We present the development of a photoacoustic tomography (PAT) imaging system with the demonstrated capability of obtaining high-throughput scans at a sustained rate of under 1 minute per animal using integrated robotics to assist in 3D PAT collection. This is a considerable achievement as there is currently no existing commercial or research PAT whole-body imaging system capable of high-throughput applications (15-20 animals per hour). High-throughput experimentation is imperative in the development, characterization, and use of rodent models of human diseases as it increases the number of animals that can be evaluated within a single experiment and may reduce the time under anesthesia for each animal, thereby improving the stability, duration, and confidence of longitudinal studies The developed system features coordinated automation for robotic animal manipulation, anesthesia distribution, temperature regulation, water management, laser excitation, and photoacoustic detection. Furthermore, as shown in validation studies using phantoms and live murine models, the prototype imaging platform demonstrates high-throughput performance while retaining high sensitivity and high resolution.
SignificanceDynamic photoacoustic computed tomography (PACT) is a valuable imaging technique for monitoring physiological processes. However, current dynamic PACT imaging techniques are often limited to two-dimensional spatial imaging. Although volumetric PACT imagers are commercially available, these systems typically employ a rotating measurement gantry in which the tomographic data are sequentially acquired as opposed to being acquired simultaneously at all views. Because the dynamic object varies during the data-acquisition process, the sequential data-acquisition process poses substantial challenges to image reconstruction associated with data incompleteness. The proposed image reconstruction method is highly significant in that it will address these challenges and enable volumetric dynamic PACT imaging with existing preclinical imagers.AimThe aim of this study is to develop a spatiotemporal image reconstruction (STIR) method for dynamic PACT that can be applied to commercially available volumetric PACT imagers that employ a sequential scanning strategy. The proposed reconstruction method aims to overcome the challenges caused by the limited number of tomographic measurements acquired per frame.ApproachA low-rank matrix estimation-based STIR (LRME-STIR) method is proposed to enable dynamic volumetric PACT. The LRME-STIR method leverages the spatiotemporal redundancies in the dynamic object to accurately reconstruct a four-dimensional (4D) spatiotemporal image.ResultsThe conducted numerical studies substantiate the LRME-STIR method’s efficacy in reconstructing 4D dynamic images from tomographic measurements acquired with a rotating measurement gantry. The experimental study demonstrates the method’s ability to faithfully recover the flow of a contrast agent with a frame rate of 10 frames per second, even when only a single tomographic measurement per frame is available.ConclusionsThe proposed LRME-STIR method offers a promising solution to the challenges faced by enabling 4D dynamic imaging using commercially available volumetric PACT imagers. By enabling accurate STIRs, this method has the potential to significantly advance preclinical research and facilitate the monitoring of critical physiological biomarkers.
Previous studies of dynamic photoacoustic computed tomography (PACT) consider the case where complete data can be rapidly acquired and employed to directly reconstruct a sequence of images. However, such frame-by-frame methods do not apply to commercially available volumetric PACT imaging systems with rotating gantries because the object varies during data acquisition. Furthermore, the rotation speed and the laser repetition rate limit the number of tomographic views per frame. In this study, a low-rank matrix estimation-based spatiotemporal image reconstruction method attuned to rotating-gantry volumetric PACT systems is proposed, and its accuracy is shown by numerical and experimental studies.
The ability to perform dynamic imaging of time-varying physiological processes in small animal models is critically needed to understand the progression of human diseases and develop new therapies. Photoacoustic computed tomography (PACT) has been recognized as a promising tool for small animal imaging because of its relatively low expense, high resolution, and good signal-to-noise ratio. By exploiting the optical absorption of hemoglobin or exogenous contrast agents, dynamic PACT holds excellent potential for measuring important time-varying biomarkers like tumor vascular perfusion. Nonetheless, current dynamic PACT technologies possess several limitations. Most three-dimensional (3D) PACT imagers employ a tomographic measurement process in which a gantry containing acoustic transducers is rotated about the animal. Such a rotating gantry is advantageous for limiting the cost of the system due to the decreased number of acoustic transducers and associated electronics and for enabling convenient delivery of the light to the object. However, this presents significant challenges for dynamic image reconstruction because only a few tomographic views are available to reconstruct each temporal frame. This work presents an efficient and accurate dynamic image reconstruction method that can be deployed with widely available 3D imagers using rotating gantries. In particular, a low-rank matrix estimation based spatiotemporal image reconstruction (LRME-STIR) algorithm is proposed. In a stylized virtual dynamic contrast-enhanced imaging study, the proposed LRME-STIR algorithm is shown to accurately recover a well characterized dynamic numerical murine phantom in which tumor vascular perfusion and breathing motion are modeled.
SignificanceTo effectively study preclinical animal models, medical imaging technology must be developed with a high enough resolution and sensitivity to perform anatomical, functional, and molecular assessments. Photoacoustic (PA) tomography provides high resolution and specificity, and fluorescence (FL) molecular tomography provides high sensitivity; the combination of these imaging modes will enable a wide range of research applications to be studied in small animals.AimWe introduce and characterize a dual-modality PA and FL imaging platform using in vivo and phantom experiments.ApproachThe imaging platform’s detection limits were characterized through phantom studies that determined the PA spatial resolution, PA sensitivity, optical spatial resolution, and FL sensitivity.ResultsThe system characterization yielded a PA spatial resolution of 173 ± 17 μm in the transverse plane and 640 ± 120 μm in the longitudinal axis, a PA sensitivity detection limit not less than that of a sample with absorption coefficient μa = 0.258 cm − 1, an optical spatial resolution of 70 μm in the vertical axis and 112 μm in the horizontal axis, and a FL sensitivity detection limit not <0.9 μM concentration of IR-800. The scanned animals displayed in three-dimensional renders showed high-resolution anatomical detail of organs.ConclusionsThe combined PA and FL imaging system has been characterized and has demonstrated its ability to image mice in vivo, proving its suitability for biomedical imaging research applications.
KEYWORDS: Spatial resolution, Imaging systems, Ultrasonography, Photoacoustic imaging, In vivo imaging, Signal detection, Video, Functional imaging, Doppler effect, Data acquisition
We present the continued development a clinical ultrasound (US) imaging device with a photoacoustic (PA) mode. The combined USPA imaging platform is designed around a compact US component capable of B-mode, M-mode, color Doppler, and pulsed wave Doppler US imaging modes with a 128 element US probe. The PA mode can support a 256 element PA probe with real-time 2D imaging up to 20 Hz. The PA signals are amplified by a 40 dB pre-amplifier while US mode signals bypass the circuit, resulting in high quality PA images. We demonstrate the USPA platform’s capabilities using tissue-mimicking phantoms.
Dynamic reconstruction of three-dimensional (3D) photoacoustic tomography (PAT) recovers a sequence of 3D optical contrast distributions and enables to monitor time-varying changes of the chromophore concentrations in biological tissues. To achieve a high frame rate, only a limited-angle few-view (even a single-view) acoustic measurements can be collected at each time frame. These sparse incomplete data represent a formidable challenge to obtain sequences of reconstructed images with both high spatial and temporal resolution. Furthermore, dynamic PAT reconstruction is extremely computationally and memory expensive. High-resolution spatiotemporal reconstruction of 3D objects is, in fact, computationally unfeasible using naïve extensions of classical PAT reconstruction methods for static images. In this study, we present a fast and accurate randomized algorithm for dynamic PAT reconstruction from few tomographic views. Our method is based on the fact that, for many applications of clinical interest, dynamic PAT images are semi-separable in space and time. That is the sequence of PAT images can be expanded using a relatively small number r of basis functions in space and time. Under this assumption, the dynamic PAT reconstruction problem is reformulated as a penalized least squares model, where the nuclear norm of the space-time image is used for the regularization term. By use of a randomized truncated singular value thresholding method, our approach can be implemented in a memory-efficient (only need to store the r spatial and temporal basis functions) and computationally-scalable (only r PAT reconstructions per iteration) manner. The effectiveness of the proposed method is demonstrated using numerical simulation and experimental data of a 3D phantom.
We present the results on development of the 3D imaging platform combining photoacoustic tomography and fluorescence (PAFT) for preclinical and biological research. This combined multimodal imaging instrument addresses known deficiencies in sensitivity, spatial resolution, and anatomical registration of the individual imaging components. Multiangle photoacoustic projections, excited by an OPO operating in the near-infrared window, of a live anesthetized animal are used to reconstruct large volumes (30 cm3) that show deep anatomical vasculature and blood-rich tissues with resolutions exceeding 150 μm. A sCMOS camera is used for simultaneous co-registered multi-angle optical imaging. The images of a fluorescent dual-contrast agent are then reconstructed into a 3D volume using a tomographic algorithm. A separate 532-nm low-energy pulsed laser excitation is used for skin topography and imaging of superficial vasculature. All three imaging channels can be combined to produce spatially accurate in vivo volumes showing an animal’s skin, deep anatomical structures, and distribution of photosensitive molecular contrast agents. PAFT’s photoacoustic sensitivity was assessed using contrast agents in a phantom study. We demonstrate biomedical imaging application of PAFT’s combined imaging modalities by observing biodistribution of a dual-contrast agent injected intravenously to in vivo preclinical murine models.
PhotoSound Technologies specializes in the development of electronics solutions for massive parallel data acquisition applicable to the fields of photoacoustics (PA), X-ray acoustics, including 3D dosimetry, and ultrasound. PhotoSound’s Legion ADC256 R1.1, released in 2018, is a 256-channel 12-bit ADC with a sampling rate of 40 MHz. The ADC256’s average data bandwidth is limited by its USB3 PC interface, which has a data rate up to 3 Gbps per board. Multiple ADC256 boards can operate fully in parallel. On software level configurations, multiple ADC256 boards are represented as a single ADC board with increased number of channels. The incoming ultrasound (US) upgrades and modifications of ADC256 will enable combination and alternation of US and PA modes using the same probe. PhotoSound MoleculUS is a medical-grade Telemed US system combined with a PA-optimized ADC. MoleculUS utilizes clinical US probes to produce US images which can be interleaved with PA imaging by enabling optical fiber illumination. The other ADC256 modification, advanced PAUS oriented for research, will have PCIe PC interface for raw PA and US data and arbitrary software control over beamformer profiles, limited by high-voltage power only. The data in ultrasound and photoacoustics modes is user accessible in raw format and can be delivered to CUDA GPU using MATLAB parallel computing (CUDA) toolbox or other tools. Multiple PAUS boards can work in parallel in both PA and US modes.
KEYWORDS: Skin, Luminescence, Photoacoustic spectroscopy, Tissues, 3D image processing, Animal model studies, Preclinical imaging, Spatial resolution, In vivo imaging, Acquisition tracking and pointing
We report on the development of a preclinical 3D imaging platform integrating photoacoustic tomography and fluorescence (PAFT). The proposed multimodal imaging concept addresses known deficiencies in sensitivity, anatomical registration, and spatial resolution of the individual imaging modalities. Multi-view photoacoustic and optical projections of the studied animal are utilized to reconstruct large (27 cm3) volumes showing vascular network and blood-rich tissues, as well as regions with induced optical/fluorescence contrast with 3D resolution exceeding 150 μm. An additional 532-nm low-energy pulsed laser excitation is implemented as a separate imaging channel for registration over skin topography and superficial vasculature. PAFT technology enables functional and molecular volumetric imaging using wide range of fluorescent and luminescent biomarkers, nanoparticles, and other photosensitive constructs mapped with high fidelity over robust anatomical structures of the studied animal model. We demonstrated the PAFT performance using phantoms and by in vivo imaging of preclinical murine models.
KEYWORDS: Data acquisition, Clocks, Capacitance, Interfaces, Electromagnetic coupling, Amplifiers, Connectors, Field effect transistors, Signal to noise ratio, Human-machine interfaces
Large number of simultaneously acquired spatially distinct pressure signals is required to improve quality of real-time photoacoustic and x-ray acoustic biomedical images [1]. In the past this approach was limited by availability of commercial multi-channel analog-to-digital converter (ADC) systems and ability to operate multiple ADC boards with synchronized clock and trigger source. The new Legion series single-board 256-channel ADC (ADC256) was designed by PhotoSound for massive parallel data acquisition utilized in photoacoustic, laser-induced ultrasound, and X-ray acoustic real-time imaging applications. ADC256 is a 12-bit ADC with a sampling rate up to 40 MHz and a USB3 computer interface. It can run at 200 Hz frame rate with 4096 points per trigger acquired by each channel. Higher trigger rates without data loss are possible with smaller number of points per trigger. ADC256 has an integrated amplifier with programmable gain up to 51 dB. Additionally, it can be equipped with a matching photoacoustic preamplifier. The system architecture is scalable to 1024 channels using four synchronized boards with a single trigger source. The clock and the trigger can be delivered from the master ADC256 board (daisy chain) or from the clock and trigger server (star topology). The data collected by each ADC board has trigger and board stamps allowing to (a) use multiple computers for data acquisition, and (b) detection of lost data events, even if the trigger rate exceeds its maximum allowed value.
We have developed a preclinical 3D imaging instrument integrating photoacoustic tomography and fluorescence (PAFT) addressing known deficiencies in sensitivity and spatial resolution of the individual imaging components. PAFT is designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct registration of the two imaging modalities. Orthogonal photoacoustic projections are utilized to reconstruct large (21 cm3 ) volumes showing vascularized anatomical structures and regions of induced optical contrast with spatial resolution exceeding 100 µm. The major advantage of orthogonal fluorescence projections is significant reduction of background noise associated with transmitted or backscattered photons. The fluorescence imaging component of PAFT is used to boost detection sensitivity by providing low-resolution spatial constraint for the fluorescent biomarkers. PAFT performance characteristics were assessed by imaging optical and fluorescent contrast agents in tissue mimicking phantoms and in vivo. The proposed PAFT technology will enable functional and molecular volumetric imaging using fluorescent biomarkers, nanoparticles, and other photosensitive constructs mapped with high fidelity over robust anatomical structures, such as skin, central and peripheral vasculature, and internal organs.
We introduce a preclinical imaging platform – a 3D photoacoustic/fluorescence tomography (PAFT) instrument augmented with an environmentally responsive dual-contrast biocompatible nanoprobe. The PAFT instrument was designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct co-registration of the two imaging modalities. The nanoprobe was based on liposomes loaded with J-aggregates of indocyanine green (PAtrace). Once PAtrace interacts with the environment, a transition from J-aggregate to monomeric ICG is induced. The subsequent recovery of monomeric ICG is characterized by dramatic changes in the optical absorption spectrum and reinstated fluorescence. In the activated state, PAtrace can be simultaneously detected by both imaging modes of the PAFT instrument using 780 nm excitation and fluorescence detection at 810 nm. The fluorescence imaging component is used to boost detection sensitivity by providing lowresolution map of activated nanoprobes, which are then more precisely mapped in 3D by the photoacoustic imaging component. Activated vs non-activated particles can be distinguished based on their different optical absorption peaks, removing the requirements for complex image registration between reference and detection scans. Preliminary phantom and in vivo animal imaging results showed successful activation and visualization of PAtrace with high sensitivity and resolution. The proposed PAFT-PAtrace imaging platform could be used in various functional and molecular imaging applications including multi-point in vivo assessment of early metastasis.
Noninvasive real-time temperature imaging during thermal therapies is able to significantly improve clinical outcomes. An optoacoustic (OA) temperature monitoring method is proposed for noninvasive real-time thermometry of vascularized tissue during cryotherapy. The universal temperature-dependent optoacoustic response (ThOR) of red blood cells (RBCs) is employed to convert reconstructed OA images to temperature maps. To obtain the temperature calibration curve for intensity-normalized OA images, we measured ThOR of 10 porcine blood samples in the range of temperatures from 40°C to −16°C and analyzed the data for single measurement variations. The nonlinearity (ΔTmax) and the temperature of zero OA response (T0) of the calibration curve were found equal to 11.4±0.1°C and −13.8±0.1°C, respectively. The morphology of RBCs was examined before and after the data collection confirming cellular integrity and intracellular compartmentalization of hemoglobin. For temperatures below 0°C, which are of particular interest for cryotherapy, the accuracy of a single temperature measurement was ±1°C, which is consistent with the clinical requirements. Validation of the proposed OA temperature imaging technique was performed for slow and fast cooling of blood samples embedded in tissue-mimicking phantoms.
We describe the ongoing development and performance of a high-pulse-energy wavelength-cycling laser system for three-dimensional optoacoustic tomography of the breast. Joule-level energies are desired for achieving the required penetration depths while maintaining safe fluence levels. Wavelength cycling provides a pulse sequence which repeatedly alternates between two wavelengths (approximately 756 and 797 nm) that provide differential imaging. This improves co-registration of captured differential images and quantification of blood oxygen saturation. New design features have been developed for and incorporated into a clinical prototype laser system, to improve efficacy and ease of use in the clinic. We describe the benefits of these features for operation with a clinical pilot optoacoustic / ultrasound dual-modality three-dimensional imaging system.
We developed a new and improved Laser Optoacoustic Imaging System, LOIS-3D for preclinical research applications in small animal models. The advancements include (i) a new stabilized imaging module with a more homogeneous illumination of the mouse yielding a better spatial resolution (<0.2 mm) and (ii) a new low noise amplifier incorporated into the ultrasonic probe and providing the noise equivalent pressure around 2 Pa resulting in increased signal-to-noise ratio and the optical absorption sensitivity of about 0.15 cm-1. We also improved scan time and the image reconstruction times. This prototype has been commercialized for a number of biomedical research applications, such as imaging vascularization and measuring hemoglobin / oxyhemoglobin distribution in the organs as well as imaging exogenous or endogenous optoacoustic contrast agents. As examples, we present in vivo experiments using phantoms and mice with and without tumor injected with contrast agents with indocyanine green (ICG). LOIS-3D was capable of detecting ~1-2 pmole of the ICG, in tissues with relatively low blood content. With its high sensitivity and excellent spatial resolution LOIS-3D is an advanced alternative to fluorescence and bioluminescence based modalities for molecular imaging in live mice.
KEYWORDS: Prostate, Tissues, Ultrasonography, Temperature metrology, Blood, In vivo imaging, Prostate cancer, Image filtering, Data acquisition, Calibration
Cryoablation of prostate cancer is an FDA approved clinical procedure, which involves repetitive rapid cooling of a lesion to lethal temperatures of -40°C and below. The major drawback of the technique is the insufficient control over the fast thermal processes that may result in severe complications (impotence, incontinence, perforation of the rectal wall) and morbidity. The developed optoacoustic imaging technique provides non-invasive real-time temperature mapping of tissue adjacent to prostate and enables more efficient control over the procedure, which is necessary to reduce side effects and accelerate the physician’s learning curve. In these studies we successfully demonstrated real-time transrectal optoacoustic imaging during prostate cryoablation in live canine model focused on optoacoustic thermography of the rectal wall within the depth of 1cm. Our method utilized previously discovered universal thermal dependence of the normalized optoacoustic response of blood. Nanosecond-pulse radiation of Ti-Sapphire laser tuned to the isosbestic point of hemoglobin (802±3 nm) was delivered via fiberoptic illuminators assembled on both sides of the linear array of the 128-channel transrectal ultrasound probe. Temperature readouts at discrete locations inside and nearby prostate were also performed using standard transperineal needle sensors. The effect of homeostasis on optoacoustic imaging in live tissue was examined during cooling and shown to be significant only within the range of ±1.5°C in respect to the body temperature. Accuracy of in vivo optoacoustic temperature measurements was determined as ±2°C for the range of temperature from +35 to -15°C, which is more than sufficient for tracking the essential isotherms in the course of clinical procedures.
Optoacoustic tomography (OAT) is a promising imaging modality for human breast cancer imaging, with higher resolution and deeper penetration compared to other optical imaging modalities such as diffuse optical tomography or optical coherence tomography. It yields a resolution of 1 mm at depth up to 2 cm. But there is an inherent conflict between the limitations imposed on laser power and the need to adequately penetrate a substantial portion of the breast. To achieve sufficient penetration at every view angle, instead of illuminating the whole breast all at once, sometimes illumination is focused onto a small region of the breast and rotated along with the transducer array to cover the entire object. This paper evaluates the effect of this rotating partial illumination design on OAT image reconstruction. The optical process is simulated by conducting Monte Carlo simulations on a numerical phantom mimicking a real breast, with various specially designed illumination schemes. The acoustic process is simulated by incorporating the transducer's spatial impulse response. Iterative reconstruction is applied to estimate the OAT image. We conclude that rotating partial illumination introduces inconsistency into the system equation, and the degree of inconsistency determines the reconstruction quality.
In this work, we introduce an improved prototype of the imaging system that combines three-dimensional optoacoustic tomography (3D-OAT) and laser ultrasound tomography slicer (2D-LUT) to obtain coregistered maps of tissue optical absorption and speed of sound (SOS). The imaging scan is performed by a 360 degree rotation of a phantom/mouse with respect to a static 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. For the LUT slicer scans, the array is pivoted by 90 degrees with respect to the central transducers providing accurate registration of optoacoustic and SOS maps, the latter being reconstructed using waveform inversion with source encoding (WISE) technique. The coregistered OAT-LUT modality is validated by imaging a phantom and a live mouse. SOS maps acquired in the imaging system can be employed by an iterative optoacoustic reconstruction algorithm capable of compensating for acoustic wavefield aberrations. The most promising applications of the imaging system include 3D angiography, cancer research, and longitudinal studies of biological distributions of optoacoustic contrast agents (carbon nanotubes, metal plasmonic nanoparticles, fluorophores, etc.).
We found and interpreted the universal temperature-dependent optoacoustic (photoacoustic) response (ThOR) in blood; the normalized ThOR is invariant with respect to hematocrit at the hemoglobin’s isosbestic point. The unique compartmentalization of hemoglobin, the primary optical absorber at 805 nm, inside red blood cells (RBCs) explains the effect. We studied the temperature dependence of Gruneisen parameter in blood and aqueous solutions of hemoglobin and for the first time experimentally observed transition through the zero optoacoustic response at temperature T0, which was proved to be consistent for various blood samples. On the other hand, the hemoglobin solutions demonstrated linear concentration function of the temperature T0. When this function was extrapolated to the average hemoglobin concentration inside erythrocytes, the temperature T0 was found equivalent to that measured in whole and diluted blood. The obtained universal curve of blood ThOR was validated in both transparent and light scattering media. The discovered universal optoacoustic temperature dependent blood response provides foundation for future development of non-invasive in vivo temperature monitoring in vascularized tissues and blood vessels.
Optoacoustic (photoacoustic) imaging is being adopted for monitoring tissue temperature during hypothermic and hyperthermic cancer treatments. The technique’s accuracy benefits from the knowledge of speed of sound (SoS) and acoustic coefficient of attenuation (AcA) as they change with temperature in biological tissues, blood, and acoustic lens of an ultrasound probe. In these studies we measured SoS and AcA of different ex vivo tissues and blood components (plasma and erythrocyte concentrates) in the temperature range from 5°C to 60°C. We used the technique based on measurements of time-delay and spectral amplitude of pressure pulses generated by wideband planar acoustic waves propagating through the interrogated medium. Water was used as a reference medium with known acoustic properties. In order to validate our experimental technique, we measured the temperature dependence of SoS and AcA for aqueous NaCl solution of known concentration and obtained the results in agreement with published data. Similar to NaCl solution and pure water, SoS in blood and plasma was monotonously increasing with temperature. However, SoS of erythrocyte concentrates displayed abnormalities at temperatures above 45°C, suggesting potential effects from hemoglobin denaturation and/or hemolysis of erythrocytes. On the contrary to aqueous solutions, the SoS in polyvinyl-chloride (plastisol) – a material frequently used for mimicking optical and acoustic properties of tissues – decreased with temperature. We also measured SoS and AcA in silicon material of an acoustic lens and did not observe temperature-related changes of SoS.
Iterative image reconstruction algorithms can model complicated imaging physics, compensate for imperfect data acquisition systems, and exploit prior information regarding the object. Hence, they produce higher quality images than do analytical image reconstruction algorithms. However, three-dimensional (3D) iterative image reconstruction is computationally burdensome, which greatly hinders its use with applications requiring a large field-of-view (FOV), such as breast imaging. In this study, an improved GPU-based implementation of a numerical imaging model and its adjoint have been developed for use with general gradient-based iterative image reconstruction algorithms. Both computer simulations and experimental studies are conducted to investigate the efficiency and accuracy of the proposed implementation for optoacoustic tomography (OAT). The results suggest that the proposed implementation is more than five times faster than the previous implementation.
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.
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.
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.
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.
A three-dimensional mouse imaging system combining optoacoustic tomography and laser ultrasound (LOUIS-
3DM) has been developed. It features broadband laser ultrasound emitters positioned opposite an array of
transducers. This imaging geometry allows reconstruction of images that either depicts the speed of sound
distribution from measured time of flight data, or acoustic attenuation from the measured signal amplitude. We have
investigated the performance of two laser ultrasound source designs, both easily adaptable to a commercial imaging
system: small diameter source (600 μm) generated off a flat surface, and larger diameter (3 mm) spherical source.
Laser energy requirements are modest, well below 1 mJ per pulse for either design. Their performance at normal
incidence is comparable both in amplitude and frequency response. However, off-axis generation differs
dramatically and the shortcomings of the simple flat emitter design are evident. We show that, in order to achieve
optimal performance through proper illumination of the detector array, spherical wave front characteristics are
desired.
KEYWORDS: Ultrasonography, Imaging systems, Acoustics, 3D image processing, Tomography, Visualization, Signal attenuation, 3D modeling, Ultrasound tomography, Pre-clinical research
In this work, we introduce a novel three-dimensional imaging system for in vivo high-resolution anatomical and functional whole-body visualization of small animal models developed for preclinical or other type of biomedical research. The system (LOUIS-3DM) combines a multi-wavelength optoacoustic and ultrawide-band laser ultrasound tomographies to obtain coregistered maps of tissue optical absorption and acoustic properties, displayed within the skin outline of the studied animal. The most promising applications of the LOUIS-3DM include 3D angiography, cancer research, and longitudinal studies of biological distribution of optoacoustic contrast agents (carbon nanotubes, metal plasmonic nanoparticles, etc.).
We developed the first prototype of dual-modality imager combining optoacoustic tomography (OAT) and laser
ultrasound tomography (UST) using computer models followed by experimental validation. The system designed
for preclinical biomedical research can concurrently yield images depicting both the absorbed optical energy
density and acoustic properties (speed of sound) of an object. In our design of the UST imager, we seek to
replace conventional electrical generation of ultrasound waves by laser-induced ultrasound (LU). While earlier
studies yielded encouraging results [Manohar, et al., Appl. Phys. Lett, 131911, 2007], they were limited to
two-dimensional (2D) geometries. In this work, we conduct computer-simulation studies to investigate different
designs for the 3D LU UST imager. The number and location of the laser ultrasound emitters, which are
constrained to reside on the cylindrical surface opposite to the arc of detectors, are optimized. In addition to
the system parameters, an iterative image reconstruction algorithm was optimized. We demonstrate that high
quality volumetric maps of the speed of sound can be reconstructed when only 32 emitters and 128 receiving
transducers are employed to record time-of-flight data at 360 tomographic view angles. The implications of the
proposed system for small animal and breast-cancer imaging are discussed.
In this report, we demonstrate the feasibility of using optoacoustic tomography for deducing biodistributions of
nanoparticles in animal models. The redistribution of single-walled carbon nanotubes (SWCNTs) was visualized in
living mice. Nanoparticle concentrations in harvested organs were measured spectroscopically using the intrinsic optical
absorption and fluorescence of SWCNTs. Observed increases in optoacoustic signal brightness in tissues were compared
with increases in optical absorptivity coefficients caused by SWCNT accumulation. The methodology presented in this
report paves the way for measuring concentrations of optically absorbing agents in small animals using optoacoustic
tomography.
We report on findings from the clinical feasibility study of the ImagioTM. Breast Imaging System, which acquires two-dimensional opto-acoustic (OA) images co-registered with conventional ultrasound using a specialized duplex hand-held probe. Dual-wavelength opto-acoustic technology is used to generate parametric maps based upon total hemoglobin and its oxygen saturation in breast tissues. This may provide functional diagnostic information pertaining to tumor metabolism and microvasculature, which is complementary to morphological information obtained with conventional gray-scale ultrasound. We present co-registered opto-acoustic and ultrasonic images of malignant and benign tumors from a recent clinical feasibility study. The clinical results illustrate that the technology may have the capability to improve the efficacy of breast tumor diagnosis. In doing so, it may have the potential to reduce biopsies and to characterize cancers that were not seen well with conventional gray-scale ultrasound alone.
We used a 3-D optoacoustic (OA) tomography system to create maps of optical absorbance of mice tissues contrasted with gold nanorods (GNRs). Nude mice were scanned before and after injection of GNRs at time periods varying from 1 to 192 h. Synthesized GNRs were purified from hexadecyltrimethylammonium bromide and coated with polyethylene glycol (PEG) to obtain GNR-PEG complexes suitable for in vivo applications. Intravenous administration of purified GNR-PEG complexes resulted in enhanced OA contrast of internal organs and blood vessels compared to the same mouse before injection of the contrast agent. Maximum enhancement of the OA images was observed 24 to 48 h postinjection, followed by a slow clearance trend for the remaining part of the studied period (eight days). We demonstrate that OA imaging with two laser wavelengths can be used for noninvasive, long-term studies of biological distribution of contrast agents.
We developed a new optoacoustic microangiography system (OmAS) intended for in-vivo vascular imaging of a
human finger. The system employs an arc-shaped acoustic array that is rotated 360 degrees around the finger
providing optoacoustic data necessary for tomographic reconstruction of the three-dimensional images of a finger. A
near-infrared Q-switched laser is used to generate optoacoustic signals with increased contrast of blood vessels. The
laser is coupled through two randomized fiberoptic bundles oriented in orthogonal optoacoustic mode. To
demonstrate OmAS capabilities, we present a time-series of optoacoustic images of a human finger taken after the
hypothermia stress test. The images show a detailed vascular anatomy of a finger down to the capillary level. A
series of quick 30s scans allowed us to visualize the thermoregulatory response within the studied finger as it was
manifested via vasomotor activity during the hypothermia recovery. We propose that the developed system can be
used for diagnostics of various medical conditions that are manifested in change of the peripheral (finger) blood
flow. Examples of the medical conditions that could be diagnosed and staged using the OmAS include the peripheral
arterial disease (PAD), thrombosis, frostbite, and traumas.
Two-dimensional optoacoustic imaging with a hand-held probe operated in backward mode is being developed
for diagnostic imaging of breast cancer to evaluate the feasibility of a dual-modality optoacoustic plus ultrasonic
system that maps functional information of anatomical tissue structures with ultrasonic resolution. Tissue is
illuminated at 757nm and 1064nm for optical contrast between hypoxic blood of breast carcinomas and normally
oxygenated blood in benign masses. The system is optimized and calibrated in phantoms for a pilot clinical
study of patients with breast masses suspected for malignancy. Capability of the non-invasive system to improve
detection and diagnosis of breast tumors is discussed.
Using the method of 3D optoacoustic tomography, we studied changes in tissues of the whole body of nude mice as the
changes manifested themselves from live to postmortem. The studies provided the necessary baseline for optoacoustic
imaging of necrotizing tissue, acute and chronic hypoxia, and reperfusion. They also establish a new optoacoustic model
of early postmortem conditions of the whole mouse body. Animals were scanned in a 37°C water bath using a three-dimensional
optoacoustic tomography system previously shown to provide high contrast maps of vasculature and organs
based on changes in the optical absorbance. The scans were performed right before, 5 minutes after, 2 hours and 1 day
after a lethal injection of KCl. The near-infrared laser wavelength of 765 nm was used to evaluate physiological features
of postmortem changes. Our data showed that optoacoustic imaging is well suited for visualization of both live and
postmortem tissues. The images revealed changes of optical properties in mouse organs and tissues. Specifically, we
observed improvements in contrast of the vascular network and organs after the death of the animal. We associated these
with reduced optical scattering, loss of motion artifacts, and blood coagulation.
We report on the optoacoustic (OA) imaging of the whole mouse body using a biocompatible contrast agent - highly
purified, pegylated gold nanorods (GNR) - which has strong optical absorption in the near-infrared region and low level
of toxicity. In vitro toxicity studies showed no significant change in survival rates of the cultured normal epithelium
IEC-6 cells when incubated for 24 hours with up to 1 nM of GNR. In vivo toxicity studies in nude mice showed no
pathological changes in liver 1 month after the IV injection of GNR with intra-body concentration around 0.25-0.50 nM.
In order to study the enhancement of the OA contrast and accumulation of GNR in different tissues, we performed 3D
OA imaging of live nude mice with IV-injected GNR. The enhancement of the OA contrast in comparison with the
images of the untreated mice was visible starting 1 hour after the GNR injection. The OA contrast of kidneys, liver, and
spleen peaked at about 2-3 days after the administration of the GNR, and then was gradually reducing.
Coregistered optoacoustic (OA) and ultrasound (US) images obtained using a dual modality optoacoustic/ultrasonic
breast imaging system are used together for enhanced diagnostic capabilities in comparison to each individual
technology. Therefore, an operator-independent delineation of diagnostically relevant objects (in our case breast tumors)
with subsequent automatic analysis of image features is required. We developed the following procedure: 1) Image
filtering is implemented on a US image to minimize speckle noise and simultaneously preserve the sharpness of the
boundaries of the extended objects; 2) Boundaries of the objects of interest are automatically delineated starting with an
initial guess made by an operator; 3) Both US and OA images are analyzed using the detected boundaries (contrast,
boundary sharpness, homogeneity of the objects and background, boundary morphology parameters are calculated).
Calculated image characteristics can be used for statistically independent evaluation of structural information (US data)
and vascularization (OA data) of the studied breast tissues. Operator-independent delineation of the objects of interest
(e.g. tumors and blood vessels) is essential in clinical OA spectroscopy (using multiple laser wavelengths to quantify
concentrations of particular tissue chromophores, such as oxy- and deoxy- hemoglobin, water, and lipids). Another
potential application of the suggested image analysis algorithm could be in OA imaging system design, when system
performance should be evaluated in terms of quality of the images reconstructed from the well-defined objects of
interest. The discussed principles of image analysis are illustrated by using real clinical US and OA data.
We introduce a novel experimental design for non-invasive scanning optoacoustic microscopy that utilizes a parabolic
surface for ultrasound focusing. We demonstrate that off-axis parabolic mirrors made of sufficiently high acoustic
impedance materials work as ideal reflectors in a wide range of apertures and provide lossless conversion of a spherical
acoustic wavefront into a plane wave. We further test the performance of a custom optoacoustic imaging setup which
was developed and built based on these principles. The achieved resolution limit of 0.3 mm, with NA of 0.5 and the
transducer bandwidth of 5 MHz, matches the resolution limit defined by diffraction. Although further improvements of
current experimental setup are required to achieve resolution similar to leading microscopy systems, this proof-of-concept
work demonstrates the viability of the proposed design for optoacoustic microscopy applications.
We used a three-dimensional optical tomogaphy system that was previously developed to create high contrast maps of
optical absorbance of mice tissues. In this study, animals were scanned before and after injection of gold nanorods
(GNRs) at different time periods. As-synthesized GNRs were purified from hexadecyltrimethylammonium bromide
(CTAB) and coated with polyethylene glycol (PEG) to obtain GNR-PEG complexes suitable for in vivo applications.
Intravenous administration of the purified GNR-PEG complexes to mice resulted in an enhanced contrast of normal
tissues and blood vessels as compared to ordinary nude mice. In parallel with optoacoustic imaging we investigated the
accumulation of GNRs in liver using invasive analytical techniques. Maximum levels of GNRs in liver macrophages
were observed after 48-72 hours post-injection, followed by slow clearance trend after 8 days. Optoacoustic imaging
revealed redistribution of GNR in mouse organ and tissues: in the initial hours, accumulation of GNRs is seen
predominantly in the periphery of the mouse, while a gradual increase of GNR levels in liver, spleen and kidneys is seen
in 1 and 24 hours.
We developed an optoacoustic biosensor intended for the detection of bloodborne microorganisms using
immunoaffinity reactions of antibody-coupled gold nanorods as contrast agents specifically targeted to the antigen
of interest. Optoacoustic responses generated by the samples are detected using a wide band ultrasonic transducer.
The sensitivity of the technique has been assessed by determining minimally detectable optical density which
corresponds to the minimum detectable concentration of the target viral surface antigens. Both ionic solutions and
gold nanorods served as the contrast agent generating the optoacoustic response. The sensitivity of Nano-LISA is at
least OD=10-6 which allows reliable detection of 1 pg/ml (depending on the commercial antibodies that are used).
Adequate detection sensitivity, as well as lack of non-specific cross-reaction between antigens favors NanoLISA as
a viable technology for biosensor development.
We present our findings from a real-time laser optoacoustic imaging system (LOIS). The system utilizes a Q-switched Nd:YAG laser; a standard 128-channel ultrasonic linear array probe; custom electronics and custom software to collect, process, and display optoacoustic (OA) images at 10 Hz. We propose that this system be used during preoperative mapping of forearm vessels for hemodialysis treatment. To demonstrate the real-time imaging capabilities of the system, we show OA images of forearm vessels in a volunteer and compare our results to ultrasound images of the same region. Our OA images show blood vessels in high contrast. Manipulations with the probe enable us to locate and track arteries and veins of a forearm in real time. We also demonstrate the ability to combine a series of OA image slices into a volume for spatial representation of the vascular network. Finally, we use frame-by-frame analysis of the recorded OA video to measure dynamic changes of the crossection of the ulnar artery.
Optoacoustic (OA) tomography has demonstrated utility in identifying blood-rich malignancies in breast tissue. We describe the development and characterization of a laser OA imaging system for the prostate (LOIS-P). The system consists of a fiber-coupled Q-switched laser operating at 757 nm, a commercial 128-channel ultrasonic probe, a digital signal processor, and software that uses the filtered radial back-projection algorithm for image reconstruction. The system is used to reconstruct OA images of a blood-rich lesion induced in vivo in a canine prostate. OA images obtained in vivo are compared to images acquired using ultrasound, the current gold standard for guiding biopsy of the prostate. Although key structural features such as the urethra could be identified with both imaging techniques, a bloody lesion representing a highly vascularized tumor could only be clearly identified in OA images. The advantages and limitations of both forward and backward illumination modes are also evaluated by collecting OA images of phantoms simulating blood vessels within tissue. System resolution is estimated to be 0.2 mm in the radial direction of the acoustic array. The minimum detectable pressure signal is 1.83 Pa. Our results encourage further development toward a dual-modality OA/ultrasonic system for prostate imaging and image-guided biopsy.
In this work we modified light illumination of the laser optoacoustic (OA) imaging system to improve the 3D
visualization of human forearm vasculature. The computer modeling demonstrated that the new illumination design that
features laser beams converging on the surface of the skin in the imaging plane of the probe provides superior OA
images in comparison to the images generated by the illumination with parallel laser beams. We also developed the
procedure for vein/artery differentiation based on OA imaging with 690 nm and 1080 nm laser wavelengths. The
procedure includes statistical analysis of the intensities of OA images of the neighboring blood vessels. Analysis of the
OA images generated by computer simulation of a human forearm illuminated at 690 nm and 1080 nm resulted in
successful differentiation of veins and arteries. In vivo scanning of a human forearm provided high contrast 3D OA
image of a forearm skin and a superficial blood vessel. The blood vessel image contrast was further enhanced after it was
automatically traced using the developed software. The software also allowed evaluation of the effective blood vessel
diameter at each step of the scan. We propose that the developed 3D OA imaging system can be used during
preoperative mapping of forearm vessels that is essential for hemodialysis treatment.
In this report we demonstrate improved three-dimensional optoacoustic tomography in test samples. High quality
tomographic data and images were obtained from phantom of mice being 2.5 cm in diameter. Capillaries filled with
cupric sulfate, ferrous sulfate and nickel sulfate solutions, and immersed in a scattering medium were used for these
tests. The brightness of reconstructed phantom images was found to match accurately the absorption profiles of test
solutions. Hence, optoacoustic imaging can be applied in preclinical research to perform in vivo absorptivity
measurements to deduce functional information on blood oxygen levels or concentration of contrast agents.
We present results from a clinical case study on imaging breast cancer using a real-time interleaved two laser
optoacoustic imaging system co-registered with ultrasound. The present version of Laser Optoacoustic Ultrasonic
Imaging System (LOUIS) utilizes a commercial linear ultrasonic transducer array, which has been modified to include
two parallel rectangular optical bundles, to operate in both ultrasonic (US) and optoacoustic (OA) modes. In OA mode,
the images from two optical wavelengths (755 nm and 1064 nm) that provide opposite contrasts for optical absorption of
oxygenated vs deoxygenated blood can be displayed simultaneously at a maximum rate of 20 Hz. The real-time aspect of
the system permits probe manipulations that can assist in the detection of the lesion. The results show the ability of
LOUIS to co-register regions of high absorption seen in OA images with US images collected at the same location with
the dual modality probe. The dual wavelength results demonstrate that LOUIS can potentially provide breast cancer
diagnostics based on different intensities of OA images of the lesion obtained at 755 nm and 1064 nm. We also present
new data processing based on deconvolution of the LOUIS impulse response that helps recover original optoacoustic
pressure profiles. Finally, we demonstrate the image analysis tool that provides automatic detection of the tumor
boundary and quantitative metrics of the optoacoustic image quality. Using a blood vessel phantom submerged in a
tissue-like milky background solution we show that the image contrast is minimally affected by the phantom distance
from the LOUIS probe until about 60-65 mm. We suggest using the image contrast for quantitative assessment of an OA
image of a breast lesion, as a part of the breast cancer diagnostics procedure.
Optoacoustic Tomography (OAT) is a hybrid imaging modality that combines the advantages of both optical
imaging and ultrasound imaging techniques. Most existing reconstruction algorithms for OAT assume pointlike
transducers, which may result in conspicous image blurring and distortions in certain applications. In this
work, a new imaging model that incorporates the transducer response is employed for image reconstruction.
Computer-simulation studies demonstrate that the new reconstruction method can effectively compensate for
image resolution degradation associated with the transducer response.
We have investigated the limitations of our laser ultrasonic plane wave δ-source. In theory, the device is capable of
producing an acoustic impulse with a bandwidth exceeding 30 MHz. However, a bandwidth of 12 MHz is measured with
a calibrated wideband hydrophone. A test setup was designed and built. It permits the investigation of experimental
parameters that alter the generated acoustic impulse: laser pulse duration, laser spatial profile, and absorber opacity.
Laser energy spatial profile is the main contributor to the narrowing of the frequency band. Our findings are presented,
along with further justifications for a device with very large effective area.
We develop a system for three-dimensional whole-body optoacoustic tomography of small animals for applications in preclinical research. The tomographic images are obtained while the objects of study (phantoms or mice) are rotated within a sphere outlined by a concave arc-shaped array of 64 piezocomposite transducers. Two pulsed lasers operating in the near-IR spectral range (755 and 1064 nm) with an average pulsed energy of about 100 mJ, a repetition rate of 10 Hz, and a pulse duration of 15 to 75 ns are used as optical illumination sources. During the scan, the mouse is illuminated orthogonally to the array with two wide beams of light from a bifurcated fiber bundle. The system is capable of generating images of individual organs and blood vessels through the entire body of a mouse with spatial resolution of ~0.5 mm.
We designed, fabricated and tested the laser optoacoustic imaging system for breast cancer detection (LOIS-64), which fuses optical and acoustic imaging techniques in one modality by utilizing pulsed optical illumination and ultrawide-band ultrasonic detection of resulting optoacoustic (OA) signals. The system was designed to image a single breast slice in craniocaudal or mediolateral projection with an arc-shaped array of 64 ultrawide-band acoustic transducers. The system resolution on breast phantoms was at least 0.5 mm. The single-channel sensitivity of 1.66 mV/Pa was estimated to be sufficient for single-pulse imaging of 6 to 11 mm tumors through the whole imaging slice of the breast. The implemented signal processing using the wavelet transform allowed significant reduction of the low-frequency (LF) acoustic noise, allowed localization of the optoacoustic signals from tumors, and enhanced the contrast and sharpened the boundaries of the optoacoustic images of the tumors. During the preliminary clinical studies on 27 patients, the LOIS-64 was able to visualize 18 out of 20 malignant lesions suspected from mammography and ultrasound images and confirmed by the biopsy performed after the optoacoustic tomography (OAT) procedure.
KEYWORDS: Transducers, Acoustics, Sensors, Calibration, Wavefronts, Ultrasonography, Signal attenuation, Pulsed laser operation, Image restoration, Signal to noise ratio
Comprehensive characterization of optoacoustic transducers is achieved through the analysis of their frequency
response using a procedure of measuring angular dependence of the transducer sensitivity to the ultrawide-band
delta pulse. The testing was performed under standard repeatable operating conditions. Back-illumination of a
blackened, acoustically matched, planar surface with a short laser pulse creates an acoustic impulse which was used
as an ultrawide-band ultrasonic source. The bandwidth of such a source extends well over 10 MHz (6dB point at 16
MHz for illumination with a 16 ns pulse) and the low frequency
roll-off is around 300 kHz. Analysis of the angular
dependence of the frequency response yields invaluable directivity information about the detector under study,
which in turn permits accurate forward and inverse problem models.
We demonstrated the ability to detect surface antigens, associated with pathogens, utilizing laser optoacoustic
spectroscopy with antibody-coupled gold nanorods (GNR) as a contrast agent specifically targeted to the antigen of
interest. The sensitivity of the technique has been assessed by determining the minimum detectable concentration of a
surface antigen from biological samples. We compared the sensitivity and applicability of two different methods for
detecting optoacoustic responses, using either optical or piezoelectric sensors. Biological samples were adsorbed to the
inside walls of detachable, flat-bottomed plastic micro-wells, and then probed with appropriate antibodies conjugated
with gold nanorods. If the target antigens were present, the antibody-nanoparticle conjugates were bound, while any
nonadsorbed nanoparticles were washed out of the wells. Optoacoustic signals were generated from the bound nanorods
using a pulsed pump laser at wavelengths corresponding to one of the peak absorptions of the nanorods. Optoacoustic
responses were obtained from the samples using both detection modalities. The sensitivity, suitability, ease of use of
each method were assessed and compared. Initial results indicate that optical detection gives comparable sensitivity as
the piezoelectric method, and further enhancement of the detection sensitivity is possible for both methods. An
advantage of the piezoelectric detection method is that it may be implemented in a more compact assembly, compared to
the optical method, however, the optical method may be less sensitive to external electromagnetic and acoustic noise.
Further evaluation will be required to refine these measurements. The results with both methods indicate that the use of
antibody-targeted nanorod contrast agents, with laser-optoacoustic detection, is a promising technology for the
development of rapid in vitro diagnostic tests.
We developed a 3D whole-body optoacoustic tomography system for applications in preclinical research on mice. The
system is capable of generating images with resolution better than 0.6 mm. Two pulsed lasers, an Alexandrite laser
operating at 755 nm and a Nd:YAG laser operating at 532 nm and 1064nm were used for light delivery. The
tomographic images were obtained while the objects of study (phantoms or mice) were rotated within a sphere outlined
by a concave arc-shaped array of 64 piezo-composite transducers. During the scan, the mouse was illuminated
orthogonally to the array with two wide beams of light from a bifurcated fiber bundle. Illumination at 532 nm showed
superficial vasculature, but limited penetration depth at this wavelength prevented the detection of deeper structures.
Illumination at 755 and 1064 nm showed organs and blood vessels, respectively. Filtering of the optoacoustic signals
using high frequency enhancing wavelets further emphasized the smaller blood vessels.
We describe two laser optoacoustic imaging systems for breast cancer detection based on arrays of acoustic detectors
operated manually in a way similar to standard ultrasonic breast imaging. The systems have the advantages of standard
light illumination (regardless of the interrogated part of the breast), the ability to visualize any part of the breast, and
convenience in operation. The first system could work in both ultrasonic and optoacoustic mode, and was developed
based on a linear ultrasonic breast imaging probe with two parallel rectangular optical bundles. We used it in a pilot
clinical study to provide for the first time demonstration that the boundaries of the tumors visualized on the optoacoustic
and ultrasonic images matched. Such correlation of coregistered images proves that the objects on both images
represented indeed the same tumor. In the optoacoustic mode we were also able to visualize blood vessels located in the
neighborhood of the tumor. The second system was proposed as a circular array of acoustic transducers with an axisymmetric
laser beam in the center. It was capable of 3D optoacoustic imaging with minimized optoacoustic artifacts
caused by the distribution of the absorbed optical energy within the breast tissue. The distribution of optical energy
absorbed in the bulk tissue of the breast was removed from the image by implementing the principal component analysis
on the measured signals. The computer models for optoacoustic imaging using these two handheld probes were
developed. The models included three steps: (1) Monte Carlo simulations of the light distribution within the breast
tissue, (2) generation of optoacoustic signals by convolving
N-shaped pressure signals from spherical voxels with the
shape of individual transducers, and (3) back-projecting processed optoacoustic signals onto spherical surfaces for image
reconstruction. Using the developed models we demonstrated the importance of the included spatial impulse response of
the optoacoustic imaging system.
We developed an improved signal and image processing of optoacoustic data collected by our laser optoacoustic
imaging system designed for breast cancer detection (LOIS-B). The implemented wavelet-based signal processing
allowed significant reduction of the low-frequency acoustic noise, improved contrast and localization of the optoacoustic
sources of interest. The system was able to differentiate phantoms mimicking breast tumors based on the contrast and
morphology of their images. The implemented wavelet-based signal processing also facilitated high (0.5 mm) resolution
of the phantoms mimicking parallel blood vessels in the presence of large-amplitude low-frequency acoustic artifacts.
The application of the 3D radial back projection image reconstruction algorithm allowed visulaization of the tumor
phantoms located beyond the imaging slice of the arc-shaped array of transducers. The visualization of slices parallel to
the array of transducers with individual adjustment of the image palette for each particular slice eliminated the image
artifacts caused by the large gradient of the laser fluence orthogonal to the array of transducers.
A three-dimensional laser optoacoustic imaging system was developed, which combines the advantages of optical
spectroscopy and high resolution ultrasonic detection, to produce high contrast maps of optical absorbance in tissues.
This system was tested in a nude mouse model of breast cancer and produced tissue images of tumors and vasculature.
The imaging can utilize either optical properties of hemoglobin and oxyhemoglobin, which are the main endogenous
tissue chromophores in the red and near-infrared spectral ranges, or exogenous contrast agent based on gold nanorods.
Visualization of tissue molecules targeted by the contrast agent provides molecular information. Visulization of blood at
multiple colors of light provides functional information on blood concentration and oxygen saturation. Optoacoustic
imaging, using two or more laser illumination wavelengths, permits an assessment of the angiogenesis-related
microvasculature, and thereby, an evaluation of the tumor stage and its metastatic potential.
The optoacoustic imaging system was also used to generate molecular images of the malignancy-related receptors
induced by the xenografts of BT474 mammary adenocarcinoma cells in nude mice. The development of the latter images
was facilitated by the use of an optoacoustic contrast agent that utilizes gold nanorods conjugated to monoclonal
antibody raised against HER2/neu antigens. These nanorods possess a very strong optical absorption peak that can be
tuned in the near-infrared by changing their aspect ratio. The effective conversion of the optical energy into heat by the
gold nanorods, followed by the thermal expansion of the surrounding water, makes these nanoparticles an effective
optoacoustic contrast agent. Optical scattering methods and x-ray tomography may also benefit from the application of
this contrast agent. Administration of the gold nanorod bioconjugates to mice resulted in an enhanced contrast of breast
tumors relative the background of normal tissues in the nude mouse model. The combination of this novel contrast agent
and optoacoustic imaging has the potential to become a useful imaging modality, for preclinical research in murine
models of cancer and other human diseases.
Ultrasound imaging is the current gold standard for guiding biopsy of prostate. Optoacoustic imaging yields higher
contrast in detection of malignant tissues. The two techniques provide complementary information. We are currently
developing a hybrid laser optoacoustic and ultrasound imaging system for prostate tumor detection (LOUIS-P). The
optoacoustic part consists of a fiber-coupled Q-switched laser operating at either 757 nm or 1064 nm attached to a
commercially-available 128-channel ultrasonic probe modified for optimal detection of optoacoustic signals, a digital
signal processor with 128 independent channels, and software that uses the radial (filtered) backprojection algorithm to
reconstruct tomographic images. We evaluated system-imaging performance using test objects submerged in milky
water, and poly(vinyl-chloride) plastisol tissue phantoms simulating malignant lesions. LOUIS-P demonstrates potential
as a clinical technique for minimally invasive imaging and diagnosis of prostate cancer.
We have designed, fabricated and tested a new laser optoacoustic imaging system (LOIS-64/16) for quantitative
optoacoustic tomography of breast cancer. The system was designed to create a single slice of an optoacoustic image of
the breast with 64 ultrawide band acoustic transducers. Other 16 transducers on the back of the acoustic probe were used
to reconstruct the light distribution inside the breast. The system resolution was at least 0.5 mm for high-aspect-ratio
objects. Maximum system sensitivity was 4.8 mV/Pa and the RMS noise of 3.1 mV, which allowed imaging of small
(less than 1 cm) tumors at depths over 3 cm. The directivity of the optoacoustic transducers used in LOIS-64/16 assured
that the signal detection was better than 70% of the maximum for about 75% of the imaging slice and reduced quickly
for signals coming from out of the imaging slice. Implemented signal processing allowed significant reduction of the
low-frequency acoustic noise and localizing the small OA signals. The system was able to differentiate phantoms
mimicking tumors and malformations visualized in clinics based on the contrast and morphology of their images
obtained at 1064 nm and 757 nm.
We have designed, fabricated and tested a new 128-channel laser optoacoustic imaging system (LOIS-128) for cancer
diagnostics consisting of an acoustic probe with 128 PVDF transducers, a digital signal processor with 128 independent
channels, and software for reconstruction of optoacoustic images. The system was capable of continuous planar imaging
(at rates up to 1 Hz) of small (less than 1 cm) tumors at depths over 6 cm. The directivity of the optoacoustic transducers
used in LOIS-128 assured that signal detection at all angles within a narrow imaging slice was at least 40% of the
maximal signal. The signal detection was better than 70% of the maximum for about 75% of the image in the image
slice and close to zero for signals coming from out of the image slice. LOIS-128 could image high-aspect-ratio objects
with about 0.5 mm resolution. Finally, with the designed image reconstruction algorithm we were able to estimate
absorption coefficients for test objects with accuracy of at least 5%.
A contrast agent for optoacoustic imaging and laser therapy of early tumors is being developed based on gold
nanocolloids strongly absorbing visible and near-infrared light. The optoacoustic signals obtained from gold nanospheres
and gold nanorods solutions are studied. In the case of 100 nm nanospheres as an example, a sharp increase in the total
area under the curve of the optoacoustic signal is observed when the laser fluence is increased beyond a threshold value
of about 0.1 J/cm2. The change in the optoacoustic signal profile is attributed to the formation of water vapor bubbles
around heated nanoparticles, as evidenced via thermoacoustic microscopy experiments. It has been determined that,
surprisingly, gold nanoparticles fail to generate detectable nanobubbles upon irradiation at the laser fluence of ~2
mJ/cm2, which heats the nanoparticles up to 374°C, the critical temperature of water. Only when the estimated
temperature of the particle reaches about 10,000°C, a marked increase of the optoacoustic pressure amplitude and a
changed profile of the optoacoustic signals indicate nanobubble formation. A nanoparticle based contrast agent is the
most effective if it can be activate by laser pulses with low fluence attainable in the depth of tissue. With this goal in
mind, we develop targeting protocols that form clusters of gold nanocolloid in the target cells in order to lower the
bubble formation threshold below the level of optical fluence allowed for safe laser illumination of skin. Experiments
and modeling suggest that formation of clusters of nanocolloids may improve the sensitivity of optoacoustic imaging in
the detection of early stage tumors.
We have developed and used a laser optoacoustic imaging system with transrectal probe (LOIS-P) for detection of
mechanical lesions in canine prostates in vivo. LOIS images have been acquired with a 128-channel transrectal probe
and a 32-channel data acquisition system. Optoacoustic images showed a strong contrast enhancement for a blood
containing lesion, when compared with ultrasound images. Our studies demonstrated that sufficient optoacoustic
contrast exists between blood containing lesion and prostate tissue, although the lesion has been undetectable with
ultrasound. The imaging results have been compared with visual examination of surgically excised prostates. Although
axial resolution of the wide-band transducers employed in the transrectal probe provides good axial resolution of 0.5
mm, the convex arc geometry of the this array of transducers provides lateral resolution degrading with depth in tissue.
A two step algorithm has been developed to improve the lateral resolution of deeply located objects. This algorithm
employs optoacoustic image reconstruction based on radial
back-projection to determine location and shape of the target
object, then a procedure, we call Maximum Angular Amplitude Probability (MAAP), to determine true brightness of the
object and simultaneously remove arc-shaped artifacts associated with radial back-projection. A laser optoacoustic
imaging system (LOIS-P) with transrectal probe operating in backward detection mode empowered with the new image
reconstruction algorithm seems promising as a modality for detection of prostate cancer and guiding prostate biopsy.
Information on plasma membrane (PM) and cell wall mechanical properties is important for many biophysical applications, especially for those, which involve cells, undergoing significant mechanical stress (red blood cells, outer hair cells, fibrocytes, etc.). Optical tweezers is frequently used to study PM mechanics, particularly by pulling long PM tethers. One of the limitations on using optical tweezers to study cell wall mechanics is associated with transillumination technique of the trapped object position sensing, which prevents accurate mechanical testing in the proximity to the cell. In this work we use an optical tweezers in conjunction with a position-sensing system, which spectrally separates signals from the trapped fluorescent microsphere and imaging background. We have used this setup to study mechanics of the cell wall and PM separated from the underlying cytoskeleton on human embryonic kidney cells. We measured the force exerted by the cell on the trapped microsphere as a function of the cell wall displacement during the process of tether formation, and as a function of time during the process of tether growth and relaxation. Tethering force - cell wall displacement profiles have shown a behavior, implying that tether formation process starts with elastic deformation of the intact cell wall, followed by the plastic deformations and sliding of the PM over the underlying cytoskeleton, and ends with the local separation of a PM. Tethering force - cell wall displacement profiles have been used to estimate tether formation force, stiffness parameter of the cell wall and the works of tether formation, elastic and plastic deformations of the cell wall, related to the mechanical properties of a composite cell wall and cell wall - plasma membrane association strength. Temporal steady-state and relaxation tethering force profiles have been similar to the ones measured using transillumination position sensing, however average force values have been smaller in our case, due to the methodological differences. Our results demonstrate that measurements of cell wall and PM mechanical properties using optically-trapped fluorescent microspheres presents a versatile technology for studying of the cellular mechanics, especially effective in the proximity of the trapped microsphere to the cell.
The plasma membrane (PM) of mammalian outer hair cells (OHCs) generates mechanical forces in response to changes in the transmembrane electrical potential. The resulting change in the cell length is known as electromotility. Salicylate (Sal), the anionic, amphipathic derivative of aspirin induces reversible hearing loss and decreases electromotile response of the OHCs. Sal may change the local curvature and mechanical properties of the PM, eventually resulting in reduced electromotility or it may compete with intracellular monovalent anions, particularly Cl-, which are essential for electromotility. In this work we have used optical tweezers to study the effects of Sal on viscoelastic properties of the OHC PM when separated from the underlying composite structures of the cell wall. In this procedure, an optically trapped microsphere is brought in contact with PM and subsequently pulled away to form a tether. We measured the force exerted on the tether as a function of time during the process of tether growth at different pulling rates. Effective tether viscosity, steady-state tethering force extrapolated to zero pulling rate, and the time constant for tether growth were estimated from the measurements of the instantaneous tethering force. The time constant for the tether growth measured for the OHC basal end decreased 1.65 times after addition of 10 mM Sal, which may result from an interaction between Sal and cholesterol, which is more prevalent in the PM of OHC basal end. The time constants for the tether growth calculated for the OHC lateral wall and control human embryonic kidney cells as well as the other calculated viscoelastic parameters remained the same after Sal perfusion, favoring the hypothesis of competitive inhibition of electromotility by salicylate.
An optical tweezers system was used to study the mechanical characteristics of the outer hair cell (OHC) lateral wall by forming plasma membrane tethers. A 2nd order generalized Kelvin model was applied to describe the viscoelastic behavior of OHC membrane tethers. The measured parameters included equilibrium tethering force, (Feq), force relaxation times (τ), stiffness values (κ), and coefficients of friction (μ). An analysis of force relaxation in membrane tethers indicated that the force decay is a biphasic process containing both an elastic and a viscous phase. In general, we observed an overall negative trend in the measured parameters upon application of the cationic amphipath chlorpromazine (CPZ). CPZ was found to cause up to a 40 pN reduction in Feq in OHCs. A statistically significant reduction in relaxation times and coefficients of friction was also observed, suggesting an increase in rate of force decay and a decrease in plasma membrane viscosity.
Outer hair cells contribute an active mechanical feedback to the vibrations of the cochlear structures resulting in the high sensitivity and frequency selectivity of normal hearing. We have designed and implemented a novel experimental setup that combines optical tweezers with patch-clamp apparatus to investigate the electromechanical properties of cellular plasma membranes. A micron-size bead trapped by the optical tweezers is brought in contact with the membrane of a voltage-clamped cell, and subsequently moved away to form a plasma membrane tether. Bead displacement during tether elongation is monitored by a quadrant photodetector to obtain time-resolved measurements of the tethering force. Salient information associated with the mechanical properties of the membrane tether can thus be obtained. Tethers can be pulled from the cell membrane at different holding potentials, and the tether force response can be measured while changing transmembrane potential. Experimental results from outer hair cells and human embryonic kidney cells are presented.
An optical tweezers system was used to study the mechanical characteristics of outer hair cell (OHC) and human embryonic kidney (HEK) cell plasma membranes. The effect of the cationic amphipath chlorpromazine (CPZ) on the equilibrium tethering force, (Feq) force relaxation time constant,(τ) and effective membrane viscosity (ηeff) was measured. The Feq for the OHC lateral wall plasma membrane was ~60 pN and was unchanged by addition of CPZ. A significantly greater τ value was observed in CPZ-treated OHCs (30.5 ± 12.6 s) than in control OHCs (19.0 ± 13.2 s). The Feq and τ values for control HEK cells were >60% lower than the respective OHC values but increased by ~3 times following CPZ addition. Effective viscosity ranged between 1.49-1.81 pN•s/μm for CPZ-treated OHCs. This represents a decrease from reported control OHC membrane viscosities.
In this work we analyzed the calibration of optical trapping forces. One calibration technique utilizes the controlled motion of a trapped object in a fluid with known viscosity where the trapping force is calculated from the Stokes’ Law based on inertia-free assumptions (i.e., neglecting velocity and acceleration of the trapped object). In our study, we calculated the displacement of the trapped object from the trapping center using Fourier analysis of the equation of motion. Waveforms of different frequencies were used both in theoretical modeling and experiments to control the motion of the trapped object in an aqueous solution. Calibration data obtained experimentally were compared to theoretical results. The dynamic analysis of the trapped object showed that trapping force can significantly differ from theoretically predicted values under inertia-free assumptions. Various factors including type of the waveform used to control the motion of a trapped object during calibration, its frequency, viscosity of the calibration fluid, mass and dimensions of the trapped object, stiffness of the optical trap and frequency response of the equipment used to control the motion of the trapped object contribute to the differences.
The experimental evidences for photochemical mechanism of light activation of immune system are presented. The experiments were made in vitro using serums from normal blood, from blood of people with secondary immunodeficiency containing low-avidity IgG (up to 75 - 90%), and pure IgG buffer solutions obtained from the same serums. LED arrays and halogen lamps with narrow spectral filters from UV to IR were used as optical monochromatic sources. We have discovered that light can activate immune system by IgG transformation from low-avidity state to high-avidity one. This change has multistage irreversible character and depends on light wavelength and intensity. The estimations of optical effects were made by determination of IgG functional activity and quaternary antibody structure through the number of accessible functional protein residues in the IgG buffer solutions. Both methods showed very correlated results. Each IgG avidity transformation stage correlated with definite change of antibody spatial structure that, to our opinion, corresponds to the phenomenon pass through the several intermediate metastable forms, which are maintained by different intermolecular bonds. The bioaction spectrum of discovered effects is also presented.
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