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This PDF file contains the front matter associated with SPIE Proceedings Volume 8946, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.
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Shear wave elastography measures the stiffness of soft tissues from the speed of propagating shear waves induced in tissue. Optical coherence tomography (OCT) is a promising detection modality given its high sensitivity and spatial resolution, making it suitable for elastic characterization of skin, peripheral vasculature or ocular tissues. For clinical applications, it would be valuable to use a non-contact shear source. Thus, we propose acoustic radiation force as a remote shear source combined with OCT for visualization. A single-element focused transducer (central frequency 7.5 MHz) was used to apply a maximal pressure of ~3 MPa for 100 μs in agar phantoms. It induced shear waves with an amplitude of several hundreds of nanometers and a broadband spectrum in the kilohertz range. Phasesensitive OCT was used to track shear waves at an equivalent frame rate of 47 kHz. We reconstructed shear modulus maps in a heterogeneous phantom. In addition, we use 3-ms long coded excitation to increase the displacement signal-to-noise ratio. We applied digital pulse compression to the resulting displacement field to obtain a gain of ~15 dB compared to standard pulse excitation while maintaining the US pressure level and the shear wave spatial and temporal resolution. This is a promising result for shear wave generation at low US pressures (~ 1 MPa).
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The characteristics of lamb wave propagation in cornea can be of great importance for the quantitative measurement of corneal viscoelasticity. Here, we report the use of shear wave imaging optical coherence tomography (SWI-OCT) to assess the dispersion of lamb wave propagation in an ex vivo rabbit cornea. The temporal displacement profile of lamb wave propagation is obtained with SWI-OCT. From the spectral analysis on the corneal lamb wave, the phase information of the wave propagation can be extracted, which is used to quantify the phase velocity at different frequencies. The dependence of the phase on the propagation distance is presented with 2-D depth-resolved mapping at typical frequency components with micro-scale spatial resolution. Results indicate that the obtained lamb wave dispersion curve agrees well with the analytical approximation and demonstrate the feasibility of using SWI-OCT to study the dispersion of lamb wave propagation in cornea.
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The combination of air-puff systems with real-time corneal imaging (i.e. Optical Coherence Tomography (OCT), or Scheimpflug) is a promising approach to assess the dynamic biomechanical properties of the corneal tissue in vivo. In this study we present an experimental system which, together with finite element modeling, allows measurements of corneal biomechanical properties from corneal deformation imaging, both ex vivo and in vivo. A spectral OCT instrument combined with an air puff from a non-contact tonometer in a non-collinear configuration was used to image the corneal deformation over full corneal cross-sections, as well as to obtain high speed measurements of the temporal deformation of the corneal apex. Quantitative analysis allows direct extraction of several deformation parameters, such as apex indentation across time, maximal indentation depth, temporal symmetry and peak distance at maximal deformation. The potential of the technique is demonstrated and compared to air-puff imaging with Scheimpflug. Measurements ex vivo were performed on 14 freshly enucleated porcine eyes and five human donor eyes. Measurements in vivo were performed on nine human eyes. Corneal deformation was studied as a function of Intraocular Pressure (IOP, 15-45 mmHg), dehydration, changes in corneal rigidity (produced by UV corneal cross-linking, CXL), and different boundary conditions (sclera, ocular muscles). Geometrical deformation parameters were used as input for inverse finite element simulation to retrieve the corneal dynamic elastic and viscoelastic parameters. Temporal and spatial deformation profiles were very sensitive to the IOP. CXL produced a significant reduction of the cornea indentation (1.41x), and a change in the temporal symmetry of the corneal deformation profile (1.65x), indicating a change in the viscoelastic properties with treatment. Combining air-puff with dynamic imaging and finite element modeling allows characterizing the corneal biomechanics in-vivo.
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Elastography is the mapping of tissues and cells by their respective mechanical properties, such as elasticity and viscosity. Our interest primarily lies in the human eye. Combining Scanning Laser Doppler Vibrometry (SLDV) with geometrically focused mechanical vibratory excitations of the cornea, it is possible to reconstruct these mechanical properties of the cornea. Experiments were conducted on phantom corneas as well as excised donor human corneas to test feasibility and derive a method of modeling. Finite element analysis was used to recreate the phantom studies and corroborate with the experimental data. Results are in close agreement. To further expand the study, lamb eyes were used in MR Elastography studies. 3D wave reconstruction was created and elastography maps were obtained. With MR Elastography, it would be possible to noninvasively measure mechanical properties of anatomical features not visible to SLDV, such as the lens and retina. Future plans include creating a more robust finite element model, improving the SLDV method for in-vivo application, and continuing experiments with MR Elastography.
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Laser coagulation of the retina is an established treatment for several retinal diseases. The absorbed laser energy and thus the induced thermal damage varies with the transmittance and scattering properties of the anterior eye media and with the pigmentation of the fundus. The temperature plays the most important role in the coagulation process. An established approach to measure a mean retinal temperature rise is optoacoustics, however it provides limited information on the coagulation. Phase sensitive OCT potentially offers a three dimensional temporally resolved temperature distribution but is very sensitive to slightest movements which are clinically hard to avoid. We develop an optical technique able to monitor and quantify thermally and coagulation induced tissue movements (expansions and contractions) and changes in the tissue structure by dynamic laser speckle analysis (LSA) offering a 2D map of the affected area. A frequency doubled Nd:YAG laser (532nm) is used for photocoagulation. Enucleated porcine eyes are used as targets. The spot is 100μm. A Helium Neon laser (HeNe) is used for illumination. The backscattered light of a HeNe is captured with a camera and the speckle pattern is analyzed. A Q-switched Nd:YLF laser is used for simultaneous temperature measurements with the optoacoustic approach. Radial tissue movements in the micrometer regime have been observed. The signals evaluation by optical flow algorithms and generalized differences tuned out to be able to distinguish between regions with and without immediate cell damage. Both approaches have shown a sensitivity of 93% and a specificity above 99% at their optimal threshold.
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The progressive loss of accommodation of the eye, called presbyopia, affects people with age and can result in a complete loss of accommodation by about age 55 years. It is generally accepted that presbyopia is due to an increase in stiffness of the lens. With increasing age, the stiffness of the crystalline lens nucleus increases faster than that of the cortex. During accommodation, the deformation of different parts of the crystalline lens is different and likely changes with age. However, a direct observation of crystalline lens deformation and strain distribution is difficult because although imaging methods such as OCT or Scheimpflug imaging can distinguish cortex and nucleus, they cannot determine their regional deformation. Here, patterns of laser-induced microbubbles were created in gelatin phantoms and different parts of excised animal crystalline lenses and their displacements in response to external deformation were tracked by ultrasound imaging. In the animal lenses, the deformation of the lens cortex was greater than that of nucleus and this regional difference is greater for a 27-month-old bovine lens than for a 6-month-old porcine lens. This approach enables visualization of localized, regional deformation of crystalline lenses and, if applied to lenses from animal species that undergo accommodation, may help to understand the mechanisms of accommodation and presbyopia, improve diagnostics, and, potentially, aid in the development of new methods of lens modifying presbyopia treatments.
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Viscoelastic models are fit to shear moduli derived from geometrically focused surface waves (GFS) on human skin using viscoelastic wave theory. Unlike in previous studies on the analytical solution and experimental measurement of radially outward traveling surface waves, measurable radially inward traveling GFS waves can be generated over a wider range of frequencies as attenuation is countered by the converging nature of the wavefront. This enables a more accurate and broader assessment of both the shear storage and loss moduli of the material, which are expected to vary with frequency. In the present study, GFS waves are applied to human skin on the posterior side of the forearm using a scanning LASER Doppler vibrometer. Surface wave measurements can then be used to estimate the complex frequency dependent viscoelastic properties of biological tissue, which are affected by numerous pathologies. Using a phantom gel this technique was validated through comparison with other studies. It was found that spring-pot and fractional Voigt models yield a potentially stable model parameter for skin, but more study is needed to confirm. [Work supported by NIH: Grant # EB012142.]
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Biometric identification systems have important applications to privacy and security. The most widely used of these, print identification, is based on imaging patterns present in the fingers, hands and feet that are formed by the ridges, valleys and pores of the skin. Most modern print sensors acquire images of the finger when pressed against a sensor surface. Unfortunately, this pressure may result in deformations, characterized by changes in the sizes and relative distances of the print patterns, and such changes have been shown to negatively affect the performance of fingerprint identification algorithms. Optical coherence tomography (OCT) is a novel imaging technique that is capable of imaging the subsurface of biological tissue. Hence, OCT may be used to obtain images of subdermal skin structures from which one can extract an internal fingerprint. The internal fingerprint is very similar in structure to the commonly used external fingerprint and is of increasing interest in investigations of identify fraud. We proposed and tested metrics based on measurements calculated from external and internal fingerprints to evaluate the amount of deformation of the skin. Such metrics were used to test hypotheses about the differences of deformation between the internal and external images, variations with the type of finger and location inside the fingerprint.
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In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.
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Autologous chondrocyte transplantation (ACT) has become a promising method for repairing large articular defects. However, dedifferentiation of chondrocytes during cell expansion remains a major limitation for ACT procedures. In this study, we explore the potential of confining cell shape for re-differentiation of dedifferentiated bovine chondrocytes. A novel culture system, combining 2D micropatterning with 3D matrix formation, was developed to control and maintain individual chondrocyte’s shape. Both collagen II synthesis and the mechanical properties of cells were monitored during re-differentiation. We show that a spherical morphology without cell spreading plays a limited role in induction of re-differentiation. Instead, isolated, dedifferentiated chondrocytes partially regain chondrogenic properties if they have an appropriate cell shape and limited spreading.
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In this study, we investigated nanomechanical properties of cell membranes in response to elongation at different rates. Membrane nanotubes (tethers) were pulled at different pulling rates by an optically-trapped fluorescent microsphere as recorded and analyzed for low (1 μm/s) and high (100 μm/s) pulling rates. The force relaxation response of membrane nanotubes exhibited a bi-phasic behavior including fast and slow relaxation processes at low and high pulling rates. The fast and slow force relaxation time constants were 0.388±0.21 s and 11.74±3.35 s, in response to pulling rate of 1 μm/s, respectively and significantly decreased at higher pulling rates. These reductions in the time constants are suggestive of reduced viscous effects and weakened adhesions between the membrane and the cytoskeleton during rapid pulling.
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Acoustic Radiation Force (ARF) stimulation is actively used in ultrasound elastography to estimate mechanical properties of tissue. Compared with ultrasound imaging, OCT provides advantage in both spatial resolution and signal-to-noise ratio. Therefore, a combination of ARF and OCT technologies can provide a unique opportunity to measure viscoelastic properties of tissue, especially when the use of high intensity radiation pressure is limited for safety reasons. In this presentation we discuss a newly developed theoretical model of the deformation of a layered viscoelastic medium in response to an acoustic radiation force of short duration. An acoustic impulse was considered as an axisymmetric force generated on the upper surface of the medium. An analytical solution of this problem was obtained using the Hankel transform in frequency domain. It was demonstrated that layers at different depths introduce different frequency responses. To verify the developed model, experiments were performed using tissue-simulating, inhomogeneous phantoms of varying mechanical properties. The Young’s modulus of the phantoms was varied from 5 to 50 kPa. A single-element focused ultrasound transducer (3.5 MHz) was used to apply the radiation force with various durations on the surface of phantoms. Displacements on the phantom surface were measured using a phase-sensitive OCT at 25 kHz repetition frequency. The experimental results were in good agreement with the modeling results. Therefore, the proposed theoretical model can be used to reconstruct the mechanical properties of tissue based on ARF/OCT measurements.
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Shear stress has been recognized as one of the biophysical methods by which to permeabilize plasma membranes of cells. In particular, high pressure transient hydrodynamic flows created by laser-induced cavitation have been shown to lead to the uptake of fluorophores and plasmid DNA. While the mechanism and dynamics of cavitation have been extensively studied using a variety of time-resolved imaging techniques, the cellular response to the cavitation bubble and cavitation induced transient hydrodynamic flows has never been shown in detail. We use time-resolved quantitative phase microscopy to study cellular response to laser-induced cavitation bubbles. Laser-induced breakdown of an optically trapped polystyrene nanoparticle (500nm in diameter) irradiated with a single nanosecond laser pulse at 532nm creates transient shear stress to surrounding cells without causing cell lysis. A bi-directional transient displacement of cytoplasm is observed during expansion and collapse of the cavitation bubble. In some cases, cell deformation is only observable at the microsecond time scale without any permanent change in cell shape or optical thickness. On a time scale of seconds, the cellular response to shear stress and cytoplasm deformation typically leads to retraction of the cellular edge most exposed to the flow, rounding of the cell body and, in some cases, loss of cellular dry mass. These results give a new insight into the cellular response to laser-induced shear stress and related plasma membrane permeabilization. This study also demonstrates that laser-induced breakdown of an optically trapped nanoparticle offers localized cavitation (70 μm in diameter), which interacts with a single cell.
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We present results of a non contacting instrument based on the confocal scanning technique for assessing the thickness and structure of collagen substrates and tissue constructs. There is an unmet need in the creation of tissue constructs to quantitatively evaluate their dimensional characteristics during manufacture. With this knowledge more effective structures can be produced. The measurement is complicated by the need to make these measurements in situ. For many processes, including the plastic compression of collagen gels for generating 3D structures, the constructs are situated in a liquid solution contained in a well plate or similar container. It is therefore necessary to perform the measurements through an interfering medium and this confounds many measurement techniques. A system has therefore been developed that utilizes a scanning confocal arrangement to accurately measure the dimensional characteristics of these constructs in situ. A fiber based optical arrangement using compact, proven components from the telecommunications industry has been integrated into a dedicated system architecture so that the constructs can be measured whilst in production. This architecture is particularly important due to the “wet” nature of the samples. The meter can measure constructs with thicknesses from a few tens of micrometers up to 0.9 millimeters with sub-micrometer resolution. Results are presented that show how the meter has been used to evaluate changes in these collagen constructs whilst in production. This was little understood prior to these measurements and the greater understanding of how the materials behave has allowed the process to be greatly improved.
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In this work, we explored the potential of measuring shear wave propagation using Optical Coherence Elastography (OCE) in MCF7 cell modules (comprised of MCF7 cells and collagen) and based on a swept-source optical coherence tomography (OCT) system. Shear waves were generated using a piezoelectric transducer transmitting sine-wave bursts of 400 μs, synchronized with an OCT swept source wavelength sweep imaging system. Acoustic radiation force was applied to the MCF7 cell constructs. Differential OCT phase maps, measured with and without the acoustic radiation force, demonstrate microscopic displacement generated by shear wave propagation in these modules. The OCT phase maps are acquired with a swept-source OCT (SS-OCT) system. We also calculated the tissue mechanical properties based on the propagating shear waves in the MCF7 + collagen phantoms using the Acoustic Radiation Force (ARF) of an ultrasound transducer, and measured the shear wave speed with the OCT phase maps. This method lays the foundation for future studies of mechanical property measurements of breast cancer structures, with applications in the study of breast cancer pathologies.
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In this study, we developed a miniaturized optical coherence tomography (OCT) probe with a diameter of 4 mm. It was integrated with an air-jet indentation and air suction to induce deformation of tissue. The deforming process of tissue under suction or indentation was continuously monitored by OCT, and deformation of tissue was then derived from the transient OCT signals. Studies on phantoms with different stiffness were conducted. Results showed that the stiffness obtained by the OCT-based suction and indention well correlated with the stiffness detected using conventional mechanical testing. The probe was small enough for endoscopic use. In addition to the elasticity, the viscoelasticity of tissues can also be detected using creep indentation and suction test.
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