conjunction with a 1.24 MHz US transducer for simultaneous laser/US excitation of nanoemulsions. Active cavitation detection from backscattered signals indicated that cavitation can be initiated at very low acoustic pressures (less than 1 MPa) when laser excitation coincides with the rarefaction phase of the acoustic wave, and that no cavitation is produced when light is delivered during the compressive phase. US can sustain cavitation activity during long acoustic bursts and stimulate diffusion of the emulsion, thus increasing treatment speed. An in vitro clot model has been used to demonstrate combined US and laser excitation of the nanoemulsion for efficient thrombolysis.

We studied the phenomenon of photothermal bleaching — a gradual reduction of contrast agent particles during repeated scans in photoacoustic microscopy. The dependence of the photothermal bleaching rate on the excitation pulse energy was determined while the laser focal diameter was held constant. Our results showed that, the dependence of the photothermal bleaching rate on the excitation pulse energy differed before and after the absorbers were raised to their melting point by the deposited laser energy. Based on this finding, we suggested an optimal excitation pulse energy, which balances the photothermal bleaching rate and signal amplitude, for time-lapse imaging applications.

We present a photoacoustic microscopy (PAM) technique with an optical sectioning capability. By combining crossoptical- beam illumination with nonlinear PAM, an axial resolution of 8.7 μm was measured, demonstrating a fourfold improvement over the acoustically determined value. Compared to methods relying on high-frequency ultrasound transducers to improve the axial resolution, our approach offers a greater working distance and a higher signal-to-noise ratio.

A setup that allows for the co-registration of photoacoustic (PA) and speed-of-sound (SOS) section images is presented. By means of ultrasound (US) transmission imaging the distribution of the acoustic speed within an object can be obtained. Our method uses the PA effect for the generation of the traversing US waves. Short near-infrared (NIR) laser pulses emitted by a Nd:YAG laser system are used to illuminate external optical absorbing targets at various distances in front of the sample. At the same time the object under investigation is illuminated by a part of the same frequency-doubled laser pulse. A free laser beam, which is part of a Mach-Zehnder interferometer, is used for the detection of the US signals coming from and passing through the sample. Due to a cascaded arrangement of absorbing targets for laser ultrasound (LUS) generation a single laser pulse yields information for a projection of the SOS distribution. The resolution is determined by the number and width of LUS sources. Separation of the signals arriving at the integrating detector is possible because of their different times of flight. After collection of the data reconstruction of a two-dimensional SOS map is accomplished by applying an inverse Radon transform to the projections. For PA section imaging a cylindrical acoustic reflector behind the detector yields an acoustic focus in the observed slice. To the data gathered by detecting the reflected PA signals also the inverse Radon transform is applied to obtain a reconstructed image of the illuminated section. In this paper a detailed description of the setup is given and the results of experiments on two- and three-dimensional phantoms are presented.

Phase-change contrast agents consisting of a perfluorocarbon (PFC) liquid core stabilized by a lipid, protein, or polymer shell have been proposed for a variety of clinical applications. Previous work has demonstrated that vaporization can be induced by laser irradiation through optical absorbers incorporated inside the droplet. In this study, Poly-lactide-coglycolic acid (PLGA) particles loaded with PFC liquid and silica-coated gold nanoparticles (GNPs) were developed and characterized using photoacoustic (PA) methods. Microsized PLGA particles were loaded with PFC liquid and GNPs (14, 35, 55nm each with a 20nm silica shell) using a double emulsion method. The PA signal intensity and optical vaporization threshold were investigated using a 375 MHz transducer and a focused 532-nm laser (up to 450-nJ per pulse). The laser-induced vaporization threshold energy decreased with increasing GNP size. The vaporization threshold was 850, 690 and 420 mJ/cm² for 5μm-sized PLGA particles loaded with 14, 35 and 55 nm GNPs, respectively. The PA signal intensity increased as the laser fluence increased prior to the vaporization event. This trend was observed for all particles sizes. PLGA particles were then incubated with MDA-MB-231 breast cancer cells for 6 hours to investigate passive targeting, and the vaporization of the PLGA particles that were internalized within cells. The PLGA particles passively internalized by MDA cells were visualized via confocal fluorescence imaging. Upon PLGA particle vaporization, bubbles formed inside the cells resulting in cell destruction. This work demonstrates that GNPs-loaded PLGA/PFC particles have potential as PA theranostic agents in PA imaging and optically-triggered drug delivery systems.

Development of techniques for continuous measurement of regional blood flow, and in particular cerebral blood flow (CBF), is essential for monitoring critical care patients. Recently, a novel technique, based on ultrasound modulation of light was developed for non-invasive, continuous CBF monitoring (termed ultrasound-tagged light (UTL or UT-NIRS)), and shown to correlate with readings of 133 Xe SPECT¹ and laser Doppler². Coherent light is introduced into the tissue concurrently with an Ultrasound (US) field. Displacement of scattering centers within the sampled volume induced by Brownian motion, blood flow and the US field affects the photons’ temporal correlation. Hence, the temporal fluctuations of the obtained speckle pattern provide dynamic information about the blood flow. We developed a comprehensive simulation, combining the effects of Brownian motion, US and flow on the obtained speckle pattern. Photons trajectories within the tissue are generated using a Monte-Carlo based model. Then, the temporal changes in the optical path due to displacement of scattering centers are determined, and the corresponding interference pattern over time is derived. Finally, the light intensity autocorrelation function of a single speckle is calculated, from which the tissue decorrelation time is determined. The simulation's results are compared with in-vitro experiments, using a digital correlator, demonstrating decorrelation time prediction within the 95% confidence interval. This model may assist in the development of optical based methods for blood flow measurements and particularly, in methods using the acousto-optic effect.

Biomedical photoacoustic tomography (PAT), as a future imaging modality, can visualize the internal structure and function of soft tissues with high spatial resolution and excellent optical contrast as well as satisfactory imaging depth. A key issue for this unique imaging technique is to recover both the optical absorption and scattering coefficients from the measured acoustic data. Previous attempts in quantitative PAT(qPAT) have been implemented to deduce the map of absorption coefficient from the absorbed energy density using either a model-based method or the invasive measurement techniques when an assumed scattering coefficient is used. However, optical scattering in biological tissue typically dominates over absorption by an order of magnitude or more. Due to this effect it is very challenging to image tissues with highly scattering and accurately recover the absorption coefficient photoacoustically. In this study we propose and validate by experiment tests that quantitative scattering map can be recovered using the measured acoustic data from one-wavelength illumination. The developed reconstruction algorithm relies on PAT and coupled diffusion equation to recover the optical absorption coefficient and energy density, and the diffusion model only to recover the optical scattering coefficients. In particular, this algorithm has the capability to resolve the crosstalk issue in diffuse optical tomography (DOT) and achieve high resolution DOT using acoustics measurements.

Noninvasive and accurate blood flow measurement is critical for medical diagnoses. We proposed a cross-correlationbased method to quantitatively measure transverse flow velocity, using an optical-resolution photoacoustic microscope with a digital micromirror device (DMD). The DMD alternately delivers two spatially separated laser beams to the target. The slow-time photoacoustic signal profiles measured from the two beams are cross-correlated. The magnitude and sign of the time shift in the cross-correlation profile are used to simultaneously calculate the speed and direction of transverse flow. The proposed method was first demonstrated in an aqueous suspension of microspheres flowing in capillary tubing. Using 10-μm-microspheres, transverse flows in the range of 0.50–6.84 mm/s were measured with a root-mean-squared accuracy of 0.22 mm/s. Using three different sizes of microspheres (3, 6, and 10 μm in diameter), we proved experimentally that the flow measurements were independent of the particle size for flows in the velocity range of 0.55–6.49 mm/s. We also observed an expected parabolic distribution of flow velocity along the depth direction. Finally, we used this method to measure blood flow in a mouse ear in vivo.

We present the photoacoustic microscopy (PAM) for evaluation of angiogenesis inhibitors on a chick embryo model. Microvasculature in the chorioallantoic membrane (CAM) of the chick embryos was imaged by PAM, and the optical microscopy (OM) images of the same set of CAMs were also acquired for comparisons, serving for validation of the results from PAM. The angiogenesis inhibitors, Sunitinib, with different concentrations applied to the CAM result in the change in microvascular density, which was quantified by both PAM and OM imaging. Similar change in microvascular density from PAM and OM imaging in response to angiogenesis inhibitor at different doses was observed, demonstrating that PAM has potential to provide objective evaluation of anti-angiogenesis medication. Besides, PAM is advantageous in three-dimensional and functional imaging compared with OM so that the emerging PAM technique may offer unique information on the efficacy of angiogenesis inhibitors and could benefit applications related to antiangiogenesis treatments.

Quantitative photoacoustic microscopy (qPAT) is challenging. We present an algorithm which consecutively reconstructs absorption and scattering coefficient distributions with an iterative scheme in multiple-illumination photoacoustic tomography (MIPAT). In each iteration, the absorption distribution is estimated with the least-squares fixed-point iteration method. Then the diffusion coefficient is estimated with an updated version of optical fluence based on the previously modified absorption information. This procedure is repeated till an acceptable results is achieved. Simulation examples demonstrate the capability of this method in faithfully recovering the absorption and diffusion coeffient distributions, and fast convergence.

Reconstruction of the absorption coefficient from photoacoustic signals is discussed. The photoacoustic (PA) signals were acquired by using a ring-shaped P(VDF-TrFE) acoustic sensor coaxially arranged with an optical fiber. The acoustic sensor scanned the measured object. The linearized image reconstruction method previously presented by the authors was modified for the measurement with the coaxial probe. The distribution of the absorption coefficient was reconstructed by solving the inverse problem based on the PA wave equation and the photon diffusion equation. The linearized forward model was formulated by solving the partial differential equations with finite element method. To eliminate the effect of the unknown background on the PA signal, the differences between the PA signals measured at different positions were used for the image reconstruction. The image reconstruction method was validated by numerical and phantom experiments. Moreover, the reconstructed images with the Tikhonov and l_p sparsity regularization methods were compared from the standpoints of spatial resolution, robustness to noise and quantification of the absorption coefficient.

In this study, we proposed a new compressed sensing (CS) based image reconstruction method for photoacoustic tomography (PAT). To eliminate the finite aperture effect, the proposed method adopts the spatial impulse responses (SIRs) of the finite-sized flat transducer into the linear discrete PAT imaging model for CS. By using the nonlinear recovery algorithm based on convex optimization, PAT can be reconstructed with highly incomplete data. Therefore, the number of measurements and the system cost needed for a given image quality can be significantly reduced. In the mean time, retrospective restoration of the tangential resolution can be achieved because the SIR effect is incorporated in the CS. Simulation results demonstrate that this method not only reduces the data acquisition time but also improves the degraded tangential resolution for PAT with finite-sized flat transducers.

Some commonly employed optoacoustic (photoacoustic) tomographic configurations make use of an array of cylindrically-focused transducers located around the imaging sample to selectively acquire the optoacoustic signals generated in the imaging plane. Thereby, the feasibility of simultaneous acquisition of signals leads to important advantages such as high-throughput performance or real-time imaging capacity. For this particular geometry, two-dimensional model-based reconstruction has showcased good performance in terms of imaging accuracy and flexibility to account for various transducer-related effects and acoustic propagation phenomena. The forward model is expressed as a linear operator (model-matrix) that maps the optical absorption in a grid containing the sample to the resulting wavefield at the sensor positions. The standard approach, however, may lead to excessive memory requirements for the storage of the model-matrix. Herein, an optoacoustic model based on a discretization of the time-domain equation in a polar grid is introduced. Due to the rotational symmetry of the acquisition geometry and the discretization grid, only the part of the model-matrix directly corresponding to one transducer position (projection) needs to be stored. As a result, inversion of the model-matrix can be done in a memory efficient manner. Performance of the method was tested in numerical simulations and experimental measurements, attaining results equivalent to Cartesian-based grids but using a much more computationally efficient implementation.

The capability to image stem cells in vivo in small animal models over extended periods of time is important to furthering our understanding of the processes involved in tissue regeneration. Photoacoustic imaging is suited to this application as it can provide high resolution (tens of microns) absorption-based images of superficial tissues (cm depths). However, stem cells are rare, highly migratory, and can divide into more specialised cells. Genetic labelling strategies are therefore advantageous for their visualisation. In this study, methods for the transfection and viral transduction of mesenchymal stem cells with reporter genes for the co-expression of tyrosinase and a fluorescent protein (mCherry). Initial photoacoustic imaging experiments of tyrosinase expressing cells in small animal models of tissue regeneration were also conducted. Lentiviral transduction methods were shown to result in stable expression of tyrosinase and mCherry in mesenchymal stem cells. The results suggest that photoacoustic imaging using reporter genes is suitable for the study of stem cell driven tissue regeneration in small animals.

Photoacoustic computed tomography (PACT) provides structural and functional information when used in small animal brain imaging. Acoustic distortion caused by bone structures largely limits the deep brain image quality. In our work, we present ex vivo PACT images of freshly excised mouse brain, intending that can serve as a gold standard for future PACT in vivo studies on small animal brain imaging. Our results show that structures such as the striatum, hippocampus, ventricles, and cerebellum can be clearly di erentiated. An artery feature called the Circle of Willis, located at the bottom of the brain, can also be seen. These results indicate that if acoustic distortion can be accurately accounted for, PACT should be able to image the entire mouse brain with rich structural information.

Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors’ knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

In this paper, alternating magnetic field is explored for inducing thermoacoustic effect on dielectric objects. Termed as magnetically mediated thermo-acoustic (MMTA) effect that provides a contrast in conductivity, this approach employs magnetic resonance for delivering energy to a desired location by applying a large transient current at radio frequency below 50MHz to a compact magnetically resonant coil. The alternating magnetic field induces large electric field inside conductive objects, which then undergoes joule heating and emanates acoustic signal thermo-elastically. The magnetic mediation approach with low radio frequency can potentially provide deeper penetration than microwave radiation due to the non-magnetic nature of human body and therefore extend thermoacoustic imaging to deep laid organs. Both incoherent time domain method that applies a pulsed radio frequency current and coherent frequency domain approach that employs a linear chirp signal to modulate the envelop of the current are discussed. Owing to the coherent processing nature, the latter approach is capable of achieving much better signal to noise ratio and therefore potential for portable imaging system. Phantom experiments are carried out to demonstrate the signal generation together with some preliminary imaging results. Ex-vivo tissue studies are also investigated.

In this paper we present multimodal non-contact photoacoustic and OCT imaging. Photoacoustic signals are acquired remotely on the surface of a specimen with a Mach-Zehnder interferometer. The interferometer is realized in a fiberoptic network using a fiber laser at 1550nm as source. In the same fiber-optic network a spectral-domain OCT system is realized. The OCT system utilizes a superluminescent diode at 1325nm as light source; imaging data are acquired using a spectrometer with an InGaAs line array. Light from the fiber laser and the superluminescent diode are multiplexed into one fiber and the same objective is used for both imaging modalities. Reflected light is demultiplexed and guided to the respective imaging systems. We demonstrate the photoacoustic and OCT imaging modalities on different phantom samples. Finally, we show multimodal imaging with both modalities simultaneously. The resulting photoacoustic and OCT images match perfectly.

In a multi-modal combination of optoacoustic (OA) and pulse-echo ultrasound (US) imaging, epi-mode irradiation with the irradiation optics integrated with the acoustic probe has the advantage of flexible clinical application on any part of the body that is already accessible to US. In epi-mode strong clutter limits the OA imaging depth to often around one centimetre. We investigated clutter in automated scanning of volunteer forearms using a real-time combined OA and US system. The results agree well with our theory that clutter arises from strong optical absorption at the location of tissue illumination. As a consequence, we show that an intermediate separation distance between imaging plane and irradiation region leads to superior OA image contrast compared to an irradiation close to the imaging plane.

Breast cancer is the most frequently diagnosed cancer type and its detection at an early stage can reduce the mortality rate substantially. With the aim to detect breast cancer early, by studying tumor progression in nude mice, a pulsed laser induced photoacoustic spectroscopy set up has been designed and developed. MCF-7 cells xenografts were developed using six to eight weeks old female nude mice and tumor tissues were extracted on different days (10th, 15th and 20th Day) post injection and the corresponding photoacoustic spectra were recorded at 281nm excitation. A total of 144 time domain spectra were recorded from 36 animals belonging to the three time points (10th, 15th and 20th day post injection) and converted into frequency domains by Fast Fourier Transform (FFT) tools of the MATLAB algorithms and analyzed. The frequency patterns of the tumor masses on 10th, 15th and 20th day of tumor development showed a gradual increase in intensity at certain frequencies, 5.93 x10³ Hz, 15.9 x10³ Hz, 29.69 x10³ Hz and 32.5 x10³ Hz in the FFT patterns indicating that these frequencies were more sensitive towards tumor development. Further analysis of the data yielded a clear variation in the spectral parameters with progression of the disease suggesting that the technique may be suitable for early detection of the disease. Thus, we expect that the developed setup may be useful in assessing the different phases of tumor development which may have clinical implications.

We conducted an approved canine study to investigate the in vivo feasibility of photoacoustic imaging for intraoperative updates to brachytherapy treatment plans. Brachytherapy seeds coated with black ink were inserted into the canine prostate using methods similar to a human procedure. A transperineal, interstitial, fiber optic light delivery method, coupled to a 1064 nm laser, was utilized to irradiate the prostate and the resulting acoustic waves were detected with a transrectal ultrasound probe. The fiber was inserted into a high dose rate (HDR) brachytherapy needle that acted as a light-diffusing sheath, enabling radial light delivery from the tip of the fiber inside the sheath. The axis of the fiber was located at a distance of 4-9 mm from the long axis of the cylindrical seeds. Ultrasound images acquired with the transrectal probe and post-operative CT images of the implanted seeds were analyzed to confirm seed locations. In vivo limitations with insufficient light delivery within the ANSI laser safety limit (100 mJ/cm²) were overcome by utilizing a short-lag spatial coherence (SLSC) beamformer, which provided average seed contrasts of 20-30 dB for energy densities ranging 8-84 mJ/cm². The average contrast was improved by up to 20 dB with SLSC beamforming compared to conventional delay-and-sum beamforming. There was excellent agreement between photoacoustic, ultrasound, and CT images. Challenges included visualization of photoacoustic artifacts that corresponded with locations of the optical fiber and hyperechoic tissue structures.

Human ovarian tissue features extracted from photoacoustic spectra data, beam envelopes and co-registered ultrasound and photoacoustic images are used to characterize cancerous vs. normal processes using a support vector machine (SVM) classifier. The centers of suspicious tumor areas are estimated from the Gaussian fitting of the mean Radon transforms of the photoacoustic image along 0 and 90 degrees. Normalized power spectra are calculated using the Fourier transform of the photoacoustic beamformed data across these suspicious areas, where the spectral slope and 0-MHz intercepts are extracted. Image statistics, envelope histogram fitting and maximum output of 6 composite filters of cancerous or normal patterns along with other previously used features are calculated to compose a total of 17 features. These features are extracted from 169 datasets of 19 ex vivo ovaries. Half of the cancerous and normal datasets are randomly chosen to train a SVM classifier with polynomial kernel and the remainder is used for testing. With 50 times data resampling, the SVM classifier, for the training group, gives 100% sensitivity and 100% specificity. For the testing group, it gives 89.68± 6.37% sensitivity and 93.16± 3.70% specificity. These results are superior to those obtained earlier by our group using features extracted from photoacoustic raw data or image statistics only.

We investigated the effects of surface charge of gold nanoparticles (Au NPs) on photoacoustic (PA) signal from cultured cancer cell. We used citrated-coated Au NPs and poly-L-lysine-coated Au NPs as the model of negatively and positively charged Au NPs, respectively. Transmission electron microscopy (TEM) were performed for cellular uptake and intracellular localization. We demonstrated PA signal measurement using ring-shaped piezopolymer (P(VDFTrFE)) film sensor coaxially arranged with an optical fiber. The PA signal intensity of the cationic Au NPs was higher than that of the anionic Au NPs over an incubation period up to 3 hours. We found that the PA signal intensities were highly dependent on the surface charge of the Au NPs because the uptake of Au NPs by cultured cancer cells was dependent on the surface charge. We also found that the aggregation of the Au NPs highly influenced the PA signal intensity. These findings are invaluable to the design and synthesis of Au NPs as PA imaging contrast agents with maximized diagnostic efficacy.

Iron oxide nanoparticles are commonly used contrast agents for theranostic nanomedicines because of their advantages of good biocompatibility, high stability in physiological conditions, low cytotoxicity and excellent safety record in clinical settings for human use. In this study, we developed a NIR-activated iron oxide (NIR-Fe₃O₄) nanoparticle as a new multi-functional contrast agent of photoacoustic (PA) imaging. Unlike traditional iron oxides, the developed NIR-Fe₃O₄ owns biocompatibility and optical tunability capable of providing strong optical absorption in the NIR range for PA signal generation. Its intrinsic magnetic property enables the active magnetic tumor targeting. Phantom experiments were performed to confirm the tunability of NIR-Fe₃O₄’s optical absorption in NIR and demonstrate its magnetic targeting capability. The PA signal response of NIR-Fe₃O₄ as a function of concentration was also investigated. The results showed that the PA signal of NIR-Fe₃O₄ with OD=1.25 was comparable to that of blood at 715 nm – the wavelength of peak absorption of the used NIR-Fe₃O₄. Moreover, the PA signal from NIR-Fe₃O₄ could be further improved by magnetic targeting. Overall, we proved that the potential of the developed NIR-Fe₃O₄ as a good tumor targeting contrast agent of PA imaging.

Many breast cancer patients receive neoadjuvant treatment to reduce tumor size and enable breast conserving therapy. Most imaging methods used to monitor response to neoadjuvant chemotherapy or hormone therapy depend on overall gross tumor morphology and size measurements, which may not be sensitive or specific, despite tumor response on a cellular level. A more sensitive and specific method of detecting response to therapy might allow earlier adjustments in treatment, and thus result in better outcomes while avoiding unnecessary morbidity. We developed an imaging system that combines spectral photoacoustic tomography and ultrasonography to predict breast neoadjuvant therapeutic response based on blood volume and blood oxygenation contrast. The system consists of a tunable dye laser pumped by a Nd:YAG laser, a commercial ultrasound imaging system (Philips iU22), and a multichannel data acquisition system which displays co-registered photoacoustic and ultrasound images in real time. Early studies demonstrate functional imaging capabilities, such as oxygen saturation and total concentration of hemoglobin, in addition to ultrasonography of tumor morphology. Further study is needed to determine if the co-registered photoacoustic tomography and ultrasonography system may provide an accurate tool to assess treatment efficacy by monitoring tumor response in vivo.

Nanoparticle contrast agents for molecular targeted imaging have widespread interest in diagnostic applications with cellular resolution, specificity and selectivity for visualization and assessment of various disease processes. Of particular interest is gold nanoparticle owing to its tunability of the surface plasmon resonance (SPR) and its relative inertness. Here we present the synthesis of anisotropic multi-branched star shaped gold nanoparticles exhibiting dual-band plasmon absorption peaks and its application as a contrast agent for multispectral photoacoustic imaging. The transverse plasmon absorption peak of the synthesised dual plasmonic gold nanostar (DPGNS) was around 700 nm and that of longitudinal plasmon absorption in the longer wavelength region around 1050-1150 nm. Unlike most reported PA contrast agent with surface plasmon absorption in the range of 700 to 800 nm showing moderate tissue penetration, 1050-1200 nm range lies in the farther region of the optical window of biological tissue where scattering and the intrinsic optical extinction of endogenous chromophores is at its minimum. We also present a proof of principle demonstration of DPGNS as contrast agent for multispectral photoacoustic animal imaging. Our results show that DPGNS are promising for PA imaging with extended-depth imaging applications.

We show acousto-optic signal generation in tissue like media using a nanosecond pulsed laser for light delivery that has a long coherence length. Our method consists of injecting a light pulse at two different phases of the ultrasound. This enables us to record two different speckle patterns with a camera. When both speckle patterns are recorded within the speckle decorrelation time is it possible to relate both speckle patterns and obtain an acousto-optic signal. We quantify the strength of the acousto-optic effect by adding both speckle patterns and investigate the reduction of speckle contrast of the resulting speckle pattern. We compare the results of this pulsed method with a traditional speckle contrast based acousto-optic experiment by performing a 2d scan on a homogenous sample. Our method makes it possible to obtain acousto-optic signals well within the speckle decorrelation times of typical tissues, making in-vivo possible. Secondly, our method is fully compatible with photo acoustics which can make use of the same laser in the same setup.

Photoacoustic (PA) measurements on confined and unconfined hemoglobin molecules are presented. In vitro experiments were performed with porcine red blood cells (RBCs) at 532 and 1064 nm at various laser fluences. Fluence was gradually changed from 8 to 21 mJ/cm²/pulse for 532 nm and 353 to 643 mJ/cm²/pulse for 1064 nm. PA signals from suspended RBCs (SRBCs) and hemolyzed RBCs (HRBCs) were measured using a needle hydrophone at hematocrits ranging from 10 to 60%. PA amplitude was found to be varied linearly with the laser fluence for each type of samples at the above two optical radiations. At 532 nm, PA signals from SRBCs and HRBCs were measured to be nearly equal, whereas, at 1064 nm, signal amplitude for SRBCs was approximately 2 times higher than that of HRBCs. The results suggest that it may be feasible to detect hemolysis with PAs.

We have enhanced our design for an all-optical high frequency ultrasound transducer consisting of a UV-absorbing polyimide film integrated into an etalon receiver operating in the NIR range. A dielectric stack having high NIR reflectivity and high UV transmittance was chosen as the first mirror for increased sensitivity and the allowance of polyimide as the etalon medium. A 13 ns, 0.7 μJ optical pulse at 355 nm and a continuous-wave NIR laser were focused onto the structure with a spot diameter of 120 and 35 μm, respectively. In receive mode the etalon had a noise-equivalent pressure of 4.1 kPa over a bandwidth of 5 – 50 MHz (0.61 _Pa/√_Hz ). The device generated a pressure of 270 kPa at a depth of 200 μm, and the -3 dB bandwidth of the emission extended from 27 to 60 MHz. In transmit/receive mode, the pulse-echo had a center frequency of 35 MHz with a -6 dB bandwidth of 49 MHz (140 %). Lastly, wire targets were imaged by scanning the UV spot to create a synthetic aperture of transmitters centered upon a single receiver.

Osteoporosis is a widespread disorder, which has a catastrophic impact on patients lives and overwhelming related to healthcare costs. Recently, we proposed a multispectral photoacoustic technique for early detection of osteoporosis. Such technique has great advantages over pure ultrasonic or optical methods as it allows the deduction of both bone functionality from the bone absorption spectrum and bone resistance to fracture from the characteristics of the ultrasound propagation. We demonstrated the propagation of multiple acoustic modes in animal bones in-vitro. To further investigate the effects of multiple wavelength excitations and of induced osteoporosis on the PA signal a multispectral photoacoustic system is presented. The experimental investigation is based on measuring the interference of multiple acoustic modes. The performance of the system is evaluated and a simple two mode theoretical model is fitted to the measured phase signals. The results show that such PA technique is accurate and repeatable. Then a multiple wavelength excitation is tested. It is shown that the PA response due to different excitation wavelengths revels that absorption by the different bone constitutes has a profound effect on the mode generation. The PA response is measured in single wavelength before and after induced osteoporosis. Results show that induced osteoporosis alters the measured amplitude and phase in a consistent manner which allows the detection of the onset of osteoporosis. These results suggest that a complete characterization of the bone over a region of both acoustic and optical frequencies might be used as a powerful tool for in-vivo bone evaluation.

Ventricular catheters are used to treat hydrocephalus by diverting the excess of the cerebrospinal fluid (CSF) to the reabsorption site so as to regulate the intracranial pressure. The failure rate of these shunts is extremely high due to the ingrown tissue that blocks the CSF flow. We have studied a method to image the occlusion inside the shunt through the skull. In this approach the pulsed laser light coupled to the optical fiber illuminate the occluding tissue inside the catheter and an external ultrasound transducer is applied to detect the generated photoacoustic signal. The feasibility of this method is investigated using a phantom made of ovis aries brain tissue and adult human skull. We were able to image the target inside the shunt located 20mm deep inside the brain through about 4mm thick skull bone. This study could lead to the development of a simple, safe and non-invasive device for percutaneous restoration of patency to occluded shunts. This will eliminate the need of the surgical replacement of the occluded catheters which expose the patients to risks including hemorrhage and brain injury.

Wavefront distortion in scattering media can be compensated for using optical wavefront shaping. In this technique, a spatial light modulator (SLM) is used to apply a spatially distributed phase shift to the optical field. A genetic optimization algorithm was used to obtain the SLM pattern which best focuses light within the medium. The target volume is defined by using a focused ultrasound beam to encode light travelling within the acoustic focus. The ultrasonically-encoded light is measured and used as feedback to the algorithm, which then searches for the pattern which maximizes the encoded light intensity. We call this technique ultrasonically-encoded wavefront shaping (SEWS). Using SEWS, we focused light into a scattering medium consisting of ground glass diffuser and a gelatin phantom. The optical intensity at the target was increased by 11 times over the original intensity. These results were validated using fluorescent imaging at the ultrasonic focus.

Photoacoustic imaging has been recently developed for biomedical imaging. This imaging technique is based on the photoacoustic effect, which includes a process involving the absorption of photons, the subsequent thermal expansion, and propagation of photoacoustic waves. The propagation of photoacoustic waves has been modeled by using linear acoustic theories although the generated photoacoustic waves are naturally shock waves. In this work, the propagation of photoacoustic shock waves are studied by using nonlinear acoustic wave solutions based on Hugoniot’s shock relation combining Earnshaw solution with Poisson solution. The non-linear solution is compared with the existing linear solution using the propagating waveforms for spherical wave. The simulation results show a discrepancy between the two solutions.

The influence of size, geometry, temporal response of finite sensors and source geometry on the detected photoacoustic pressure is explored. We started considering the transducer simulated by a mesh of point-like sensors connected in parallel, which implies that appropriate modeling of pressure depends on the discretization size of the sensing surface. On the other hand, in analogy with the approach for the finite sensor, a finite source can be considered as a set of point like sources. We simulated the pressure produced by the linear, cylindrical and spherical source geometries, located at certain position over an axis perpendicular to the sensing surface. In order to simulate the photoacoustic signal, the computed pressure was convolved with the impulse response of two kind of commercial sensors: a low frequency transducer (3.5 MHz) and a high frequency transducer (125 MHz). Taking a fixed coordinate system we investigated the signal variations when the translational degree of freedom was modified. We found that simulated pressure generated by the different geometries using the proposed approach clearly differs from the point-like detection model.

Acoustic resolution optoacoustic microscopy is a powerful modality allowing imaging morphology and function at depths up to a few centimeters in biological tissues. This optoacoustic configuration is based on a spherically-focused ultrasonic transducer raster scanned on an accessible side of the sample to be imaged. Volumetric images can then be formed by stacking up the recorded time-resolved signals at the measured locations. However, the focusing capacity of a spherically-focused transducer depends on its aperture and the acoustic spectrum of the collected signals, which may lead to image artifacts if a simplistic reconstruction approach is employed. In this work, we make use of a model-based reconstruction procedure developed in three dimensions in order to account for the shape of spherically focused transducers in acoustic resolution optoacoustic microscopy set-ups. By discretizing the transducer shape to a set of sub-sensors, the resulting model incorporates the frequency-dependent transducer sensitivity for acquisition of broadband optoacoustic signals. Inversion of the full model incorporating the effects of the transducer shape is then performed iteratively. The obtained results indicate good performance of the method for absorbers of different size emitting optoacoustic waves with different frequency spectra.

Fixed-point iteration shows promise for quantitative reconstruction of optical absorption in photoacoustic tomography. However, there are issues that prevent the technique from being practical including: non-uniqueness of scattering and absorption profiles, divergence with over-iteration, and sensitivity to noise. Multiple illumination has been proposed to deal with the first problem, and may help with the second. The issue of noise may be balanced out by increasing the regularization parameter at the expense of the exactness of the reconstruction. In a multiple-illumination setup with a circular geometry where fluence is abundant, using a patterned illumination with a decoding step may provide an alternative which will boost SNR. We present a simple sequence of patterned illuminations based on an S-sequence that serves to improve SNR. While the forward model of the iterative method may be applied directly to the patterned excitations, including the decoding step improves SNR in an individual image by a factor equal to the size of the S-sequence, thus greatly improving convergence for a given value of regularization and SNR. For example, with 15 illuminations, 50-60dB noise levels with S-sequence patterned illuminations gives similar simulated performance to the 70dB case with single-source illuminations. This technique will allow the application of fixed-point iteration techniques in a broader range of SNR conditions without resorting to averaging.

The simultaneous and/or quantitative recovery of optical absorption and scattering coefficients in ultrasound modulated optical tomography requires the use of a model-based inversion procedure. In this work we employ a linearised forward model as part of a non-linear image reconstruction process, recovering parameters with an error of less than ±3% from simulated measurements with 1% Gaussian noise and initial conditions differing by 10% from the actual background.

Patients with acute intracranial hematomas often require surgical drainage within the first four hours after traumatic brain injury (TBI) to avoid death or severe neurologic disability. CT and MRI permit rapid, noninvasive diagnosis of hematomas, but can be used only at a major health-care facility. At present, there is no device for noninvasive detection and characterization of hematomas in pre-hospital settings. We proposed to use an optoacoustic technique for rapid, noninvasive diagnosis and monitoring of hematomas, including intracranial hematomas. Unlike bulky CT and MR equipment, an optoacoustic system can be small and easily transported in an emergency vehicle. In this study we used a specially-designed blast device to inflict TBI in rats. A near-infrared OPO-based optoacoustic system developed for hematoma diagnosis and for blood oxygenation monitoring in the superior sagittal sinus (SSS) in small animals was used in the study. Optoacoustic signals recorded simultaneously from the SSS and hematomas allowed for measurements of their oxygenations. The presence of hematomas was confirmed after the experiment in gross pictures of the exposed brains. After blast the hematoma signal and oxygenation increased, while SSS oxygenation decreased due to the blastinduced TBI. The increase of the oxygenation in fresh hematomas may be explained by the leakage of blood from arteries which have higher blood pressure compared to that of veins. These results indicate that the optoacoustic technique can be used for early diagnosis of hematomas and may provide important information for improving outcomes in patients with TBI or stroke (both hemorrhagic and ischemic).

We have developed three-dimensional arbitrary trajectory (3-DAT) scanning, which can rapidly image vessels of interest over a large field of view (FOV) and maintain a high signal-to-noise ratio (SNR) along the depth direction. The concept of 3-DAT scanning was demonstrated by imaging a human hair within a FOV of 3.5 × 2.0 mm². Further, we showed that hemoglobin oxygen saturation (sO₂) and blood flow can be measured simultaneously. The frame rate was 67 times faster than a traditional two-dimensional raster scan. We also observed sO₂ dynamics in response to a switch between systemic hyperoxia and hypoxia.

In this paper, we demonstrate a temporal-domain intensity-based photoacoustic imaging method that can differentiate between fatty and non-fatty tissues. PA pressure intensity is partly dependent on the tissue’s speed of sound, which increases as temperature increases in non-fatty tissue and decreases in fatty tissue. Therefore, by introducing a temperature change in the tissue and subsequently monitoring the change of the PA intensity, it is possible to distinguish between the two types of tissue. A commercial ultrasound system with a 128-element 5-14 MHz linear array transducer and a tunable ND:YAG laser were used to produce PA images. Ex-vivo bovine fat and porcine liver tissues were precooled to below 10°C and then warmed to room-temperature over ~1 hour period. A thermocouple monitored the temperature rise while PA images were acquired at 0.5°C intervals. The averaged intensity of the illuminated tissue region at each temperature interval was plotted and linearly fitted. Liver samples showed a mean increase of 2.82 %/°C, whereas bovine fat had a mean decrease of 6.24 %/°C. These results demonstrate that this method has the potential to perform tissue differentiation in the temporal-domain.

An optical phase contrast full field detection setup in combination with a CCD-camera is presented to record acoustic fields for real-time projection and fast three-dimensional imaging. When recording projection images of the wave pattern around the imaging object, the three-dimensional photoacoustic imaging problem is reduced to a set of two-dimensional reconstructions and the measurement setup requires only a single axis of rotation. Using a 10 Hz pulse laser system for photoacoustic excitation a three dimensional image can be obtained in less than 1 min. The sensitivity and resolution of the detection system was estimated experimentally with 5 kPa mm and 75μm, respectively. Experiments on biological samples show the applicability of this technique for the imaging of blood vessel distributions.

To focus light beyond one transport mean free path, time-reversed ultrasonically encoded (TRUE) optical focusing has previously been implemented by both analog and digital devices. By allowing wavefront recording with finer resolution and larger aperture, the analog scheme, which uses photorefractive materials as the phase-conjugate mirror, generates a more complete set of time-reversed optical modes than the digital scheme. Here, we report the direct visualization of localized fluorescence excitation inside a turbid medium by photorefractive time reversal. Further, we imaged fluorescent targets embedded in a turbid phantom whose thickness was four transport mean free paths.

Sentinel lymph node biopsy (SLNB) has emerged as an accurate, less invasive alternative to axillary lymph node dissection, and it has rapidly become the standard of care for patients with clinically node-negative breast cancer. The sentinel lymph node (SLN) hypothesis states that the pathological status of the axilla can be accurately predicted by determining the status of the first (i.e., sentinel) lymph nodes that drain from the primary tumor. Physicians use radio-labeled sulfur colloid and/or methylene blue dye to identify the SLN, which is most likely to contain metastatic cancer cells. However, the surgical procedure causes morbidity and associated expenses. To overcome these limitations, we developed a dual-modality photoacoustic and ultrasound imaging system to noninvasively detect SLNs based on the accumulation of methylene blue dye. Ultimately, we aim to guide percutaneous needle biopsies and provide a minimally invasive method for axillary staging of breast cancer. The system consists of a tunable dye laser pumped by a Nd:YAG laser, a commercial ultrasound imaging system (Philips iU22), and a multichannel data acquisition system which displays co-registered photoacoustic and ultrasound images in real-time. Our clinical results demonstrate that real-time photoacoustic imaging can provide sensitive and specific detection of methylene blue dye in vivo. While preliminary studies have shown that in vivo detection of SLNs by using co-registered photoacoustic and ultrasound imaging is feasible, further investigation is needed to demonstrate robust SLN detection.

Photoacoustic imaging has great potential for identifying vascular regions for clinical imaging. In addition to assessing angiogenesis in cancers, there are many other disease processes that result in increased vascularity that present novel targets for photoacoustic imaging. Doppler imaging can provide good localization of large vessels, but poor imaging of small or low flow speed vessels and is susceptible to motion artifacts. Photoacoustic imaging can provide visualization of small vessels, but due to the filtering effects of ultrasound transducers, only shows the edges of large vessels. Thus, we have combined photoacoustic imaging with ultrasound power Doppler to provide contrast agent- free vascular imaging. We use a research-oriented ultrasound array system to provide interlaced ultrasound, Doppler, and photoacoustic imaging. This system features realtime display of all three modalities with adjustable persistence, rejection, and compression. For ease of use in a clinical setting, display of each mode can be disabled. We verify the ability of this system to identify vessels with varying flow speeds using receiver operating characteristic curves, and find that as flow speed falls, photoacoustic imaging becomes a much better method for identifying blood vessels. We also present several in vivo images of the thyroid and several synovial joints to assess the practicality of this imaging for clinical applications.

The representation theorems of the convolution type and the correlation type are used to obtain the superposition of the Green's function and its time reversal counterpart for the photoacoustic wave equation. Based on the representation theorems, an interferometry relation providing the Green's function between sources and receivers is obtained. The reciprocity theorems for a spherical geometrical system consisting of sources located on the boundary of the inner spherical region and transducers located on the outer boundary are utilized. Therefore, the measurement would be observed at one of the detectors if there were a photoacoustic point source at the other one.

Activatable photoacoustic probes have a promising future due to their ability to provide high-resolution, high-penetration depth information on enzyme activity in vivo. Spectral identification methods, however, suffer from heterogeneous optical properties and wavelength-dependent light attenuation in tissue, thereby limiting the effective suppression of background noise signal. Our approach is predicated on probing the excited-state lifetime of a dual-labeled methylene blue (MB) probe that changes its lifetime from short to long upon cleavage. Recently, we have reported on the ability of our system to probe the long triplet lifetime of free MB monomers in solution and to differentiate between monomers and dimers based on their lifetime contrast. Here we introduce an improvement to our system that significantly increases the system sensitivity to fast changes, and reduces the minimum resolvable lifetime down to a few nanoseconds. We applied this method to probe the excited-state lifetime of a covalently coupled dual methylene blue-lysine conjugate (MB2K) in a mixed MB/MB2K solution. Preliminary results show that a stable dimeric bond is formed between the chromophores within the conjugate, and that this conjugate is statically quenched. Examination of the transient absorption of MB2K reveals it does not exhibit a triplet excited-state lifetime, suggesting that it undergoes a fast deexcitation process directly from the singlet state. Finally, we demonstrate how the transient photoacoustic lifetime signal can be used to selectively detect the presence of MB monomers while improving background noise suppression by differentiating the lifetime of free MB dye with other absorbing structures.

Although molecularly targeted cancer therapies have shown great promise, it is now evident that responses are dependent upon the molecular genetic context. Spatial and temporal tumour heterogeneity renders biopsy of solid tumours unsuitable for determining the genetic profile of the disease, making adaptation of appropriate therapy difficult. We have utilized the tunable optical absorption characteristic of gold nanorods to assess the potential of photoacoustics for non-invasive multiplexed molecular imaging. Gold nanorods with resonance peaks at 700nm and 900nm were functionalised with in-house antibodies ICR55 and ICR62, targeted to HER2 and EGFR transmembrane receptors, respectively. Three human squamous carcinoma cell lines (LICR-LON-HN4 expressing high HER2 and low EGFR, LICR-LON-HN3 expressing intermediate levels of HER2 and EGFR and A431 expressing high EGFR and low HER2) were incubated with the targeted nanorods for 24 hours. Cells were then incorporated as simulated tumours in tissue-like phantoms composed of 7.5% gelatin containing 0.5% Intralipid® for optical scattering and imaged at a depth of 2.5 cm, using a new clinical in-house multi-spectral photoacoustic imaging system. Images were obtained from the cell inclusions for wavelengths ranging from 710 to 950 nm at 40 nm intervals, and the mean amplitude of the photoacoustic image was computed for each wavelength, to determine their relative receptor expression levels. The molecular profile of the cells obtained using multi-wavelength photoacoustics had substantial similarity to that obtained using flow cytometry. These preliminary results confirm selective uptake of the functionalised nanorods, which reflects the cellular expression of therapeutically important oncoproteins, and give an indication of the potential of photoacoustics for multiplexed molecular profiling.

A simple method of characterizing organic dyes and nanoparticles used as contrast agents for photoacoustic molecular imaging based on relative photoacoustic measurements is described. By acquiring just two time-resolved photoacoustic signals, one in the sample of interest and the other in water, measurements of the thermalisation efficiency and other parameters relevant to the characterization of contrast agents can be acquired. The method was validated using absorbing solutions of known thermalisation efficiency and Grüneisen coefficient. It was then used to measure the thermalisation efficiency of solutions of gold nanorods, rhodamine B, methylene blue, IR-820, fluorescein and cresyl violet. In addition, photoacoustic measurements of the photostability of these substances were acquired.

Photoacoustic (PA) tomography imaging is an emerging, versatile, and noninvasive imaging modality, which combines the advantages of both optical imaging and ultrasound imaging. It opens up opportunities for noninvasive imaging of angiogenesis, a feature of skin pathologies including cancers and psoriasis. In this study, high-density copper oleate encapsulated within a phospholipid surfactant (CuNPs) generated a soft nanoparticle with PA contrast comparable to gold. Within the near-infrared window, the copper nanoparticles can provide a signal more than 7 times higher that of blood. ανβ3-targeted of CuNPs in a Matrigel mouse model demonstrated prominent PA contrast enhancement of the neovasculature compared to mice given nontargeted or competitively inhibited CuNPs. Incorporation of a sn-2 lipase-labile fumagillin prodrug into the CuNPs produced marked antiangiogenesis in the same model, demonstrating the theranostic potential of a PA agent for the first time in vivo. With a PA signal comparable to gold-based nanoparticles yet a lower cost and demonstrated drug delivery potential, ανβ3-targeted CuNPs hold great promise for the management of skin pathologies with neovascular features.

Fusion of video and other imaging modalities is common in modern surgical procedures to provide surgeons with additional information that can provide precise surgical guidance. An example of such uses interventional guidance equipment and surgical navigation systems to register the tools and devices used in surgery with each other. In this work, we focus explicitly on registering three-dimensional ultrasound with a stereocamera system. These surgical navigation systems often use optical or electromagnetic trackers. However, both of these tracking systems have various drawbacks leading to target registration errors of approximately 3mm. Previous work has shown that photoacoustic markers can be used to register three-dimensional ultrasound with video resulting in target registration errors which are much lower than the current state of the art. This work extends this idea by generating multiple photoacoustic markers concurrently as opposed to the sequential method used in the previous work. This development greatly enhances the acquisition time by a factor equal to the number of concurrently generated photoacoustic markers. This work is demonstrated on a synthetic phantom and an ex vivo porcine kidney phantom. The resulting target registration errors for these experiments ranged from 840 to 1360 μm and standard deviations from 370 to 640 μm.

Phasoscopy is a recently proposed concept correlating electromagnetic (EM) absorption and scattering properties based on energy conservation. Phase information can be extracted from EM absorption induced acoustic wave and scattered EM wave for biological tissue characterization. In this paper, a novel imaging modality, termed photoacoustic phasoscopy (PAPS) imaging, is proposed and verified experimentally based on phasoscopy concept with laser illumination. Both endogeneous photoacoustic wave and scattered photons are collected simultaneously to extract the phase information, and phasoscopy image is then reconstructed by mapping phase distribution. The phasoscopy imaging experiments on vessel-mimicking phantom and ex vivo porcine tissues demonstrate significantly improved contrast than conventional photoacoustic imaging.

In recent years, various methods have been developed to improve ultrasound based interventional tool tracking. However, none of them has yet provided a solution that effectively solves the tool visualization and mid-plane localization accuracy problem and fully meets the clinical requirements. Our previous work has demonstrated a new active ultrasound pattern injection system (AUSPIS), which integrates active ultrasound transducers with the interventional tool, actively monitors the beacon signals and transmits ultrasound pulses back to the US probe with the correct timing. Ex vivo and in vivo experiments have proved that AUSPIS greatly improved tool visualization, and provided tool-tip localization accuracy of less than 300 μm. In the previous work, the active elements were made of piezoelectric materials. However, in some applications the high driving voltage of the piezoelectric element raises safety concerns. In addition, the metallic electrical wires connecting the piezoelectric element may also cause artifacts in CT and MR imaging. This work explicitly focuses on an all-optical active ultrasound element approach to overcome these problems. In this approach, the active ultrasound element is composed of two optical fibers - one for transmission and one for reception. The transmission fiber delivers a laser beam from a pulsed laser diode and excites a photoacoustic target to generate ultrasound pulses. The reception fiber is a Fabry–Pérot hydrophone. We have made a prototype catheter and performed phantom experiments. Catheter tip localization, mid-plan detection and arbitrary pattern injection functions have been demonstrated using the all-optical AUSPIS.

Microbubbles (MBs) are currently used as ultrasound (US) contrast agents and as delivery vehicles for site-specific US triggered drug and gene delivery. Multimodal US-based imaging methods have been applied preclinically to assess and validate the effectiveness and fate of MBs as imaging contrast agents and drug delivery vehicles. Here we present the generation of trimodality MBs generated from the dense concentration of porphyrins within a MB shell, enabled by the conjugation of a porphyrin to a phospholipid. These trimodality MBs possess US, photoacoustic and fluorescence properties with potential to expand into other imaging modalities such as MRI and nuclear imaging.

A novel method – photoacoustic recovery after photothermal bleaching (PRAP) – is proposed and implemented to study particle dynamics and medium properties at the micron scale via photoacoustic imaging. PRAP is an intuitive way to visualize as well as quantify dynamic processes in many kinds of media. We demonstrate PRAP first in a phantom study, and then in live cells. PRAP provides high signal-to-noise ratio imaging with minimal bleaching-induced artifacts during the recovery stage, ideal for monitoring the diffusive and kinetic phenomena inside a cell.

Five numerical phantoms were developed to investigate the accuracy of approximate inversion schemes in the reconstruction of oxygen saturation in photoacoustic imaging. In particular, two types of inversion are considered: Type I, an inversion that assumes fluence is unchanged between illumination wavelengths, and Type II, a method that assumes known background absorption and scattering coefficients to partially correct for the fluence. These approaches are tested in tomography (PAT) and acoustic-resolution microscopy mode (AR-PAM). They are found to produce accurate values of oxygen saturation in a blood vessel of interest at shallow depth - less than 3mm for PAT and less than 1mm for AR-PAM.

Combined optical and mechanical scanning (COMS) in optical-resolution photoacoustic microscopy (OR-PAM) has provided five scanning modes with fast imaging speed and wide field of view (FOV). With two-dimensional (2D) galvanometer-based optical scanning, we have achieved a 2 KHz B-scan rate and 50 Hz volumetric-scan rate, which enables real-time tracking of cell activities in vivo. With optical-mechanical hybrid 2D scanning, we are able to image a wide FOV (10×8 mm²) within 150 seconds, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical-based contour scanning, we can maintain the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is ideal for fast and quantitative studies of tumors and the brain.

Acoustic attenuation influences the transmission of the ultrasonic waves excited optoacoustically in biological samples, in a way that the amplitude of the waves is reduced as they propagate through acoustically attenuating tissues. Furthermore, being dependent on frequency, acoustic attenuation also causes broadening of the time-resolved optoacoustic signals, which in turn leads to blurring of features and overall deterioration of image quality. The effects of acoustic attenuation are more prominent for the high frequency components of the optoacoustic waves and they must be taken into account for high resolution imaging. In this work, we modify a model-based reconstruction algorithm to incorporate the effects of acoustic attenuation in tomographic optoacoustic imaging set-ups. As the waves propagate from the excitation until the measurement points, they undergo space and frequency dependent attenuation, which can be effectively accounted for using the suggested model-based approach. The simulation results obtained showcase a good performance of the introduced method in terms of resolution improvement.

We proposed a method of quantifying the effective attenuation coefficients of optical absorbers which uses the continuous wavelet transform to calculate the time-resolved frequency spectra of photoacoustic (PA) signals. In order to apply the method to blood oxygenation monitoring of blood vessels, this study discusses how to reduce the effects of blood vessel diameters, which influences on the time resolved frequency spectra of PA signals. Numerical simulations which calculate the PA signals produced from blood vessel phantoms with various diameters were performed. The simulations revealed that the frequency of PA signal became independent from the vessel diameters by measuring the PA signal from small area. The frequencies of simulated PA signals were proportional to the effective attenuation coefficients with a correlation coefficient of 0.99, and a slope of 0.035 MHz/cm^-1 under condition that the measurement area was 4.0 mm at a frequency of 1.5 MHz. Thus we used the focused acoustic sensor of which focusing the foregoing measurement area. It consisted of a P(VDF-TrFE) film, which was characterized by broad frequency band. As results of experiments using the focused acoustic sensor, the frequencies of PA signals produced from blood vessel phantoms were proportional to the effective attenuation coefficients with correlation coefficient of 0.96 although the frequencies were suffered from deviations of 0.135 MHz, which corresponded to the effective attenuation coefficient of 3.46 cm^-1. Since the large deviations were caused by experimental factors such as sensor alignment, it is required to improve robustness to the experimental factors.

An important and interesting question in photoacoustic computed tomography (PACT) is whether the absorbed optical energy density distribution, A(r), and the speed of sound distribution, c(r), can both be accurately determined from the measured photoacoustic data alone. However, in many cases c(r) is unknown or cannot be accurately estimated. Therefore, it would be practically beneficial if A(r) and c(r) can be jointly reconstructed from the measurements. In this work, we propose a reconstruction approach to the joint reconstruction of both properties in PACT.

Photoacoustic (PA) imaging is an emerging medical imaging modality that relies on the absorption of optical energy and the subsequent emission of acoustic waves that are detected with a conventional ultrasound probe. PA images are susceptible to background noise artifacts that reduce the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR). We investigated spatial-angular compounding of PA images to enhance these image qualities. Spatial-angular compounding was implemented by averaging multiple PA images acquired as an ultrasound probe was rotated about the elevational axis with the laser beam and PA target fixed in the same location. An external tracking system was used to provide the position and orientation (i.e. pose) information of each PA image. Based on this pose information, frames in similar elevational planes were filtered from the acquired image data and compounded using one of two methods. One method registered overlapping signals between frames prior to compounding (using the pose information), while the second method omitted this spatial registration step. These two methods were applied to pre-beamformed RF, beamformed RF, and envelope-detected data, resulting in six different compounding pipelines. Compounded PA images with similar lateral resolution to a single reference image had factors of 1.1 - 1.6, 2.0 - 11.1, and 2.0 - 11.1 improvements in contrast, CNR, and SNR, respectively, when compared to the reference image. These improvements depended on the amount of relative motion between the reference image and the images that were compounded. The inclusion of spatial registration prior to compounding preserved lateral resolution and signal location when the relative rotations about the elevation axis were 3.5° or less for images that were within an elevational distance of 2.5 mm from the reference image, particularly when the method was applied to the enveloped-detected data. Results indicate that spatial-angular compounding has the potential to improve image quality for a variety of photoacoustic imaging applications.

Quantitative detection of stochastic microstructure in turbid media remains a challenge to both optical and acoustical observation. A method of photoacoustic spectral matching is proposed to solve this problem. This method allows us to quantitatively detect the characteristic dimension of stochastic microstructures using a long wavelength. Using a working wavelength of about 375 μm, we accurately measure the dimensions (49, 94.8, and 199 μm) of particles hidden in turbid phantoms. Since stochastic microstructures composed of particles commonly appear in tissue, this method might provide an insight into the physiological and pathological processes deep within organisms.

Photoacoustic tomography (PAT) offers structural and functional imaging of living biological tissue with highly sensitive optical absorption contrast and excellent spatial resolution comparable to medical ultrasound (US) imaging. We report the development of a fully integrated PAT and US dual-modality imaging system, which performs signal scanning, image reconstruction and display for both photoacoustic (PA) and US imaging all in a truly real-time manner. The backprojection (BP) algorithm for PA image reconstruction is optimized to reduce the computational cost and facilitate parallel computation on a state of the art graphics processing unit (GPU) card. For the first time, PAT and US imaging of the same object can be conducted simultaneously and continuously, at a real time frame rate, presently limited by the laser repetition rate of 10 Hz. Noninvasive PAT and US imaging of human peripheral joints in vivo were achieved, demonstrating the satisfactory image quality realized with this system. Another experiment, simultaneous PAT and US imaging of contrast agent flowing through an artificial vessel was conducted to verify the performance of this system for imaging fast biological events. The GPU based image reconstruction software code for this dual-modality system is open source and available for download from http://sourceforge.net/projects/pat realtime .

The increasing use of mouse models for human brain disease studies, coupled with the fact that existing functional imaging modalities cannot be easily applied to mice, presents an emerging need for a new functional imaging modality. Utilizing acoustic-resolution photoacoustic microscopy (AR-PAM), we imaged spontaneous cerebral hemodynamic fluctuations and their associated functional connections in the mouse brain. The images were acquired noninvasively in B-scan mode with a fast frame rate, a large field of view, and a high spatial resolution. At a location relative to the bregma 0, correlations were investigated inter-hemispherically between bilaterally homologous regions, as well as intra-hemispherically within the same functional regions. The functional connectivity in different functional regions was studied. The locations of these regions agreed well with the Paxinos mouse brain atlas. The functional connectivity map obtained in this study can then be used in the investigation of brain disorders such as stroke, Alzheimer’s, schizophrenia, multiple sclerosis, autism, and epilepsy. Our experiments show that photoacoustic microscopy is capable to detect connectivities between different functional regions in B-scan mode, promising a powerful functional imaging modality for future brain research.

Three-dimensional photoacoustic tomography with line sensors, which integrate the pressure along their length, has shown to produce accurate images of small animals. To reduce the scanning time and to enable in vivo applications, a detection array is built consisting of 64 piezoelectric line sensors which are arranged on a semi-cylinder. When measuring line integrated pressure signals around the imaging object, the three-dimensional photoacoustic imaging problem is reduced to a set of two-dimensional reconstructions and the measurement setup requires only a single axis of rotation. The shape and size of the array were adapted to the given problem of biomedical imaging and small animal imaging in particular. The length and width of individual line elements had to be chosen in order to take advantage of the favorable line integrating properties, maintaining the requested resolution of the image. For data acquisition the signals from the 64 elements are amplified and multiplexed into a 32 channel digitizer. Single projection images are recorded with two laser pulses within 0.2 seconds, as determined by the laser pulse repetition rate of 10 Hz. Phantom experiments are used for characterization of the line-array. Compared to previous implementations with a single line sensor scanning around an object, with the developed array the data acquisition time can be reduced from about one hour to about one minute.

Gold nanorods (GNRs) were synthesized with surfactant templating and coated with IR792 to produce surface-enhanced Raman signal (SERS). Subcutaneous and orthotopic tumor models were created in nude mice using the OV2008 cell line, and a Nexus128 scanner from Endra LifeSciences was used to collect the photoacoustic data. We used GNRs with resonance at 756 nm, and the Raman signal was 10-fold larger than 60 nm gold core/silica shell nanoparticles. This signal was stable for over 24 hours in 50% serum. The batch-to-batch reproducibility was 15.5% and 3.6% in the SERS and photoacoustic modalities for n=4 batches. Animals were injected with 200 μL of 2.5, 5.4, and 16.8 nM GNRs. Relative to baseline photoacoustic signal, these concentrations increased tumor signal 1.3-, 1.6-, and 2.5-fold, respectively. The maximum signal increase occurred within 2 hours of injection persisted for at least 24 hours and was significant at p<0.05 for at least 3 animals. Assaying for gold in the tumors validated signal—we found a strong correlation (R²>0.90) between tumor gold concentration and photoacoustic signal. By 24 hours, free GNRs had been sequestered to the liver and spleen with 2%ID/g immobilized in the tumor. The same GNRs produced SERS signal, and Raman maps were created with least squares analysis. We used the Raman signal to identify tumor margins and also to monitor resection and ensure complete removal of tumor tissue. Thus, the GNRs allow pre-surgical photoacoustic visualization for tumor staging and intra-operative Raman imaging to guide resection. Future work will study GNRs targeted to cell surface proteins to increase tumor accumulation.

Red blood cell (RBC) rouleaux formation is a reversible phenomenon that occurs during low blood flow and small shearing forces in circulation. Certain pathological conditions can alter the molecular constituents of blood and properties of the RBCs leading to enhanced rouleaux formation, which results in impaired perfusion and tissue oxygenation. In this study rouleaux were artificially generated using Dextran-70 and examined using a photoacoustic (PA) microscope. Individual rouleau were irradiated with a 532 nm pulsed laser focused to a 10 μm spot size, and the resulting PA signals recorded with a 200 MHz transducer. The laser and transducer were co-aligned, with the sample positioned between them. The frequency-domain PA ultrasound spectra were calculated for rouleaux with lengths ranging from 10 to 20 μm. For the rouleaux, a single spectral minimum at 269±4 MHz was observed. The spectral minima were in good agreement with a theoretical thermoelastic expansion model using an infinite length cylindrical absorber, bearing a diameter equivalent to an average human RBC (7.8 μm). These results suggest that PA ultrasound spectroscopy can be potentially used as a tool for monitoring blood samples for the presence of rouleaux.

Gold-based contrast agents, gold nanorod (GNR), were designed for the enhancement of optoacoustic signal. After synthesis, the GNR-CTAB complexes were modified by pegylation (PEG), or replacement of CTAB (cetyl trimethylammonium bromide) with MTAB (16-mercaptohexadecyl trimethylammonium bromide) for coverage of gold nanorods with heparin (GNR-HP). Modified GNR are purified through centrifugation and filtration. GNRCTAB can be used as a model of positively charged gold surface for quantitative optoacoustic sensing in GNRbacteria interactions, whereas GNR-PEG and GNR-HP can be used as negatively charged gold surface models. We studied controlled agglomeration of contrast agents with the bacteria E.Coli and Vibrio Cholerae. For bacterial sensing, the localized plasmon resonance peak shifts as a function of electrostatic binding, which was detected with two different wavelengths through 3D optoacoustic imaging.

Photoacoustic computed tomography (PACT) holds great promise for transcranial brain imaging. However, the strong reflection, scattering and attenuation of acoustic waves in the skull present significant challenges to developing this method. We report on a systematic computer-simulation study of transcranial brain imaging using PACT. The goal of this study was to identify an effective imaging system design that can be translated for clinical use. The propagation of photoacoustic waves through a model skull was studied by use of an elastic finite-difference time-domain (FDTD) method. The acoustic radiation pattern from a photoacoustic source just beneath the skull was observed with a ring transducer array that was level with the source. The observed radiation pattern was found to contain stronger contributions from waves that were converted to shear waves in skull than longitudinal waves that did not undergo mode conversion. Images reconstructed from the pressure data that contain shear wave components possess better resolution than images reconstructed from the data that only contain the longitudinal wave signals. These observations revealed that the detection system should be designed to capture photoacoustic signals that travel through the skull in the form of shear waves as well as in the form of longitudinal waves. A preliminary investigation on the effect of the presence of absorption in the skull is also reported. This study provides an insight into the wave phenomena in transcranial PACT imaging, as well as a concrete detection design strategy that mitigates the degraded resolution of reconstructed images.

Planar sensor arrays have many advantages including ease of manufacture and low cost. However, when used for photoacoustic (PA) imaging, planar sensors have a limited view of the acoustic field, which means some of the waves from the PA source are not recorded. This results in artifacts in the reconstructed image of the PA source (the initial acoustic pressure distribution). In this paper, we describe novel sensor array configurations based on the Fabry Perot (FP) sensor and acoustic reflectors that retain its detection advantages while improving the visibility of the reconstructed PA image.