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This PDF file contains the front matter associated with SPIE Proceedings Volume 10067 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.
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Shear wave measurement enables quantitative assessment of tissue viscoelasticity. In previous studies, a transverse shear wave was measured using optical coherence elastography (OCE), which gives poor resolution along the force direction because the shear wave propagates perpendicular to the applied force. In this study, for the first time to our knowledge, we introduce an OCE method to detect a longitudinally polarized shear wave that propagates along the force direction. The direction of vibration induced by a piezo transducer (PZT) is parallel to the direction of wave propagation, which is perpendicular to the OCT beam. A Doppler variance method is used to visualize the transverse displacement. Both homogeneous phantoms and a side-by-side two-layer phantom were measured. The elastic moduli from mechanical tests closely matched to the values measured by the OCE system. Furthermore, we developed 3D computational models using finite element analysis to confirm the shear wave propagation in the longitudinal direction. The simulation shows that a longitudinally polarized shear wave is present as a plane wave in the near field of planar source due to diffraction effects. This imaging technique provides a novel method for the assessment of elastic properties along the force direction, which can be especially useful to image a layered tissue.
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In many applications of optical coherence elastography (OCE), it is necessary to rapidly acquire images in vivo, or within intraoperative timeframes, over fields-of-view far greater than can be achieved in one OCT image acquisition. For example, tumour margin assessment in breast cancer requires acquisition over linear dimensions of 4-5 centimetres in under 20 minutes. However, the majority of existing techniques are not compatible with these requirements, which may present a hurdle to the effective translation of OCE. To increase throughput, we have designed and developed an OCE system that simultaneously captures two 3D elastograms from opposite sides of a sample. The optical system comprises two interferometers: a common-path interferometer on one side of the sample and a dual-arm interferometer on the other side. This optical system is combined with scanning mechanisms and compression loading techniques to realize dual-scanning OCE. The optical signals scattered from two volumes are simultaneously detected on a single spectrometer by depth-encoding the interference signal from each interferometer. To demonstrate dual-scanning OCE, we performed measurements on tissue-mimicking phantoms containing rigid inclusions and freshly isolated samples of murine hepatocellular carcinoma, highlighting the use of this technique to visualise 3D tumour stiffness. These findings indicate that our technique holds promise for in vivo and intraoperative applications.
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Tissue properties such as elasticity and viscosity have been shown to be related to such tissue conditions as contraction, edema, fibrosis, and fat content among others. Magnetic Resonance Elastography has shown outstanding ability to measure the elasticity and in some cases the viscosity of tissues, especially in the liver, providing the ability to stage fibrotic liver disease similarly to biopsy. We discuss ultrasound methods of measuring elasticity and viscosity in tissues. Many of these methods are becoming widely available in the extant ultrasound machines distributed throughout the world. Some of the methods to be discussed are in the developmental stage. The advantages of the ultrasound methods are that the imaging instruments are widely available and that many of the viscoelastic measurements can be made during a short addition to the normal ultrasound examination time. In addition, the measurements can be made by ultrasound repetitively and quickly allowing evaluation of dynamic physiologic function in circumstances such as muscle contraction or artery relaxation. Measurement of viscoelastic tissue mechanical properties will become a consistent part of clinical ultrasound examinations in our opinion.
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Background and motivation -
Conventional Optical Coherence Elastography (OCE) methods consist in launching controlled shear waves in tissues, and measuring their propagation speed using an ultrafast imaging system. However, the use of external shear sources limits transfer to clinical practice, especially for ophthalmic applications. Here, we propose a totally passive OCE method for ocular tissues based on time-reversal of the natural vibrations.
Methods -
Experiments were first conducted on a tissue-mimicking phantom containing a stiff inclusion. Pulsatile motions were reproduced by stimulating the phantom surface with two piezoelectric actuators excited asynchronously at low frequencies (50-500 Hz). The resulting random displacements were tracked at 190 frames/sec using spectral-domain optical coherence tomography (SD-OCT), with a 10x5µm² resolution over a 3x2mm² field-of-view (lateral x depth). The shear wavefield was numerically refocused (i.e. time-reversed) at each pixel using noise-correlation algorithms. The focal spot size yields the shear wavelength. Results were validated by comparison with shear wave speed measurements obtained from conventional active OCE. In vivo tests were then conducted on anesthetized rats.
Results -
The stiff inclusion of the phantom was delineated on the wavelength map with a wavelength ratio between the inclusion and the background (1.6) consistent with the speed ratio (1.7). This validates the wavelength measurements. In vivo, natural shear waves were detected in the eye and wavelength maps of the anterior segment showed a clear elastic contrast between the cornea, the sclera and the iris.
Conclusion -
We validated the time-reversal approach for passive elastography using SD-OCT imaging at low frame-rate. This method could accelerate the clinical transfer of ocular elastography.
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It is increasingly recognized that cells measure and respond to the mechanics of their environment. We are especially interested in this mechanosensing during CNS development and pathologies. Using quantitative scanning force microscopy we have shown that various neural tissues are very compliant (shear modulus < 1 kPa) and mechanically heterogeneous. We have recreated compliant polyacrylamide gel substrate with shear moduli between 0.1 and 30 kPa to match and exceed those of CNS tissue. Various primary neurons and glial cells have been cultured on these gels and their reaction studied. Both primary microglia and astrocytes responded to increasing substrate stiffness by changes in morphology and upregulation of inflammatory genes. Upon implantation of composite hydrogel stripes into rat brains, foreign body reactions were significantly enhanced around the stiff parts of the implant. It appears that the mechanical mismatch between a neural implant and native tissue might be at the root of foreign body reactions. Also oligodendrocytes are mechanosensitive as their survival, proliferation, migration, and differentiation capacity in vitro depend on substrate stiffness. This finding might be linked to the failure of remyelination in chronic demyelinating diseases such as multiple sclerosis. And finally, we have also shown retinal ganglion axon pathfinding in the early embryonic Xenopus brain development to be instructed by stiffness gradients. These results form the basis for further investigations into the mechanobiology of cell function in the CNS. Ultimately, this research could help treating previously incurable neuropathologies such as spinal cord injuries and neurodegenerative disorders.
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Although it is well documented that abnormal levels of either intraocular (IOP) or intracranial pressure (ICP) can lead to potentially blinding conditions, such as glaucoma and papilledema, little is known about how the pressures actually affect the eye. Even less is known about potential interplay between their effects, namely how the level of one pressure might alter the effects of the other. Our goal was to measure in-vivo the pressure-induced stretch and compression of the lamina cribrosa due to acute changes of IOP and ICP. The lamina cribrosa is a structure within the optic nerve head, in the back of the eye. It is important because it is in the lamina cribrosa that the pressure-induced deformations are believed to initiate damage to neural tissues leading to blindness. An eye of a rhesus macaque monkey was imaged in-vivo with optical coherence tomography while IOP and ICP were controlled through cannulas in the anterior chamber and lateral ventricle, respectively. The image volumes were analyzed with a newly developed digital image correlation technique. The effects of both pressures were highly localized, nonlinear and non-monotonic, with strong interactions. Pressure variations from the baseline normal levels caused substantial stretch and compression of the neural tissues in the posterior pole, sometimes exceeding 20%. Chronic exposure to such high levels of biomechanical insult would likely lead to neural tissue damage and loss of vision. Our results demonstrate the power of digital image correlation technique based on non-invasive imaging technologies to help understand how pressures induce biomechanical insults and lead to vision problems.
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Age-related macular degeneration (AMD) is an eye condition that is considered to be one of the leading causes of blindness among people over 50. Recent studies suggest that the mechanical properties in retina layers are affected during the early onset of disease. Therefore, it is necessary to identify such changes in the individual layers of the retina so as to provide useful information for disease diagnosis. In this study, we propose using an acoustic radiation force optical coherence elastography (ARF-OCE) system to dynamically excite the porcine retina and detect the vibrational displacement with phase resolved Doppler optical coherence tomography. Due to the vibrational mechanism of the tissue response, the image quality is compromised during elastogram acquisition. In order to properly analyze the images, all signals, including the trigger and control signals for excitation, as well as detection and scanning signals, are synchronized within the OCE software and are kept consistent between frames, making it possible for easy phase unwrapping and elasticity analysis. In addition, a combination of segmentation algorithms is used to accommodate the compromised image quality. An automatic 3D segmentation method has been developed to isolate and measure the relative elasticity of every individual retinal layer. Two different segmentation schemes based on random walker and dynamic programming are implemented. The algorithm has been validated using a 3D region of the porcine retina, where individual layers have been isolated and analyzed using statistical methods. The errors compared to manual segmentation will be calculated.
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The collagen fibril orientation of the cornea can provide critical information about cornea tissue health because diseases such as keratoconus and therapeutic interventions such as UV-A/riboflavin corneal collagen crosslinking (CXL) can alter the ultrastructural arrangement of collagen fibrils. Here, we quantify the elastic anisotropy and hysteresis of in situ porcine corneas as a function of intraocular pressure (IOP) with noncontact optical coherence elastography. Moreover, the effects of UV-A riboflavin corneal collagen crosslinking on the elastic anisotropy and hysteresis were evaluated. The propagation of an air-pulse induced elastic wave was imaged at stepped meridional angles by a home built phasestabilized swept source OCE system. The stiffness of the cornea was translated from the velocity of the wave, and the elastic anisotropy was quantified by modifying the planar anisotropy coefficient. As the IOP increased, the stiffness of the corneas increased from ~18 kPa at 15 mmHg IOP to ~ 120 kPa at 30 mmHg IOP. While there was a measureable hysteresis, it was not significant. After CXL, the Young’s modulus of the corneas significantly increased from ~18 kPa to ~44 kPa at 15 mmHg IOP. The mechanical anisotropy also increased significantly from ~10 a.u. in the untreated corneas to ~23 a.u. in the CXL treated corneas, 15 mmHg IOP. However, CXL did not change the elastic anisotropic orientation, and the mechanical anisotropic hysteresis was not significant after CXL.
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Background and Objectives: Keratoconus is a disease characterized by progressive steepening and thinning of the cornea, altering visual acuity and sometimes potentiating the need for corneal transplant if the disease progresses.1–3 Corneal crosslinking, a procedure that uses topical riboflavin and UV light to increase the stiffness of the cornea through the creation of collagen crosslinks was recently approved by the FDA for use in the U.S. The objective of the present study was to investigate whether endogenous collagen fluorescence changes following treatment can be correlated to alterations in the stiffness of the cornea, thereby guiding treatment parameters. Study Design and Results: 78 ex-vivo rabbit eyes divided into three groups: riboflavin solution plus UV irradiation, dextran solution plus UV irradiation, and riboflavin solution only. An additional group of eyes received no treatment. The epithelium was removed from each sample and topical riboflavin was applied. Eyes were irradiated with a 365 nm black ray UV lamp for various treatment times, ranging from half the clinical treatment time to three times the length. Mechanical testing was performed to determine the force/displacement relationship for the various treatment times. Fluorescence spectral changes following treatment corresponded with changes in stiffness. In particular, a decrease in the value of fluorescence intensity at 290/340 nm excitation/emission wavelengths corresponded to an increase in corneal stiffness following treatment. It may be possible to use fluorescence spectral changes of endogenous corneal crosslinks to evaluate mechanical stiffness changes non-invasively.
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The mechanophysiology of tissues in the posterior eye have been implicated for diseases such as myopia and glaucoma. For example, the eye-globe shape, and consequently optical axial length, can be affected by scleral stiffness. In glaucoma, an elevated intraocular pressure is the primary risk factor for glaucoma, which is the 2nd most prevalent known cause of blindness. Recent work has shown that biomechanical properties of the optic nerve are critical for the onset and progression of glaucoma because weak tissues cause large displacements in the optic nerve, causing tissue damage. In this work, we utilize air-pulse optical coherence elastography (OCE) to quantify the spatial distribution of biomechanical properties of the optic nerve, its surrounding tissues, and the posterior sclera. Air-pulse measurements were made in a grid on in situ porcine eyes in the whole eye-globe configuration as various IOPs. The OCE-measured displacement process was linked to tissue stiffness by a simple kinematic equation. The results show that the optic nerve and peripapillary sclera are much stiffer than the surrounding sclera, and the stiffness of the optic nerve and peripapillary sclera increased as a function of IOP. However, the stiffness of the surrounding sclera did not dramatically increase. Our results show that understanding the dynamics of the biomechanical properties of the eye are critical to understand the aforementioned diseases and may provide additional information for assessing visual health and integrity.
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Brillouin spectroscopy is able to measure material’s mechanical properties by analyzing the optical spectrum of acoustically-induced light scattering within a sample. In the past decade, the development of high-resolution Brillouin spectrometers based on virtually-imaged phased array (VIPA) has greatly increased the spectral detection efficiency thus enabling mechanical characterization of biological tissue and biomaterials. Further improvements in spectrometer performances have enabled in vivo measurements at safe power levels and 2D/3D imaging of biological cells. However, it remains a slow technique compared to other imaging modalities, because only one point of the sample can be measured by the traditional backward-scattering configuration at a time. In this work, we demonstrate a parallel detection configuration with 90-degree geometry where the Brillouin shift of hundreds of points in a line can be measured simultaneously. In a 1.1mm-by-1.5mm samples, this novel configuration effectively shortens the acquisition time of 2D Brillouin imaging from hours to ~30 seconds with spatial resolution of ~3um, thus making it a powerful technology for label-free mechanical characterization of tissue and biomaterials.
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Brillouin spectroscopy is a noncontact technique for characterizing the mechanical properties of materials. Typically, Brillouin spectrometers have been realized using scanning Fabry–Perot spectrometers that measure, with long acquisition times, spontaneous Brillouin scattering from the samples. In the last few years, the use of virtually imaged phase array (VIPA) etalons for constructing Brillouin spectrometers has enabled to acquire spontaneous Brillouin spectra <1,000-fold faster than with scanning Fabry–Perot spectrometers, opening up new means for high-speed Brillouin analysis of materials.
In this talk, we will present a different approach for high-speed Brillouin material analysis. The method uses continuous-wave stimulated Brillouin scattering (CW-SBS) to measure stimulated Brillouin gain (SBG) spectra of materials at <100 milliseconds – up to 100-fold faster than with existing CW-SBS spectrometers. The SBS spectrometer comprises two nearly counter-propagating single-frequency lasers at 780 nm whose frequency detuning is scanned through the material Brillouin shift. SBG is detected via an ultra-narrowband hot rubidium-85 vapor notch filter and a lock-in detector, resulting in an improved signal-to-noise ratio that enables to significantly shorten acquisition times. We will show that this improvement, combined with micrometer-step-size spatial scanning of the sample, provides precise Brillouin profiles of layered liquids at 30-milliseconds pixel-dwell-time, facilitating Brillouin profilometry analysis of materials at high speed.
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Brillouin microscopy allows high–resolution mapping of the mechanical properties of a sample by measuring the spectra of acoustically induced light scattering therein, and thus has been widely investigated for biomedical application.
Measuring the Brillouin spectral shift is challenging when the light is focused onto the interfaces between two materials of different refractive index, because a sizeable portion of the incident light is Fresnel-reflected into the Brillouin spectrometer. To address this need, here, we designed a Brillouin confocal microscope in which the specular reflection at the interface between two materials is physically rejected without significant loss to the Brillouin signal.
To achieve this goal, we illuminate the sample with a small-diameter Gaussian beam focused by a high numerical aperture objective lens. In the collection path, the beam reflected from the sample has the same diameter as the incident beam, while the scattered light beam is as large as the clear aperture of the microscope objective. Therefore, using a small blocking filter allows to efficiently reject the reflected light.
We calculated the tradeoff between extinction improvement and signal loss when the diameter of the blocking filter is changed. Experimentally, we demonstrated extinction improvement of over 60dB with only 30% signal loss while achieving submicron resolutions.
This innovation can be useful for in vivo measurements of the cornea to avoid artifacts in the epithelium and anterior portions of the stroma, as well as to investigate cells cultured on glass coverslips without necessity of index-matching materials.
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Brillouin microscopy is an emerging technique in biomedical imaging capable of non-invasive assessing viscoelastic properties on a microscopic scale. In this report, we outline the latest developments in Brillouin spectroscopy instrumentation and applications in an attempt to anticipate the future impact areas of this new imaging modality.
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Obesity and overweight are accompanied by an enlargement of adipocytes, which is commonly related to the increasing number or size of lipid droplets within the cells. Some studies have shown that the accumulation of lipid droplets within adipocytes results in their increased stiffness. Recently, Brillouin microspectroscopy has been introduced as a nondestructive method of imaging the elasticity of cells. Unlike other imaging modalities, it is capable of assessing the elastic properties on both tissue- and cell levels. In this study, Brillouin spectroscopy was used to measure the elasticity changes in response to accumulation of lipid droplets within adipocyte during adipogenesis. The cell line used in the study is 3T3-L1, with chemically-induced differentiation from pre-adipocytes to mature adipocytes. The Brillouin shift measurements of the cells before and after differentiation indicate that the stiffness of adipocytes increases due to accumulation of lipid droplets. The results are in agreement with previous atomic force microscopy (AFM) nanoindentation studies. Brillouin microspectroscopy is a technique suitable for measuring the changes of elasticity of adipocytes in response to lipid droplet accumulation.
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Mechanical characterisation of biomaterials provides the basis for investigating disease-related changes in the biomechanical properties of living tissues and cells. Brillouin microscopy offers a non-invasive and label-free method to measure material properties. Briefly, Brillouin scattering involves energy exchange between photons and acoustic phonons, resulting in an optical frequency shift of the scattered light. This shift is proportional to the speed of sound in the material, and consequently to the longitudinal elastic modulus (M). However, it is unclear how Brillouin measurements, which characterize the mechanical response at GHz frequencies, relate to mechanical properties measured at much lower frequencies (~1 Hz) relevant to physiological conditions. Furthermore, as most biomaterials are hydrated, it remains unclear how the relative incompressibility of water influences the acoustic wave speed so as to affect Brillouin measurements of hydrated biomaterials.
In this study, we aim to establish the relationship between Brillouin frequency shift, acoustic wave speed and quasi-static elastic modulus of hydrogels of varying stiffness. Hydrogels are homogeneous and isotropic materials that mimic the poroelastic nature of biological tissues. Each measurement probes the mechanics of hydrogels in a significantly different frequency range: GHz for Brillouin imaging, MHz for ultrasound and Hz for unconfined compression tests. The acoustic wave speed falls into range from 1490 to 1533 m/s in both MHz (ultrasound) and GHz (Brillouin) frequency ranges. The quasi-static modulus correlates positively with Brillouin frequency shift, increasing from 6 to 54 kPa. All the results indicate the measurements obtained by Brillouin microscopy are capable of representing the material properties of hydrogels in quasi-static condition.
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Two-photon polymerization and crosslinking are commonly used methods for microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, to cellular scaffolds. However, the use of two-photon processes for precise, internal modification of biological tissues has not yet been reported. One of the major challenges has been a lack of appropriate tools to monitor and characterize crosslinked regions nondestructively.
Here, we demonstrate spatially selective two-photon collagen crosslinking (2P-CXL) in intact tissue for the first time. Using riboflavin photosensitizer and femtosecond laser irradiation, we crosslinked a small volume of tissue within animal corneas. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Using confocal Brillouin microscopy, we measured local changes in longitudinal mechanical moduli and visualized the cross-linked pattern without perturbing surrounding non-irradiated regions. 2P-CXL-induced tissue stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high spatial resolution, with broad implications in ophthalmology, laser surgery, and tissue engineering.
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VIPA (Virtually Imaged Phased Array) based spectrometers are now routinely favoured over other types of spectrometers (such as scanning Fabry-Perot) for Brillouin imaging because VIPAs permit higher data acquisition speeds as compared to others. However, higher speeds mean lower photon counts at the camera used to acquire the spectra. The quality of optical components used is also important and have profound effect on the quality of the spectrum. Yet, these issues have not been addressed by various groups doing Brillouin imaging around the world. In this talk we examine the effect of the various optical components on the overall performance of the spectrometer both in one and two stage configuration. We define information content in the measured spectra and using information theoretic approach determine system parameters under various design conditions. We show for example, the spherical aberration imparted by the plano-convex cylinder lens usually placed at the entrance of the spectrometer reduces signal quality but it otherwise does not affect the accuracy of measurements. On the other hand, aberrations introduced by lenses further down the optical train may result in significant loss in localisation accuracy of the spectra. Our approach will aid users of VIPA based spectrometers designing better quality systems.
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Brillouin imaging has recently emerged as a powerful technique for its ability to give insight to the mechanical properties of biomaterial. It exploits inelastic scattering of light by acoustic vibrations and maps the tissue stiffness point by point with micron resolution. The non-invasive, real-time nature of the measurements also makes it a potent candidate for in-vivo imaging of live cells and tissues. This, however, has to rely on a compact and flexible apparatus, a Brillouin endoscope, for remote access to specimen parts.
One of the main challenges encountered in the construction of Brillouin endoscope is that the inelastic scattering in the fibre conduit itself is orders of magnitude stronger than the Brillouin signal scattered by the specimen. This is because the length of the fibre endoscope (meters) is orders of magnitude larger than the imaging volume (microns). The problem can be overcome if the scattered light is collected by a separate fibre and does not mix with the fibre scattering inside the delivery channel.
Here we present an all-fibre integrated Brillouin microspectroscopy system that exploits the paths separation between delivery and collection channels. The experimental setup consists of a pair of standard silica single-mode fibres coupled to a graded-index lens and illuminated with a 671nm continuum wavelength source. We test our system performance on liquid samples of water and ethanol and confirm Brillouin shifts of 5.9 GHz and 4.6 GHz, respectively. More importantly, we do not observe any signals corresponding to Brillouin shift in the fibre, in agreement with expectation.
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Polarisation sensitive optical coherence tomography (PS-OCT) offers additional intrinsic contrast to probe differences between healthy tissue and cancer that are often barely visible due to limited scattering contrast in an OCT image. PS-OCT reconstructs tissue birefringence from phase-sensitive measurements of orthogonal polarisation components of backscattering. In material science, polarisation has been used to study stress distribution, including the birefringence induced by stress in an otherwise isotropic material. Similar effects in biological tissues have not been well studied yet; however, may have application to tissues subjected to stress, e.g., tendons, muscles, lens, cornea or airway smooth muscle (ASM). The objective of this work is to explore stress-induced birefringence in tissue. We employ an advanced swept source-based PS-OCT system capable of measurement of tissue local polarisation properties. The sample in both cases is illuminated with orthogonal, passively depth-encoded polarisation states. Light returning from the tissue is detected via a polarisation-diversity detection module and a Mueller formalism is used to reconstruct polarisation properties (including retardation, diattenuation, and depolarisation) of the tissue. In this study, we demonstrate the measurement of stress-induced birefringence in phantoms and in soft tissues with polarisation sensitive optical coherence tomography.
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Phase-resolved optical coherence tomography (OCT), a functional extension of OCT, provides depth-resolved phase information with extra contrast. In cardiology, changes in the mechanical properties have been associated with tissue remodeling and disease progression. Here we present the capability of profiling structural deformation of the sample in vivo by using a highly stable swept source OCT system The system, operating at 1300 nm, has an A-line acquisition rate of 200 kHz. We measured the phase noise floor to be 6.5 pm±3.2 pm by placing a cover slip in the sample arm, while blocking the reference arm. We then conducted a vibrational frequency test by measuring the phase response from a polymer membrane stimulated by a pure tone acoustic wave from 10 kHz to 80 kHz. The measured frequency response agreed with the known stimulation frequency with an error < 0.005%. We further measured the phase response of 7 fresh swine hearts obtained from Green Village Packing Company through a mechanical stretching test, within 24 hours of sacrifice. The heart tissue was cut into a 1 mm slices and fixed on two motorized stages. We acquired 100,000 consecutive M-scans, while the sample is stretched at a constant velocity of 10 um/s. The depth-resolved phase image presents linear phase response over time at each depth, but the slope varies among tissue types. Our future work includes refining our experiment protocol to quantitatively measured the elastic modulus of the tissue in vivo and building a tissue classifier based on depth-resolved phase information.
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The primary effect of scarring is the loss of function in the affected area. Scarring also leads to physical and psychological problems that could be devastating to the patient’s life. Currently, scar assessment is highly subjective and physician dependent. The examination relies on the expertise of the physician to determine the characteristics of the scar by touch and visual examination using the Vancouver scar scale (VSS), which categorizes scars depending on pigmentation, pliability, height and vascularity. In order to establish diagnostic guidelines for scar formation, a quantitative, accurate assessment method needs to be developed. An instrument capable of measuring all categories was developed; three of the aforementioned parameters will be explored. In order to look at pliability, a durometer which measures the amount of resistance a surface exerts to prevent the permanent indentation of the surface is used due to its simplicity and quantitative output. To look at height and vascularity, a profilometry system that collects the location of the scar in three-dimensions and laser speckle imaging (LSI), which shows the dynamic changes in perfusion, respectively, are used. Gelatin phantoms were utilized to measure pliability. Finally, dynamic changes in skin perfusion of volunteers’ forearms undergoing pressure cuff occlusion were measured, along with incisional scars.
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In elastography, quantitative elastograms are desirable as they are system and operator independent. Such quantification also facilitates more accurate diagnosis, longitudinal studies and studies performed across multiple sites. In optical elastography (compression, surface-wave or shear-wave), quantitative elastograms are typically obtained by assuming some form of homogeneity. This simplifies data processing at the expense of smearing sharp transitions in elastic properties, and/or introducing artifacts in these regions.
Recently, we proposed an inverse problem-based approach to compression OCE that does not assume homogeneity, and overcomes the drawbacks described above. In this approach, the difference between the measured and predicted displacement field is minimized by seeking the optimal distribution of elastic parameters. The predicted displacements and recovered elastic parameters together satisfy the constraint of the equations of equilibrium. This approach, which has been applied in two spatial dimensions assuming plane strain, has yielded accurate material property distributions.
Here, we describe the extension of the inverse problem approach to three dimensions. In addition to the advantage of visualizing elastic properties in three dimensions, this extension eliminates the plane strain assumption and is therefore closer to the true physical state. It does, however, incur greater computational costs. We address this challenge through a modified adjoint problem, spatially adaptive grid resolution, and three-dimensional decomposition techniques. Through these techniques the inverse problem is solved on a typical desktop machine within a wall clock time of ~ 20 hours. We present the details of the method and quantitative elasticity images of phantoms and tissue samples.
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Compression optical coherence elastography (OCE) enables rapid acquisition with high resolution over fields of view relevant to many clinical applications. Compression OCE typically provides a relative measure of mechanical properties; however, we have recently demonstrated a technique which quantifies stiffness via a compliant layer, termed quantitative OCE. In quantitative OCE, stiffness is reported as a tangent modulus, which is a surrogate for Young’s modulus at a given preload in non-linear elastic material. In biological tissues, which are typically non-linear elastic, values of stiffness reported through quantitative OCE could be over- or under-estimated, and are heavily biased by the arbitrary bulk preload applied to that region.
We present a method to measure tissue nonlinearity locally, by preforming compression OCE at multiple preloads ranging from 2% to 40%. We show, through presentation of 2D quantitative elastograms, that compression OCE has the potential to measure the non-linear stiffness in tissue mimicking phantoms and biological tissue. Further, intrinsic mechanical contrast in tissue is dependent upon its preload. By tailoring tissue preload, we demonstrate improved contrast between benign and tumor tissue in a murine liver carcinoma model.
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We demonstrate a computationally-efficient optical coherence elastography (OCE) method based on fringe washout. By introducing ultrasound in alternating depth profile, we can obtain information on the mechanical properties of a sample within acquisition of a single image. This can be achieved by simply comparing the intensity in adjacent depth profiles in order to quantify the degree of fringe washout. Phantom agar samples with various densities were measured and quantified by our OCE technique, the correlation to Young’s modulus measurement by atomic force micrscopy (AFM) were observed. Knee cartilage samples of monoiodo acetate-induced arthiritis (MIA) rat models were utilized to replicate cartilage damages where our proposed OCE technique along with intensity and birefringence analyses and AFM measurements were applied. The results indicate that our OCE technique shows a correlation to the techniques as polarization-sensitive OCT, AFM Young’s modulus measurements and histology were promising. Our OCE is applicable to any of existing OCT systems and demonstrated to be computationally-efficient.
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Phase-sensitive optical coherence tomography (OCT) is used for visualizing dynamic and cumulative strains and corneashape changes during laser-produced tissue heating. Such non-destructive (non-ablative) cornea reshaping can be used as a basis of emerging technologies of laser vision correction. In experiments with cartilaginous samples, polyacrilamide phantoms and excised rabbit eyes we demonstrate ability of the developed OCT system to simultaneously characterize transient and cumulated strain distributions, surface displacements, scattering tissue properties and possibility of temperature estimation via thermal-expansion measurements. The proposed approach can be implemented in perspective real-time OCT systems for ensuring safety of new methods of laser reshaping of cornea.
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Embryos undergo dramatic changes in size, shape, and mechanical properties during development, which is regulated by both genetic and environmental factors. Quantifying mechanical properties of different embryonic tissues may represent good metrics for the embryonic health and proper development. Alternations and structure coupled with biomechanical information may provide a way for early diagnosis and drug treatment of various congenital diseases. Many methods have been developed to determine the mechanical properties of the embryo, such as atomic force microscopy (AFM), ultrasound elastography (UE), and optical coherent elastography (OCE). However, AFM is invasive and time-consuming. While UE and OCE are both non-invasive methods, the spatial resolutions are limited to mm to sub-mm, which is not enough to observe the details inside the embryo. Brillouin microscopy can potentially enable non-invasive measurement of the mechanical properties of a sample by measuring the spectra of acoustically induced light scattering therein. It has fast speed (~0.1 second per point) and high resolution (sub-micron), and thus has been widely investigated for biomedical application, such as single cell and tissue. In this work, we utilized this technique to characterize the mechanical property of an embryo. A 2D elasticity imaging of the whole body of an E8 embryo was acquired by a Brillouin microscopy, and the stiffness changes between different organs (such as brain, heart, and spine) were shown. The elasticity maps were correlated with structural information provided by OCT.
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Cellular Biomechanics and Applications: Joint Session with Conferences 10067 and 10074
At the cell - extracellular matrix interface, physiologically important traction forces exerted by angiogenic sprouts can be investigated indirectly by mapping the consecutive matrix deformations. In this paper we present an approach to study these forces in three dimensions and with high time resolution. The technique employs lightsheet microscopy, in which a sheet of light is used to illuminate the sample - resulting in z-sectioning capability, superior image recording speed and reduced phototoxicity.
For this study, human umbilical vein endothelial cells (HUVEC) are transduced with a LifeAct adenoviral vector to visualize the actin cytoskeleton during live sprouting into a collagen type I hydrogel. The calculation of the matrix deformations is formulated as a B-spline-based 3D non-rigid image registration process that warps the image of beads inside the stressed gel to match the image after stress relaxation.
Using this approach we study the role of fast moving actin filaments for filopodia- and tip-cell dynamics in 3D under chemically defined culture conditions such as inhibited acto-myosin force generation. With a time resolution in the range of ten seconds, we find that our technique is at least 20 times faster than conventional traction force microscopy based on confocal imaging. Ultimately, this approach will shed light on rapid mechano-chemical feedback mechanisms important for sprouting angiogenesis.
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There is an unmet need in tissue engineering for non-invasive, label-free monitoring of cell mechanical behaviour in their physiological environment. Here, we describe a novel optical coherence phase microscopy (OCPM) set-up which can map relative cell mechanical behaviour in monolayers and 3D systems non-invasively, and in real-time. 3T3 and MCF-7 cells were investigated, with MCF-7 demonstrating an increased response to hydrostatic stimulus indicating MCF-7 being softer than 3T3. Thus, OCPM shows the ability to provide qualitative data on cell mechanical behaviour. Quantitative measurements of 6% agarose beads have been taken with commercial Cell Scale Microsquisher system demonstrating that their mechanical properties are in the same order of magnitude of cells, indicating that this is an appropriate test sample for the novel method described.
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Optical Clearing and Biomechanics: Joint Session with Conferences 10063 and 10067
The skull bone, a curved solid multilayered plate protecting the brain, constitutes a big challenge for the use of ultrasound-mediated techniques in neuroscience. Ultrasound waves incident from water or soft biological tissue are mostly reflected when impinging on the skull. To this end, skull properties have been characterized for both high-intensity focused ultrasound (HIFU) operating in the narrowband far-field regime and optoacoustic imaging applications. Yet, no study has been conducted to characterize the near-field of water immersed skulls. We used the thermoelastic effect with a 532 nm pulsed laser to trigger a wide range of broad-band ultrasound modes in a mouse skull. In order to capture the waves propagating in the near-field, a thin hydrophone was scanned in close proximity to the skull's surface. While Leaky pseudo-Lamb waves and grazing-angle bulk water waves are clearly visible in the spatio-temporal data, we were only able to identify skull-guided acoustic waves after dispersion analysis in the wavenumber-frequency space. The experimental data was found to be in a reasonable agreement with a flat multilayered plate model.
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A robust method to measure viscosity of microquantities of biological samples, such as blood and mucus, could lead to a better understanding and diagnosis of diseases. Microsamples have presented persistent challenges to conventional rheology, which requires bulk quantities of a sample. Alternatively, fluid viscosity can be probed by monitoring microscale motion of particles. Here, we present a decorrelation-based method using M-mode phase-sensitive optical coherence tomography (OCT) to measure particle Brownian motion. This is similar to previous methods using laser speckle decorrelation but with sensitivity to nanometer-scale displacement. This allows for the measurement of decorrelation in less than 1 millisecond and significantly decreases sensitivity to bulk motion, thereby potentially enabling in vivo and in situ applications. From first principles, an analytical method is established using M-mode images obtained from a 47 kHz spectral-domain OCT system. A g(1) first-order autocorrelation is calculated from windows containing several pixels over a time frame of 200-1000 microseconds. Total imaging time is 500 milliseconds for averaging purposes. The autocorrelation coefficient over this short time frame decreases linearly and at a rate proportional to the diffusion constant of the particles, allowing viscosity to be calculated. In verification experiments using phantoms of microbeads in 200 µL glycerol-water mixtures, this method showed insensitivity to 2 mm/s lateral bulk motion and accurate viscosity measurements over a depth of 400 µm. In addition, the method measured a significant decrease of the apparent diffusion constant of soft tissue after formalin fixation, suggesting potential applications in mapping tissue stiffness.
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Modern dentistry can not exist without dental implantation. This work is devoted to study of the "bone-implant" system and to optimization of dental prostheses installation. Modern non-invasive methods such as MRI an 3D-scanning as well as numerical calculations and 3D-prototyping allow to optimize all of stages of dental prosthetics. An integrated approach to the planning of implant surgery can significantly reduce the risk of complications in the first few days after treatment, and throughout the period of operation of the prosthesis.
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Over 2 million dermal filler procedures are performed each year in the USA alone, and this figure is only expected to increase as the aging population continues to grow. Dermal filler treatments can last from a few months to years depending on the type of filler and its placement. Although adverse reactions are rare, they can be quite severe due to ischemic events and filler migration. Previously, techniques such as ultrasound or magnetic resonance imaging have been used to evaluate the filler injections. However, these techniques are not practical for real-time filler injection guidance due to limitations such as the physical presence of the transducer. In this work, we propose the use of optical coherence tomography (OCT) for image-guided dermal filler injections due to the high spatial and temporal resolution of OCT. In addition, we utilize a noncontact optical coherence elastography (OCE) technique, to evaluate the efficacy of the dermal filler injection. A grid of air-pulse OCE measurements was taken, and the dynamic response of the skin to the air-pulse was translated to the Young’s modulus and shear viscosity. Our results show that OCT was able to visualize the dermal filler injection process, and that OCE was able to localize the dermal filler injection sites. Combined with functional techniques such as optical microangiography, and recent advanced in OCT hardware, OCT may be able to provide real-time injection guidance in 3D by visualizing blood vessels to prevent ischemic events.
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Photothermal optical coherence tomography (PT-OCT) employs a secondary intensity-modulated photothermal laser to create modulated thermal strains that cause variations of the refractive index in the proximity of absorbing chromophores. These variations are directly detected with phase-sensitive OCT and offer insight to the molecular composition and thermo-elastic properties of the sample. Here, we define optimal PT laser modulation parameters by investigating the effect of PT laser power and modulation frequency on the ensuing thermal waves and thermal waves’ impact on the spatial resolution of PT-OCT imaging based on numerical simulations of PT-OCT and samples containing point absorbers.
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The directionality of collagen fibers across the anterior cruciate ligament (ACL) as well as the insertion of this key ligament into bone are important for understanding the mechanical integrity and functionality of this complex tissue. Quantitative analysis of three-dimensional fiber directionality is of particular interest due to the physiological, mechanical, and biological heterogeneity inherent across the ACL-to-bone junction, the behavior of the ligament under mechanical stress, and the usefulness of this information in designing tissue engineered grafts. We have developed an algorithm to characterize Optical Coherence Tomography (OCT) image volumes of the ACL. We present an automated algorithm for measuring ligamentous fiber angles, and extracting attenuation and backscattering coefficients of ligament, interface, and bone regions within mature and immature bovine ACL insertion samples. Future directions include translating this algorithm for real time processing to allow three-dimensional volumetric analysis within dynamically moving samples.
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This study investigates the feasibility of quantifying the viscoelasticity of soft tissues with a dynamic noncontact optical coherence elastography (OCE) technique coupled with a Rayleigh wave model. Spectral analysis of an air-pulse induced elastic wave as measured by OCE provided the elastic wave dispersion curve. The dispersion curve was fitted to an analytical solution of the Rayleigh wave model to determine the Young’s modulus and shear viscosity of samples. In order to validate the method, 10% gelatin phantoms with and without different concentrations of oil were prepared and tested by OCE and mechanical testing. Results demonstrated that the elasticities as assessed by the Rayleigh wave model generally agreed well with mechanical testing, and that the viscosity in the phantom with oil samples was higher than the phantoms without oil, which is in agreement with the literature. Further, this method was applied to quantify the viscoelasticity of chicken liver. The Young’s modulus was E=2.04±0.88 kPa and the shear viscosity was η=1.20±0.13 Pa·s with R2=0.96±0.04 between the OCE-measured dispersion curve and Rayleigh wave model analytical solution. Combining OCE and the Rayleigh wave model shows promise as an effective tool for noninvasively quantifying the viscoelasticity of soft tissues.
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