Spread Spectrum Photoacoustic Tomography With Image Optimization.

Photoacoustic (PA) imaging technology is expected to be applied to clinical assessment for peripheral vascularity. We started a clinical evaluation with the prototype PA imaging system we recently developed. Prototype PA imaging system was composed with in-house Q-switched Alexandrite laser system which emits short-pulsed laser with 750 nm wavelength, handheld ultrasound transducer where illumination optics were integrated and signal processing for PA image reconstruction implemented in the clinical ultrasound (US) system. For the purpose of quantitative assessment of PA images, an image analyzing function has been developed and applied to clinical PA images. In this analyzing function, vascularity derived from PA signal intensity ranged for prescribed threshold was defined as a numerical index of vessel fulfillment and calculated for the prescribed region of interest (ROI). Skin surface was automatically detected by utilizing B-mode image acquired simultaneously with PA image. Skinsurface position is utilized to place the ROI objectively while avoiding unwanted signals such as artifacts which were imposed due to melanin pigment in the epidermal layer which absorbs laser emission and generates strong PA signals. Multiple images were available to support the scanned image set for 3D viewing. PA images for several fingers of patients with systemic sclerosis (SSc) were quantitatively assessed. Since the artifact region is trimmed off in PA images, the visibility of vessels with rather low PA signal intensity on the 3D projection image was enhanced and the reliability of the quantitative analysis was improved.

Photoacoustic (PA) tomography enables imaging of optical absorption property in deep scattering tissue by listening to the PA wave. However, it is an open challenge that the conversion efficiency from light to sound based on PA effect is extremely low. The consequence is the poor signal-to-noise ratio (SNR) of PA signal especially in scenarios of low laser power and deep penetration. The conventional way to improve PA signal's SNR is data averaging, which however severely limits the imaging speed. In this paper, we propose a new adaptive wavelet threshold de-noising (aWTD) algorithm, and apply it in photoacoustic tomography to increase the PA signal's SNR without sacrificing the signal fidelity and imaging speed. PA image quality in terms of contrast is also significantly improved. The proposed method provides the potential to develop real-time low-cost PA tomography system with low-power laser source.

Molecular fluorescence-guided surgery has shown promise for tumor margin delineation but is limited by its depth profiling capability. Interestingly, most fluorophores, either clinically approved or in clinical trials, can also be used as photoacoustic contrast agents, yet their use is limited due to the low light fluence permitted for clinical use and the limited sensitivity of current photoacoustic imaging systems. There is therefore an urgent unmet need to establish methods for enhancing contrast in molecular targeted PA imaging which could potentially complement and overcome limitations in molecular fluorescence guided therapies. In this study, we compare the photoacoustic (PA) and fluorescence imaging capabilities of a cetuximab-IRDye800 conjugate in a subcutaneous tumor xenograft model. We demonstrate that while the fluorescence signal increases steadily over time after administration of cetuximab-IRDye800, PA signal peaks early (~2 fold higher at 6-hour as compared to pre-injection controls) and then decreases (~1.3 fold higher at 24-hour as compared to pre-injection controls). This pattern aligns with previous findings using other antibody-conjugated PA contrast agents. Mechanistically, we demonstrate that the formation of H-aggregates upon antibody conjugation enhances PA contrast of the IRDye800. The disruption of these H-aggregates, as the antibody-dye conjugate is degraded post receptor-mediated endocytosis, decreases PA signal intensity. The timeframe of maximum PA signal and decrease thereafter is consistent with the time frame of receptor-mediated endocytosis of cetuximab-IRDye800. Our data suggests that tumor cell surface binding results in peak PA signal while lysosomal localization and degradation results in a significant drop in PA signal. Our study sheds light on the distinct temporal dynamics of PA and fluorescence signals of Cetuximab-IRDye800 conjugate and we propose that optimizing IRDye800 conjugation to antibodies can further enhance PA signal intensity when timed to precisely to capture IRDye800 in an H-aggregate form.

As a molecular imaging modality, photoacoustic (PA) imaging has been in the spotlight because it can provide an optical contrast image of physiological information and a relatively deep imaging depth. However, its sensitivity is limited despite the use of exogenous contrast agents due to the background PA signals generated from nontargeted absorbers, such as blood and boundaries between different biological tissues. In addition, clutter artifacts generated in both in-plane and out-of-plane imaging region degrade the sensitivity of PA imaging. We propose a method to eliminate the nontargeted PA signals. For this study, we used a dual-modal ultrasound (US)-PA contrast agent that is capable of generating both the backscattered US and PA signals in response to the transmitted US and irradiated light, respectively. The US images of the contrast agents are used to construct a masking image that contains the location information about the target site and is applied to the PA image acquired after contrast agent injection. In vitro and in vivo experimental results demonstrated that the masking image constructed using the US images makes it possible to completely remove nontargeted PA signals. The proposed method can be used to enhance the clear visualization of the target area in PA images.

Photoacoustic (PA) imaging is advantageous in contrast agent imaging because of high spatial resolution at depth more than several millimeter inside biological tissues. To detect small tumors specifically, we are developing small organic molecule-based activatable PA probe with mechanism similar to that of the enzyme-activatable fluorescence probe that have successfully used for rapid fluorescence imaging of small tumors. The probe can be imaged also by fluorescence imaging and the fluorescence image can be merged onto the PA images. To extend the imaging depth by increasing PA signal intensity, PA probe that produce PA signals efficiently is required. To select small organic molecules suitable for PA probe, we synthesized small-organic molecule-based contrast agents with various absorption spectra and fluorescence quantum yields and then we exhaustively evaluated their PA signal generation characteristics including PA signal generation efficiencies. To analyze PA signal generation efficiencies precisely, the absolute values of PA signal pressures produced from aqueous solutions of the contrast agents were measured by P(VDF-TrFE) piezoelectric film acoustic sensor. As a result, small organic molecule with low fluorescence quantum yield produced PA signals efficiently. Thus, as opposed to fluorescence probes, PA probes should have low fluorescence quantum yields. By considering the result and other characteristics including excitation wavelengths, we could single out the small organic molecule suitable for PA probe. We synthesized the new activatable PA probe with low fluorescence quantum yield and excitation wavelength longer than 600 nm and its specificity was examined in in vitro experiment.

Photoacoustic (PA) imaging is an emerging technology that could map the functional contrasts in deep biological tissues in high resolution by “listening” to the laser induced thermoelastic waves. Almost all of the current studies in PA imaging are focused on the intensity of the PA signals as an indication of the optical absorbance of the biological tissues. Our group has for the first time demonstrated that the frequency domain power distribution of the broadband PA signals encode the texture information within the regions-of-interest (ROI). Following the similar method of ultrasound spectral analysis (USSA), photoacoustic spectrum analysis (PASA) could evaluate the relative concentrations and, more importantly, the dimensions of microstructures of the optically absorbing materials in biological tissues, including lipid, collagen, water and hemoglobin. By providing valuable insights into tissue pathology, PASA should benefit basic research and clinical management of many diseases, and may help achieve eventual “noninvasive biopsy”. In this work, taking advantage of the optical absorption contrasts contributed by lipid and hemoglobin at 1200-nm and 532-nm wavelengths respectively, we investigated the capability of PASA in identifying histological changes corresponding to fat accumulation livers through the study on ex vivo and in situ mouse models. The PA signals from the mouse livers were acquired using our PA and US dual-modality imaging system, and analyzed in the frequency domain. After quantifying the power spectrum by fitting it to a first order model, three spectral parameters, including the intercept, the midband fit and the slope, were extracted and used to differentiate fatty livers from normal livers. The comparison between the PASA parameters from the normal and the fatty livers supports our hypotheses that PASA can quantitatively identify the microstructure changes in liver tissues for differentiating normal and fatty livers.

Photoacoustic (PA) imaging holds great promise for preclinical research and clinical practice. However, most studies rely on the laser wavelength in the first near-infrared (NIR) window (NIR-I, 650-950 nm), while few studies have been exploited in the second NIR window (NIR-II, 1000-1700 nm), mainly due to the lack of NIR-II absorbing contrast agents. We herein report the synthesis of a broadband absorbing PA contrast agent based on semiconducting polymer nanoparticles (SPN-II) and apply it for PA imaging in NIR-II window. SPN-II can absorb in both NIR-I and NIR-II regions, providing the feasibility to directly compare PA imaging at 750 nm with that at 1064 nm. Because of the weaker background PA signals from biological tissues in NIR-II window, the signal-to-noise ratio (SNR) of SPN-II resulted PA images at 1064 nm can be 1.4-times higher than that at 750 nm when comparing at the imaging depth of 3 cm. The proof-of-concept application of NIR-II PA imaging is demonstrated in in vivo imaging of brain vasculature in living rats, which showed 1.5-times higher SNR as compared with NIR-I PA imaging. Our study not only introduces the first broadband absorbing organic contrast agent that is applicable for PA imaging in both NIR-I and NIR-II windows but also reveals the advantages of NIR-II over NIR-I in PA imaging.

Compared to piezoelectric based photoacoustic (PA) scanners, the planar Fabry-Perot (FP) scanner has several advantages. It can provide small element size with high sensitivity, a smooth broadband frequency response, and is transparent to excitation light. This enables the FP scanner to provide excellent high-resolution in vivo PA images of soft tissue to depths up to approximately 10 mm. However, unlike piezoelectric scanners, the FP scanner in its current form cannot provide a pulse-echo ultrasound (US) as well as a PA image, which is useful because of the additional tissue contrast it provides. To address this, a dual mode FP scanner-based system that, for the first time, can acquire co-registered 3D PA and US images has been developed. In order to provide an optical US generation capability, the FP ultrasound sensor was coated with a novel Gold-Nanoparticle-PDMS composite which was excited with nanosecond laser pulses to generate plane wave US pulses. By modifying the FP sensor in this way, it now acts as an US transmitter as well as a receiver. The coating is highly absorbing at the US generation wavelength (>95%) but transparent at the PA excitation wavelength, the latter to allow the system to also operate in PA imaging mode as before. The generated US pulses exhibited peak pressures in the MPa range, which is comparable to the output of conventional piezoelectric based medical US scanners. The pulses had a broad bandwidth (>40 MHz) and the emitted wavefront was planar to within λ/10 at 10 MHz. PA and pulse-echo US signals were mapped in turn by the FP scanner over centimetre scale areas with a step size of 100 μm and an element size of 64 μm. The -3dB bandwidth of the FP sensor was 30 MHz. Reconstruction methods using a k-space formulation recovered co-registered 3D PA and US images. The system’s lateral spatial resolution was evaluated by imaging a line target at depths up to 10 mm and ranged between 50 and 120 μm for both modes. Arbitrarily shaped 3D objects were imaged to demonstrate the volumetric US imaging capability of the scanner. Tissue mimicking phantoms, with impedance mismatches representative of soft tissues, and ex vivo tissue samples were imaged with the system as well as a conventional clinical US scanner for comparison. Finally, the system obtained promising high-resolution 3D dual mode PA-US images for a variety of phantoms with contrast based on both optical absorption and acoustic impedance. This novel all-optical system has the potential to add complementary morphological contrast to photoacoustic vascular images which could aid the clinical assessment of superficial tumours, lymph node disease and other conditions.

Photoacoustic (PA) imaging provides excellent optical contrast with decent penetration and high spatial resolution, making it attractive for a variety of neural applications. We evaluated optical contrast agents with high absorption in the near infrared (NIR) as potential enhancers for PA neuroimaging: optical dyes, gold nanorods (GNRs) and PEBBLEs loaded with indocyanine green. Two PA systems were developed to test these agents in excised neural tissue and in vivo mouse brain. Lobster nerves were stained with the agents for 30 minutes and placed in a hybrid nerve chamber capable of electrical stimulation and recording, optical spectroscopy and PA imaging. Contrast agents boosted the PA signal by at least 30 dB using NIR illumination from a tunable pulsed laser. Photobleaching may be a limiting factor for optical dyes-the PA signal decreased steadily with laser illumination. The second setup enabled in vivo transcranial imaging of the mouse brain. A custom clinical ultrasound scanner and a 10-MHz linear array provided near real-time images during and after an injection of 2 nM gold nanorods into the tail vein. The peak PA signal from the brain vasculature was enhanced by up to 2 dB at 710 nm. Temporal dynamics of the PA signal were also consistent with mixing of the GNRs in the blood. These studies provide a baseline for enhanced PA imaging in neural tissue. The smart contrast agents employed in this study can be further engineered for molecular targeting and controlled drug delivery with potential treatment for a myriad of neural disorders.

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.

The lateral imaging characteristic of an acoustic lens is similar to the well-known object-image relationship of an optical lens. In this paper, axial signal analysis is carried on and reconstruction methods for a sample's axial photoacoustic (PA) image are researched based on the acoustic-lens PA imaging system. The integral and envelope methods are both proposed to reconstruct a sample's axial PA image, which has only been achieved approximately by the PA signals' peak values obtained by a peak-holding circuit in previous reports. And the envelope method is proposed for the first time. Hilbert transform was used to obtain the envelope of the sample's PA signals and axial PA images. Simulation results show that the integral method is appropriate for low frequency samples, and that the envelope reconstruction method can provide low frequency information of the PA images of samples. So for a sample, it would be best to combine the two methods together.

<p>The tumour vasculature supplies the tumour with oxygen and nutrition that are vital for the survival and growth of cancer cells. Vascular disrupting agents (VDAs) are a category of cancer therapeutic drugs that target the endothelial cells lining the vessels and damage tumour vasculature. Photoacoustic (PA) imaging has high sensitivity in detecting vascular changes due to the high optical absorption of hemoglobin. PA quantitative analysis can be used to further extract structural and biochemical changes of hemoglobin through ultrasound spectral analysis and multi-spectral PA imaging. I proposed the use of simulations, in-vitro, and in-vivo PA imaging to assess the efficacy of VDAs in cancer therapy.</p> <p>PA signals acquired from single and a collection of red blood cells (RBCs) were examined before and after acid sphingomyelinase exposure (SMase is a signaling molecule excreted due to endothelial cells damage) to investigate changes at the cellular level. Bleeding of tumour vasculature was simulated using a fractal-based model of bifurcating cylinders and the diffusion of blood to the tumour interstitium. Wavelength selection and fluence matching approaches were proposed to improve chromophore quantification in real time for PA imaging. In-vivo experiments were conducted to examine how PA imaging can be used to monitor the effect of the VDA 5,6-dimethylxanthenone-4-acetic acid (DMXAA). PA ultrasound frequency analysis and multi-spectral PA imaging were used to detect structural and biochemical changes to the tumour vasculature.</p> <p>PA quantitative analysis of the in-vivo data demonstrates a decrease in the total hemoglobin 72 hrs post DMXAA injection and an increase in Hb 24 hrs post DMXAA injection. The changes in the chromophores’ composition are due to changes in the environment as the RBCs extravasate to the surrounding tissues as seen from the in-vitro experiments. A decrease in the average spectral slope at 24 hrs post injection was measured. The decrease in the spectral slope was correlated to an increase in the effective vessel size due to bleeding, which was also supported by the vascular tree simulations. The combination of simulations, in-vitro and in-vivo studies demonstrate the capability of PA signal analysis in monitoring VDA at early time points.</p>

Brachytherapy seed therapy is an increasingly common way to treat prostate cancer through localized radiation. The current standard of care relies on transrectal ultrasound (TRUS) for imaging guidance during the seed placement procedure. As visualization of individual metallic seeds tends to be difficult or inaccurate under TRUS guidance, guide needles are generally tracked to infer seed placement. In an effort to improve seed visualization and placement accuracy, the use of photoacoustic (PA) imaging, which is highly sensitive to metallic objects in soft tissue, was investigated for this clinical application. The PA imaging properties of bare (i.e., embedded in pure gelatin) and tissue-embedded (at depths of up to 13 mm) seeds were investigated with a multi-wavelength (750 to 1090 nm) PA imaging technique. Results indicate that, much like ultrasonic (US) imaging, an angular dependence (i.e., seed orientation relative to imaging transducer) of the PA signal exists. Despite this shortcoming, however, PA imaging offers improved contrast, over US imaging, of a seed in prostate tissue if sufficient local fluence is achieved. Additionally, although the PA signal of a bare seed is greatest for lower laser wavelengths (e.g., 750 nm), the scattering that results from tissue tends to favor the use of higher wavelengths (e.g., 1064 nm, which is the primary wavelength of Nd:YAG lasers) when the seed is located in tissue. A combined PA and US imaging approach (i.e., PAUS imaging) shows strong potential to visualize both the seed and the surrounding anatomical environment of the prostate during brachytherapy seed placement procedures.

This study examines the application of backscattered ultrasound (US) and photoacoustic (PA) signals for assessment of bone structure and density variations. Both methods were applied in the frequency-domain, employing linear frequency modulation chirps. A near-IR laser (800 nm) was used for inducing the PA signal. The backscattered pressure waves were detected with a 2.2-MHz US transducer. Experiments were focused on detection and evaluation of PA and US signals from in-vitro animal and human bones with cortical and trabecular sublayers. It was shown that PA signals can be detected as deep as a few millimeters below trabecular and cortical layers. The occurrence of multiple scattering was demonstrated in PA detected signals from cancellous bone. Osteoporotic changes in the bone were simulated by using a very mild demineralization ethylenediaminetetraacetic acid solution. Changes in the time-domain signals as well as integrated backscattering spectra were compared for the samples before and after demineralization. The results demonstrated the sensitivity of PA to variations in bone minerals. In comparison to PA, US was capable of generating detectable signals from deeper bone sublayers (few centimeters). However, while US signal variations with changes in the cortical layer were insignificant, PA proved to be sensitive even to minor variations of the cortical bone density.

In order to improve efficiency and security in heat applications during hyperthermia, it is important to monitor tissue temperature during treatments. Photoacoustic (PA) pressure wave amplitude has a temperature dependence given by the Gruenesein parameter. Therefore, changes in photoacoustic signal amplitude carry information about temperature variation in tissue. Therefore, PA has been proposed as an imaging technique to monitor temperature during hyperthermia. However, no study has compared the performance of different algorithms to generate PA-based thermal images. Here, four methods to estimate variations in PA signal amplitude for thermal PA image formation were investigated: peak-to-peak, integral of the modulus, autocorrelation maximum value, and energy of the signal. Changes in PA signal amplitude were evaluated using a 1D window moving across the entire image. PA images were acquired for temperatures ranging from 36°C to 42°C using a phantom immersed in a temperature controlled thermal bath. In terms of signal to noise ratio (SNR), the performance was similar for all algorithms and using larger windows resulted in a higher SNR. Bias varied from 1.5°C to −1.5°C for temperatures 3°C higher than the baseline. In conclusion, four methods were evaluated to optimize PA thermal images formation.