Skeletonization algorithm-based blood vessel quantification using in vivo 3D photoacoustic imaging.

Blood vessels are the only system to provide nutrients and oxygen to every part of the body. Many diseases can have significant effects on blood vessel formation, so that the vascular network can be a cue to assess malicious tumor and ischemic tissues. Various imaging techniques can visualize blood vessel structure, but their applications are often constrained by either expensive costs, contrast agents, ionizing radiations, or a combination of the above. Photoacoustic imaging combines the high-contrast and spectroscopic-based specificity of optical imaging with the high spatial resolution of ultrasound imaging, and image contrast depends on optical absorption. This enables the detection of light absorbing chromophores such as hemoglobin with a greater penetration depth compared to purely optical techniques. We present here a skeletonization algorithm for vessel architectural analysis using non-invasive photoacoustic 3D images acquired without the administration of any exogenous contrast agents. 3D photoacoustic images were acquired on rats (n = 4) in two different time points: before and after a burn surgery. A skeletonization technique based on the application of a vesselness filter and medial axis extraction is proposed to extract the vessel structure from the image data and six vascular parameters (number of vascular trees (NT), vascular density (VD), number of branches (NB), 2D distance metric (DM), inflection count metric (ICM), and sum of angles metric (SOAM)) were calculated from the skeleton. The parameters were compared (1) in locations with and without the burn wound on the same day and (2) in the same anatomic location before (control) and after the burn surgery. Four out of the six descriptors were statistically different (VD, NB, DM, ICM, p < 0.05) when comparing two anatomic locations on the same day and when considering the same anatomic location at two separate times (i.e. before and after burn surgery). The study demonstrates an approach to obtain quantitative characterization of the vascular network from 3D photoacoustic images without any exogenous contrast agent which can assess microenvironmental changes related to disease progression.

Sensing the delivery and endocytosis of nanoparticles using magneto-photo-acoustic imaging.

Many biomedical applications necessitate a targeted intracellular delivery of the nanomaterial to specific cells. Therefore, a non-invasive and reliable imaging tool is required to detect both the delivery and cellular endocytosis of the nanoparticles. Herein, we demonstrate that magneto-photo-acoustic (MPA) imaging can be used to monitor the delivery and to identify endocytosis of magnetic and optically absorbing nanoparticles. The relationship between photoacoustic (PA) and magneto-motive ultrasound (MMUS) signals from the in vitro samples were analyzed to identify the delivery and endocytosis of nanoparticles. The results indicated that during the delivery of nanoparticles to the vicinity of the cells, both PA and MMUS signals are almost linearly proportional. However, accumulation of nanoparticles within the cells leads to nonlinear MMUS-PA relationship, due to non-linear MMUS signal amplification. Therefore, through longitudinal MPA imaging, it is possible to monitor the delivery of nanoparticles and identify the endocytosis of the nanoparticles by living cells.

A Dual Gold Nanoparticle System for Mesenchymal Stem Cell Tracking.

Stem cell-based therapies have demonstrated improved outcomes in preclinical and clinical trials for treating cardiovascular ischemic diseases. However, the contribution of stem cells to vascular repair is poorly understood. To elucidate these mechanisms, many have attempted to monitor stem cells following their delivery in vivo, but these studies have been limited by the fact that many contrast agents, including nanoparticles, are commonly passed on to non-stem cells in vivo. Specifically, cells of the reticuloendothelial system, such as macrophages, frequently endocytose free contrast agents, resulting in the monitoring of macrophages instead of the stem cell therapy. Here we demonstrate a dual gold nanoparticle system which is capable of monitoring both delivered stem cells and infiltrating macrophages using photoacoustic imaging. In vitro analysis confirmed preferential labeling of the two cell types with their respective nanoparticles and the maintenance of cell function following nanoparticle labeling. In addition, delivery of the system within a rat hind limb ischemia model demonstrated the ability to monitor stem cells and distinguish and quantify macrophage infiltration. These findings were confirmed by histology and mass spectrometry analysis. This work has important implications for cell tracking and monitoring cell-based therapies.

Enhanced pulsed magneto-motive ultrasound imaging using superparamagnetic nanoclusters.

Recently, pulsed magneto-motive ultrasound (pMMUS) imaging augmented with ultra-small magnetic nanoparticles has been introduced as a tool capable of imaging events at molecular and cellular levels. The sensitivity of a pMMUS system depends on several parameters, including the size, geometry and magnetic properties of the nanoparticles. Under the same magnetic field, larger magnetic nanostructures experience a stronger magnetic force and produce larger displacement, thus improving the sensitivity and signal-to-noise ratio (SNR) of pMMUS imaging. Unfortunately, large magnetic iron-oxide nanoparticles are typically ferromagnetic and thus are very difficult to stabilize against colloidal aggregation. In the current study we demonstrate improvement of pMMUS image quality by using large size superparamagnetic nanoclusters characterized by strong magnetization per particle. Water-soluble magnetic nanoclusters of two sizes (15 and 55 nm average size) were synthesized from 3 nm iron precursors in the presence of citrate capping ligand. The size distribution of synthesized nanoclusters and individual nanoparticles was characterized using dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM). Tissue mimicking phantoms containing single nanoparticles and two sizes of nanoclusters were imaged using a custom-built pMMUS imaging system. While the magnetic properties of citrate-coated nanoclusters are identical to those of superparamagnetic nanoparticles, the magneto-motive signal detected from nanoclusters is larger, i.e. the same magnetic field produced larger magnetically induced displacement. Therefore, our study demonstrates that clusters of superparamagnetic nanoparticles result in pMMUS images with higher contrast and SNR.

The influence of viscosity on the shear strain remotely induced by focused ultrasound in viscoelastic media.

Shear wave elasticity imaging (SWEI), an emerging acoustic technology for medical diagnostics, is based on remote generation of shear waves in tissue by radiation force in the focal region of an ultrasonic beam. In this study, the feasibility of Doppler ultrasonic technique to visualize the remotely induced shear waves was demonstrated. The generation of shear displacement in the focal region of a pulsed 1-MHz ultrasound beam with pulse duration of approximately about 2 ms and intensity levels on the order of 145 W/cm2, and consequent propagation of shear wave in tissue-mimicking and muscle tissue in vitro, were measured. The analysis of temporal behavior of shear displacement within the focal plane allowed estimation of shear wave velocities. The velocities were 4 and 7 m/s in hard phantom and tissue containing phantom, respectively. The measured shear displacements on the order of micrometers in gel-based phantoms are in reasonable agreement with theoretical estimates derived from an earlier developed model of shear wave generation by radiation force of focused ultrasound. The study revealed significant dependence of shear strain on the medium viscosity. The complex oscillatory character of shear strain relaxation in viscoelastic phantom and muscle tissue in vitro was observed.

Doppler ultrasound detection of shear waves remotely induced in tissue phantoms and tissue in vitro.

In shear wave elasticity imaging (SWEI), mechanical excitation within the tissue is remotely generated using radiation force of focused ultrasound. The induced shear strain is subsequently detected to estimate visco-elastic properties of tissue and thus aid diagnostics. In this paper, the mechanical response of tissue to radiation force was detected using a modified ultrasound Doppler technique. The experiments were performed on tissue mimicking and tissue containing phantoms using a commercial diagnostic scanner. This scanner was modified to control both the pushing and probing beams. The pushing beam was fired repetitively along a single direction while interlaced probing beams swept the surrounding region of interest to detect the induced motion. The detectability of inhomogeneous inclusions using ultrasonic Doppler SWEI method has been demonstrated in this study. The displacement fields measured in elastic phantoms clearly reveal the oscillatory nature of the mechanical relaxation processes in response to impulsive load due to the boundary effects. This relaxation dynamics was also present in cooked muscle tissue, but was not detected in more viscous and less elastic phantom and raw muscles. Presence of a local heterogeneity in the vicinity of the focal region of the pushing beam results in generation of a standing wave field pattern which is manifested in the oscillatory response of the excited region of the tissue. There has been made an assumption that dynamic characteristics of the relaxation process may be used for visualization of inhomogeneities.

Triplex ultrasound: elasticity imaging to age deep venous thrombosis.

Deep venous thrombosis (DVT), and its sequela, pulmonary embolism (PE), is the leading cause of preventable in-hospital mortality in the USA and other developed countries. After it is detected, acute clots must be differentiated from chronic DVT for appropriate treatment. However, there are no reliable thrombus staging methods presently available in clinical practice. In this study, we tested the hypothesis that blood clots can be detected and staged using a triplex ultrasound (US) test. Triplex US is based on a "gold standard" duplex US technique augmented by US-based reconstructive elasticity imaging. Fibrin-composed blood clots harden with development and organization. By imaging clot elasticity, it may be possible to both detect and differentiate clots and, therefore, provide an urgently needed noninvasive means of DVT staging.

Strain rate imaging using two-dimensional speckle tracking.

Strain rate images (SRI) of the beating heart have been proposed to identify non-contracting regions of myocardium. Initial attempts used spatial derivatives of tissue velocity (Doppler) signals. Here, an alternate method is proposed based on two-dimensional phase-sensitive speckle tracking applied to very high frame rate, real-time images. This processing can produce high resolution maps of the time derivative of the strain magnitude (i.e., square root of the strain intensity). Such images complement traditional tissue velocity images (TVI), providing a more complete description of cardiac mechanics. To test the proposed approach, SRI were both simulated and measured on a thick-walled, cylindrical, tissue-equivalent phantom modeling cardiac deformations. Real-time ultrasound images were captured during periodic phantom deformation, where the period was matched to the data capture rate of a commercial scanner mimicking high frame rate imaging of the heart. Simulation results show that SRI with spatial resolution between 1 and 2 mm are possible with an array system operating at 5 MHz. Moreover, these images are virtually free of angle-dependent artifacts present in TVI and simple strain rate maps derived from these images. Measured results clearly show that phantom regions of low deformation, which are difficult to identify on tissue velocity-derived SRI, are readily apparent with SRI generated from two-dimensional phase-sensitive speckle tracking.

Three-dimensional static displacement, stimulated echo NMR elasticity imaging.

This article presents a method for measuring three-dimensional mechanical displacement and strain fields using stimulated echo MRI. Additional gradient pulses encode internal displacements in response to an externally applied deformation. By limiting the mechanical transition to the stimulated echo mixing time, a more accurate static displacement measurement is obtained. A three-dimensional elasticity reconstruction within a region of interest having a uniform shear modulus along its boundary is performed by numerically solving discretized elasticity equilibrium equations. Data acquisition, strain measurements and reconstruction were performed using a silicone gel phantom containing an inclusion of known elastic properties. A comparison between two-dimensional and three-dimensional reconstructions from simulated and experimental displacement data shows higher accuracy from the three-dimensional reconstruction. The long-term objective of this work is to provide a method for remotely palpating and elastically quantitating manually inaccessible tissues.

High-resolution elasticity imaging for tissue engineering.

An elasticity microscope provides high resolution images of tissue elasticity. With this instrument, it may be possible to monitor cell growth and tissue development in tissue engineering. To test this hypothesis, elasticity micrographs were obtained in two model systems commonly used for tissue engineering. In the first, strain images of a tissue-engineered smooth muscle sample clearly identified a several hundred micron thick cell layer from its supporting matrix. Because a one-dimensional mechanical model was appropriate for this system, strain images alone were sufficient to image the elastic properties. In contrast, a second system was investigated in which a simple one-dimensional mechanical model was inadequate. Uncultured collagen microspheres embedded in an otherwise homogeneous gel were imaged with the elasticity microscope. Strain images alone did not clearly depict the elastic properties of the hard spherical cell carriers. However, reconstructed elasticity images could differentiate the hard inclusion from the background gel. These results strongly suggest that the elasticity microscope may be a valuable tool for tissue engineering and other applications requiring the elastic properties of soft tissue at high spatial resolution (75 microm or less).

Reconstructive ultrasound elasticity imaging for renal transplant diagnosis: kidney ex vivo results.

It may be possible to diagnose and monitor scarring, inflammation and edema in transplant kidney using reconstructive ultrasound elasticity imaging. Kidney elasticity is expected to change dramatically with scar, and to a lesser degree, with acute inflammation and edema. The hypothesis that changes in kidney elasticity can be imaged using a clinical ultrasound scanner was experimentally tested with an ex vivo canine kidney model, and results on a single pair of kidneys are reported in this paper. A cross-linking agent affected kidney elasticity both globally and locally. Elasticity changes were monitored with accurate estimates of internal displacement and strain followed by Young's modulus reconstruction. The results of this study strongly suggest that ultrasound elasticity imaging can detect elasticity changes in complex structures such as the kidney. Moreover, it has the potential to become an important clinical tool for renal transplant diagnosis.

Speckle tracking methods for ultrasonic elasticity imaging using short-time correlation.

In ultrasound elasticity imaging, strain decorrelation is a major source of error in displacements estimated using correlation techniques. This error can be significantly decreased by reducing the correlation kernel. Additional gains in signal-to-noise ratio (SNR) are possible by filtering the correlation functions prior to displacement estimation. Tradeoffs between spatial resolution and estimate variance are discussed, and estimation in elasticity imaging is compared to traditional time-delay estimation. Simulations and experiments on gel-based phantoms are presented. The results demonstrate that high resolution, high SNR strain estimates can be computed using small correlation kernels (on the order of the autocorrelation width of the ultrasound signal) and correlation filtering.

Adaptive strain estimation using retrospective processing [medical US elasticity imaging.

Because errors in displacement and strain estimates depend on the magnitude of the induced strain, the strain signal-to-noise ratio (SNR) will be a function of the applied deformation. If deformation is applied at the body surface, it is difficult during data acquisition to select a single surface displacement providing the highest strain SNR throughout the image. By applying continuous deformation and capturing data in real-time, the surface displacement providing the highest strain SNR can be selected retrospectively. A method to adaptively optimize strain SNR over the image plane using retrospective processing is presented and demonstrated with experimental results.

Reconstructive elasticity imaging for large deformations.

A method is presented to reconstruct the elastic modulus of soft tissue based on ultrasonic displacement and strain images for comparatively large deformations. If the average deformation is too large to be described with a linear elastic model, nonlinear displacement-strain relations must be used and the mechanical equilibrium equations must include high order spatial derivatives of the displacement. Numerical methods were developed to reduce error propagation in reconstruction algorithms, including these higher order derivatives. Problems arising with the methods, as well as results using ultrasound measurements on gel-based, tissue equivalent phantoms, are given. Comparison to reconstructions using a linear elastic model shows that equivalent image quality can be produced with algorithms appropriate for finite amplitude deformations.

Measuring the elastic modulus of small tissue samples.

Independent measurements of the elastic modulus (Young's modulus) of tissue are necessary step in turning elasticity imaging into a clinical tool. A system capable of measuring the elastic modulus of small tissue samples was developed. The system tolerates the constraints of biological tissue, such as limited sample size (< or = 1.5 cm3) and imperfections in sample geometry. A known deformation is applied to the tissue sample while simultaneously measuring the resulting force. These measurements are then converted to an elastic modulus, where the conversion uses prior calibration of the system with plastisol samples of known Young's modulus. Accurate measurements have been obtained from 10 to 80 kPa, covering a wide range of tissue modulus values. In addition, the performance of the system was further investigated using finite element analysis. Finally, preliminary elasticity measurements on canine kidney samples are presented and discussed.

Elasticity reconstructive imaging by means of stimulated echo MRI.

A method is introduced to measure internal mechanical displacement and strain by means of MRI. Such measurements are needed to reconstruct an image of the elastic Young's modulus. A stimulated echo acquisition sequence with additional gradient pulses encodes internal displacements in response to an externally applied differential deformation. The sequence provides an accurate measure of static displacement by limiting the mechanical transitions to the mixing period of the simulated echo. Elasticity reconstruction involves definition of a region of interest having uniform Young's modulus along its boundary and subsequent solution of the discretized elasticity equilibrium equations. Data acquisition and reconstruction were performed on a urethane rubber phantom of known elastic properties and an ex vivo canine kidney phantom using <2% differential deformation. Regional elastic properties are well represented on Young's modulus images. The long-term objective of this work is to provide a means for remote palpation and elasticity quantitation in deep tissues otherwise inaccessible to manual palpation.

Nonlinear estimation of the lateral displacement using tissue incompressibility.

Using the incompressibility property of soft tissue, high quality lateral displacement distributions can be reconstructed from accurate axial displacement measurements and noisy lateral displacement estimates. Previous methods appropriate for small deformations have been extended for high magnitude deformations requiring a nonlinear model. Problems arising in incompressibility processing for large deformations are considered. Applications of nonlinear incompressibility methods to ultrasonic measurements on gel-based, tissue equivalent phantoms are given. Lateral displacement images reconstructed with nonlinear methods are compared to those reconstructed with linear methods for both small and large deformations.

Shear wave elasticity imaging: a new ultrasonic technology of medical diagnostics.

Shear wave elasticity imaging (SWEI) is a new approach to imaging and characterizing tissue structures based on the use of shear acoustic waves remotely induced by the radiation force of a focused ultrasonic beam. SWEI provides the physician with a virtual "finger" to probe the elasticity of the internal regions of the body. In SWEI, compared to other approaches in elasticity imaging, the induced strain in the tissue can be highly localized, because the remotely induced shear waves are attenuated fully within a very limited area of tissue in the vicinity of the focal point of a focused ultrasound beam. SWEI may add a new quality to conventional ultrasonic imaging or magnetic resonance imaging. Adding shear elasticity data ("palpation information") by superimposing color-coded elasticity data over ultrasonic or magnetic resonance images may enable better differentiation of tissues and further enhance diagnosis. This article presents a physical and mathematical basis of SWEI with some experimental results of pilot studies proving feasibility of this new ultrasonic technology. A theoretical model of shear oscillations in soft biological tissue remotely induced by the radiation force of focused ultrasound is described. Experimental studies based on optical and magnetic resonance imaging detection of these shear waves are presented. Recorded spatial and temporal profiles of propagating shear waves fully confirm the results of mathematical modeling. Finally, the safety of the SWEI method is discussed, and it is shown that typical ultrasonic exposure of SWEI is significantly below the threshold of damaging effects of focused ultrasound.

Strain imaging of coronary arteries with intraluminal ultrasound: experiments on an inhomogeneous phantom.

In coronary arteries, knowing the relative stiffness of atherosclerotic lesions can help physicians select the most appropriate therapeutic modality. Because soft material supports larger strains than hard, measurements of this quantity can distinguish tissue of differing stiffness. In a previous paper, we described techniques for computing displacements and strains in coronary arteries using an integrated angioplasty and imaging catheter. Here, we demonstrate that hard and soft materials in a tissue-mimicking phantom can be differentiated with this device. Because tissue motion cannot be distinguished from catheter motion a priori, we perform all computations in the coordinate system centered at the balloon's geometric center. This reference frame depends only on balloon shape and is independent of catheter motion. A specialized correlation-based, phase-sensitive speckle tracking algorithm has been developed to compute strain. Maximum phantom displacement was about 25 microns, and the maximum radial, normal strain was about 1.5 percent.

Magnetic-resonance imaging techniques for detection of elasticity variation.

The relative success of manual palpation in the detection of breast cancer would suggest that a method for remote palpation resulting in a measurement of tissue elasticity could provide a diagnostic tool for detecting cancerous lesions deeper within the breast. This presumption is based in part on the excellent contrast between neoplastic and normal tissue due to the large (orders of magnitude) relative variation in the shear elastic modulus. By comparison, the bulk deformational modulus maintains the same value to within 20% for most soft tissues. A specific method of magnetic-resonance imaging (MRI) which measures tissue displacements has been used in experiments with a phantom containing regions of increased Young's modulus as a demonstration. The spatial modulation of magnetization technique uses the displacement of a spatial grid pattern caused by spin saturation to track regional motion. Mathematical reconstruction of the distribution of elastic moduli is shown for select examples. Any modality, e.g., MRI, ultrasound, etc., which can detect local tissue motion with sufficient spatial resolution can be used and therefore the results presented here should give an indication of the utility of such motion tracking techniques to future measurement of tissue elasticity.