Characterizing Mechanical Properties of Soft Tissues Using Non-contact Displacement Measurements: How Should We Assess the Uncertainty?

Techniques aimed at the non-invasive characterization of soft tissues according to elastic properties are rapidly evolving. Virtual touch-based elastographic methods including acoustic radiation force imaging (ARFI) and optical elastography measure the peak axial displacement (PD) and time-to-peak-displacement (TTP) of tissue in response to a localized force. These measurements have been used clinically to differentiate tissues, albeit with mixed results. However, to date, the reason has not been fully understood. In this study, we apply a novel modeling approach to explore the mechanistic link between simplistic displacement measurements and tissue viscoelasticity in the application of virtual touch-based elastographic methods to staging chronic liver disease (CLD). To our knowledge, such a study has not been reported in the literature. Specifically, a numerical screening study was first conducted to identify factors that most strongly determine PD and TTP. Response surface experimental designs were then applied to these factors to produce meta-models of expected PD and TTP probability density functions (PDFs) as functions of identified factors. Results from the screening study suggest that both PD and TTP measurements are primarily influenced by three factors: the initial Young's modulus of the tissue, the first viscoelastic Prony series time constant, and pre-compression applied during acquisition. To investigate the implications of these results, stochastic inputs for these three factors associated were used to determine a robust response surface. The identified response surface methodology can be used to determine optimal cutoff values for PD and TTP that could be used in order to stage chronic liver disease.

Comparison of optical microscopy and optical coherence tomography as quality assurance methods for evaluating lubricious hydrophilic coatings surrounding catheter shafts.

Cardiac catheters are a vital tool in medicine due to their widespread use in many minimally invasive procedures. To aid in advancing the catheter within the patient's vasculature, many catheters are coated with a lubricious hydrophilic coating (HPC). Although HPCs benefit patients, their delamination during use is a serious safety concern. Adverse health effects associated with HPC delamination include pulmonary and myocardial embolism, embolic stroke, infarction, and death. In order to improve patient outcomes, more consistent manufacturing methods and improved quality assurance techniques are needed to evaluate HPC medical devices. The present work investigates the efficacy of two novel methods to image and evaluate HPCs post-manufacturing, relative to industry-standard scanning electron microscopy (SEM)-based methods. We have shown that novel evaluation approaches based on optical microscopy (OM) and optical coherence tomography (OCT) are capable of imaging HPC layers and quantifying HPC thickness, saving hours of time relative to SEM sample preparation and imaging. Additionally, the nondestructive nature of OCT avoids damage and alteration to the HPC prior to imaging, leading to more reliable HPC thickness measurements. Overall, the work demonstrated the feasibility and advantages of using OM and OCT to image and measure HPC thickness relative to industry-standard SEM methods.

Upgrading prevascularization in tissue engineering: A review of strategies for promoting highly organized microvascular network formation.

Functional and perfusable vascular network formation is critical to ensure the long-term survival and functionality of engineered tissues after their transplantation. Although several vascularization strategies have been reviewed in past, the significance of microvessel organization in three-dimensional (3D) scaffolds has been largely ignored. Advances in high-resolution microscopy and image processing have revealed that the majority of tissues including cardiac, skeletal muscle, bone, and skin contain highly organized microvessels that orient themselves to align with tissue architecture for optimum molecular exchange and functional performance. Here, we review strategies to develop highly organized and mature vascular networks in engineered tissues, with a focus on electromechanical stimulation, surface topography, micro scaffolding, surface-patterning, microfluidics and 3D printing. This review will provide researchers with state of the art approaches to engineer vascularized functional tissues for diverse applications. STATEMENT OF SIGNIFICANCE: Vascularization is one of the critical challenges facing tissue engineering. Recent technological advances have enabled researchers to develop microvascular networks in engineered tissues. Although far from translational applications, current vascularization strategies have shown promising outcomes. This review emphasizes the most recent technological advances and future challenges for developing organized microvascular networks in vitro. The next critical step is to achieve highly perfusable, dense, mature and organized microvascular networks representative of native tissues.

Laser speckle contrast imaging is sensitive to advective flux.

Unlike laser Doppler flowmetry, there has yet to be presented a clear description of the physical variables that laser speckle contrast imaging (LSCI) is sensitive to. Herein, we present a theoretical basis for demonstrating that LSCI is sensitive to total flux and, in particular, the summation of diffusive flux and advective flux. We view LSCI from the perspective of mass transport and briefly derive the diffusion with drift equation in terms of an LSCI experiment. This equation reveals the relative sensitivity of LSCI to both diffusive flux and advective flux and, thereby, to both concentration and the ordered velocity of the scattering particles. We demonstrate this dependence through a short series of flow experiments that yield relationships between the calculated speckle contrast and the concentration of the scatterers (manifesting as changes in scattering coefficient), between speckle contrast and the velocity of the scattering fluid, and ultimately between speckle contrast and advective flux. Finally, we argue that the diffusion with drift equation can be used to support both Lorentzian and Gaussian correlation models that relate observed contrast to the movement of the scattering particles and that a weighted linear combination of these two models is likely the most appropriate model for relating speckle contrast to particle motion.

Combined effects of scattering and absorption on laser speckle contrast imaging.

Several variables may affect the local contrast values in laser speckle contrast imaging (LSCI), irrespective of relative motion. It has been suggested that the optical properties of the moving fluid and surrounding tissues can affect LSCI values. However, a detailed study of this has yet to be presented. In this work, we examined the combined effects of the reduced scattering and absorption coefficients on LSCI. This study employs fluid phantoms with different optical properties that were developed to mimic whole blood with varying hematocrit levels. These flow phantoms were imaged with an LSCI system developed for this study. The only variable parameter was the optical properties of the flowing fluid. A negative linear relationship was seen between the changes in contrast and changes in reduced scattering coefficient, absorption coefficient, and total attenuation coefficient. The change in contrast observed due to an increase in the scattering coefficient was greater than what was observed with an increase in the absorption coefficient. The results indicate that optical properties affect contrast values and that they should be considered in the interpretation of LSCI data.

Assessment of incident intensity on laser speckle contrast imaging using a nematic liquid crystal spatial light modulator.

Before laser speckle contrast imaging (LSCI) can be used reliably and quantitatively in a clinical setting, there are several theoretical and practical issues that still must be addressed. In order to address some of these issues, an electro-optical system that utilizes a nematic liquid crystal spatial light modulator (SLM) to mimic LSCI experiments was assembled. The focus of this paper is to address the issue of how incident intensity affects LSCI results. Using the SLM-based system, we systematically adjusted incident intensity on the SLM and assessed the resulting first- and second-order statistics of the imaged speckle to explain the corresponding spatial contrast values in both frozen and time-integrated speckle patterns. The SLM-based system was used to generate speckle patterns with a controlled minimum speckle size, probability intensity distribution, and temporal decorrelation behavior. By eliminating many experimental parameters, this system is capable of serving as a useful intermediary tool between computer simulation and physical experimentation for further developing LSCI as a quantitative imaging modality.

Laser speckle contrast imaging: theoretical and practical limitations.

When laser light illuminates a diffuse object, it produces a random interference effect known as a speckle pattern. If there is movement in the object, the speckles fluctuate in intensity. These fluctuations can provide information about the movement. A simple way of accessing this information is to image the speckle pattern with an exposure time longer than the shortest speckle fluctuation time scale-the fluctuations cause a blurring of the speckle, leading to a reduction in the local speckle contrast. Thus, velocity distributions are coded as speckle contrast variations. The same information can be obtained by using the Doppler effect, but producing a two-dimensional Doppler map requires either scanning of the laser beam or imaging with a high-speed camera: laser speckle contrast imaging (LSCI) avoids the need to scan and can be performed with a normal CCD- or CMOS-camera. LSCI is used primarily to map flow systems, especially blood flow. The development of LSCI is reviewed and its limitations and problems are investigated.

Optical vortex behavior in dynamic speckle fields.

The dynamic behavior of phase singularities, or optical vortices, in the pseudo-phase representation of dynamic speckle patterns is investigated. Sequences of band-limited, dynamic speckle patterns with predetermined Gaussian decorrelation behavior were generated, and the pseudo-phase realizations of the individual speckle patterns were calculated via a two-dimensional Hilbert transform algorithm. Singular points in the pseudo-phase representation are identified by calculating the local topological charge as determined by convolution of the pseudo-phase representations with a series of 2×2 nabla filters. The spatial locations of the phase singularities are tracked over all frames of the speckle sequences, and recorded in three-dimensional space (x,y,f), where f is frame number in the sequence. The behavior of the phase singularities traces 'vortex trails' which are representative of the speckle dynamics. Slowly decorrelating speckle patterns results in long, relatively straight vortex trails, while rapidly decorrelating speckle patterns results in tortuous, relatively short vortex trails. Optical vortex analysis such as described herein can be used as a descriptor of biological activity, flow, and motion.

A dynamic light scattering approach for monitoring dental composite curing kinetics.

OBJECTIVES: The purpose of this study was to develop and evaluate a dynamic light scattering-based method for monitoring the polymerization reaction of a light activated dental composite. METHODS: Laser light back-scattered from thin disk-shaped composite samples was used to study the curing reaction kinetics. Samples were irradiated simultaneously on opposite surfaces with a 633 nm laser beam and a halogen curing lamp (320, 160, or 100 mW/cm(2)). Dynamic laser speckle patterns were imaged onto a CCD camera at a rate of 32 frames/s for 2 min. The intensity decorrelation rate calculated from sequential speckle patterns was used to assess the rate of motion within the samples during the reaction. RESULTS: Motion within the composite increased immediately upon the onset of light exposure for all trials. This was followed by a brief period characterized by a relatively constant high rate of motion. Finally the rate of motion decreased exponentially. The reaction acceleration, deceleration, and maximum rate were dependent upon the irradiance of the curing light source. SIGNIFICANCE: This method monitors reaction rate and the change in reaction rate at high temporal resolution without contact. Reaction kinetics was shown to begin immediately after light exposure suggesting limited opportunity for viscous flow and stress relief.

Doppler optical coherence tomography imaging of local fluid flow and shear stress within microporous scaffolds.

Establishing a relationship between perfusion rate and fluid shear stress in a 3D cell culture environment is an ongoing and challenging task faced by tissue engineers. We explore Doppler optical coherence tomography (DOCT) as a potential imaging tool for in situ monitoring of local fluid flow profiles inside porous chitosan scaffolds. From the measured fluid flow profiles, the fluid shear stresses are evaluated. We examine the localized fluid flow and shear stress within low- and high-porosity chitosan scaffolds, which are subjected to a constant input flow rate of 0.5 ml min(-1). The DOCT results show that the behavior of the fluid flow and shear stress in micropores is strongly dependent on the micropore interconnectivity, porosity, and size of pores within the scaffold. For low-porosity and high-porosity chitosan scaffolds examined, the measured local fluid flow and shear stress varied from micropore to micropore, with a mean shear stress of 0.49+/-0.3 dyn cm(-2) and 0.38+/-0.2 dyn cm(-2), respectively. In addition, we show that the scaffold's porosity and interconnectivity can be quantified by combining analyses of the 3D structural and flow images obtained from DOCT.

Distinct extracellular matrix microenvironments of progenitor and carotid endothelial cells.

Endothelial cells (ECs) produce and maintain the local extracellular matrix (ECM), a critical function that contributes to EC and blood vessel health. This function is also crucial to vascular tissue engineering, where endothelialization of vascular constructs require a cell source that readily produces and maintains ECM. In this study, baboon endothelial progenitor cell (EPC) deposition of ECM (laminin, collagen IV, and fibronectin) was characterized and compared to mature carotid ECs, evaluated in both elongated and cobblestone morphologies typically found in vivo. Microfluidic micropatterning was used to create 15-microm wide adhesive lanes with 45-microm spacing to reproduce the elongated EC morphology without the influence of external forces. Both EPCs and ECs elongated on micropatterned lanes had aligned actin cytoskeleton and readily deposited ECM. EPCs deposited and remodeled the ECM to a greater extent than ECs. Since a readily produced ECM can improve graft patency, EPCs are an advantageous cell source for endothelializing vascular constructs. Furthermore, EC deposition of ECM was dependent on cell morphology, where elongated ECs deposited more collagen IV and less fibronectin compared to matched cobblestone controls. Thus micropatterned surfaces controlled EC shape and ECM deposition, which ultimately has implications for the design of tissue-engineered vascular constructs.

Detrimental effects of speckle-pixel size matching in laser speckle contrast imaging.

Through a series of simulations and experiments, we demonstrate that the frequently cited criterion of matching speckle size to detector element (pixel) size in laser speckle contrast imaging (LSCI) has the detrimental effect of reducing the contrast and thereby decreasing the variation in the laser speckle contrast image. Unlike quasi-elastic light scattering, where this matching condition has been shown to maximize the signal-to-noise ratio, in LSCI, the minimum speckle size must exceed the Nyquist criterion in order to maximize the contrast of the speckle patterns.

Can laser speckle flowmetry be made a quantitative tool?

The ultimate objective of laser speckle flowmetry (and a host of specific implementations such as laser speckle contrast analysis, LASCA or LSCA; laser speckle spatial contrast analysis, LSSCA; laser speckle temporal contrast analysis, LSTCA; etc.) is to infer flow velocity from the observed speckle contrast. Despite numerous demonstrations over the past 25 years of such a qualitative relationship, no convincing quantitative relationship has been proven. One reason is a persistent mathematical error that has been propagated by a host of workers; another is a misconception about the proper autocorrelation function for ordered flow. Still another hindrance has been uncertainty in the specific relationship between decorrelation time and local flow velocity. Herein we attempt to dispel some of these errors and misconceptions with the intent of turning laser speckle flowmetry into a quantitative tool. Specifically we review the underlying theory, explore the impact of various analytic models for relating measured intensity fluctuations to scatterer motion, and address some of the practical issues associated with the measurement and subsequent data processing.

Endothelial cell cytoskeletal alignment independent of fluid shear stress on micropatterned surfaces.

Endothelial cells (ECs) in athero-protective regions are elongated with actin and microtubule fibers aligned parallel to the direction of blood flow. Fluid shear stress (FSS) affects EC shape and functions, but little is known about shape-dependent EC properties that are independent of FSS. To evaluate these properties, ECs were elongated on micropatterned (MP) 25mum wide collagen-coated lanes (MPECs) and characterized by cell shape index, actin and microtubule alignment, and polarization of the microtubule-organizing center (MTOC). ECs on non-patterned surfaces were also exposed to FSS. MPEC elongation was microtubule-dependent (and actin-independent); shape indices and cytoskeletal alignment were comparable to FSS-elongated ECs. Cytoskeletal alignment was lost when MPECs were exposed to perpendicular FSS, but not parallel FSS. MTOC polarization was FSS-dependent. Thus, by isolating EC elongation and cytoskeletal alignment from FSS, micropatterning creates a platform for studying EC shape-related cellular functions that are independent of FSS.

Statistics of local speckle contrast.

In describing the first-order properties of laser speckle under polarized illumination conditions, it is almost an article of faith that the contrast is unity. In many processing schemes, however, the contrast defined as the quotient of the standard deviation and the mean is calculated over a localized spatial region. In such cases, this local contrast displays a distribution of values that can depart substantially from unity. Properties of this distribution depend on details of the data acquisition and on the size of the local neighborhood over which the contrast is calculated. We demonstrate that this local contrast can be characterized in terms of a log-normal distribution. Further, we show that the two defining parameters of this model can in turn be expressed in terms of the minimum speckle size and the extent of the local neighborhood. Performance of the model is illustrated with some typical optical coherence tomography data.

The copula: a tool for simulating speckle dynamics.

Use of a copula for generating a sequence of correlated speckle patterns is introduced. The chief characteristic of this algorithm is that it generates a continuous speckle sequence with a specified evolution of the correlation and does so with just two arrays of random numbers. Thus, physically realistic temporally varying speckle patterns with proper first- and second-order statistics are easily realized. We illustrate use of the algorithm for generating sequences with prescribed Gaussian, exponential, and equal-interval correlations and demonstrate how correlation times can be specified independently. This approach to generating sequences of random realizations with prescribed correlations should prove useful in modeling such phenomena as dynamic light scatter, flow-dependent laser speckle contrast, and propagation of spatial coherence.

Quantitative temporal speckle contrast imaging for tissue mechanics.

We demonstrate through a series of simulations that by parameterizing the temporal speckle contrast statistic from a sequence of translating speckle images on a number of experimental constants, the local temporal contrast can be used to quantitatively assess local motion, provided that the spatial and temporal Nyquist sampling criteria are both met. We develop a simple exponential model for quantifying speckle motion for speckle patterns that display arbitrary intensity statistics and provide suggestions for optimizing both the experimental acquisition of speckle data and the temporal contrast analysis of the data. The confounding effects of uncorrelated noise are also discussed. The model is demonstrated by applying it to an optical coherence tomography image sequence of an engineered tissue construct undergoing dynamic compression. Applications to tissue mechanics are shown, although the discussion is equally relevant for fluid motion studies.

Development of a reinforced porcine elastin composite vascular scaffold.

Elastin, a principal structural component of native arteries, has distinct biological and mechanical advantages when used as a biomaterial; however, its low ultimate tensile strength has limited its use as an arterial conduit. We have developed a scaffold, consisting of a purified elastin tubular conduit strengthened with fibrin bonded layers of acellular small intestinal submucosa (aSIS) for potential use as a small diameter vascular graft. The addition of aSIS increased the ultimate tensile strength of the elastin conduits nine-fold. Burst pressures for the elastin composite vascular scaffold (1,396 +/- 309 mmHg) were significantly higher than pure elastin conduits (162 +/- 36 mmHg) and comparable to native saphenous veins. The average suture pullout strength of the elastin composite vascular scaffolds was 14.612 +/- 3.677 N, significantly higher than the pure elastin conduit (0.402 +/- 0.098 N), but comparable to native porcine carotid arteries (13.994 +/- 4.344 N). Cyclic circumferential strain testing indicated that the composite scaffolds were capable of withstanding physiological loading conditions for at least 83 h. Implantation of the elastin composites as carotid interposition grafts in swine demonstrated its superiority to clinically acceptable ePTFE with significantly longer average patency times of 5.23 h compared to 4.15 h. We have developed a biologically based elastin scaffold with suitable mechanical properties and low thrombogenicity for in vivo implantation, and with the potential for cellular repopulation and host integration reestablishing an appropriate elastic artery.

Imaging the mechanical stiffness of skin lesions by in vivo acousto-optical elastography.

Optical elastography is an imaging modality that relies on variations in the local mechanical properties of biological tissues as the contrast mechanism for image formation. Skin lesions, such as melanomas and other invasive conditions, are known to alter the arrangement of collagen fibers in the skin and thus should lead to alterations in local skin mechanical properties. We report on an acousto-optical elastography (AOE) imaging modality for quantifying the mechanical behavior of skin lesions. The method relies upon stimulating the tissue with a low frequency acoustic force and imaging the resulting strains in the tissue by means of quantifying the magnitude of the dynamic shift in a back-reflected laser speckle pattern from the skin. The magnitude of the shift reflects the local stiffness of the tissue. We demonstrate AOE on a tissue-mimicking phantom, an in vivo mouse melanoma lesion and two types of in vivo human melanocytic nevi. The skin lesions we examined were found to have distinct mechanical properties that appear to correlate with the varying degrees of dermal involvement of the lesions.

OCT-based elastography for large and small deformations.

We present two approaches to speckle tracking for optical coherence tomography (OCT)-based elastography, one appropriate for small speckle motions and the other for large, rapid speckle motions. Both approaches have certain advantages over traditional cross-correlation based motion algorithms. We apply our algorithms to quantifying the strain response of a mechanically inhomogeneous, bi-layered polyvinyl alcohol tissue phantom that is subjected to either small or large dynamic compressive forces while being imaged with a spectral domain OCT system. In both the small and large deformation scenarios, the algorithms performed well, clearly identifying the two mechanically disparate regions of the phantom. The stiffness ratio between the two regions was estimated to be the same for the two scenarios and both estimates agreed with the expected stiffness ratio based on earlier mechanical testing. No single numerical approach is appropriate for all cases and the experimental conditions dictate the proper choice of speckle shift algorithm for OCT-based elastography studies.