This PDF file contains the front matter associated with SPIE Proceedings Volume 10880, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

In a typical experiment in compression elastography a sample is compressed to an overall strain of about 1-5%, and then perturbed with a much smaller strain in the range of 0.05%-0.1%. The displacement field corresponding to this perturbative excitation is measured using phase-sensitive OCT. This three-dimensional perturbative displacement data carries within it a wealth of information regarding the volumetric distribution of linear elastic properties of tissue. In this talk we will describe a class of iterative algorithms that use this data input and generate volumetric maps of linear elastic properties of biological specimens. The main idea behind these algorithms is to pose this inverse problem as a constrained minimization problem and use adjoint equations, spatially adaptive resolution and domain decomposition techniques to solve this problem. We will also consider the case when the overall compression and the perturbative excitation steps are repeated several times while increasing the overall strain. For example, a sequence wherein the overall strain varies as 2, 4, 6, 8, and 10%, and each increment is followed by a small perturbative excitation. The measured displacement field corresponding to this small excitation is sensitive to the nonlinear elastic properties of the specimen, which determine how its elastic modulus varies with increasing strain. We will extend the algorithms designed to infer the linear elastic properties of biological specimens to infer these non-linear elastic properties. We will demonstrate our ability to infer linear and nonlinear elastic properties on tissue-phantom, and ex-vivo and in-vivo tissue samples.

Myocardial infarction (MI) is a leading cause of death and decrease of quality of life in the USA. An immense amount of research and development has been focused on the molecular mechanisms associated with MI, which have led to numerous therapies for treating and repairing cardiac tissue after MI. However, there is a relative lack of information about the changes in cardiac tissue biomechanical properties due to MI. Therefore, there is a direct need for techniques that can measure cardiac tissue biomechanical properties, which would help further develop our understanding of tissue biomechanical dynamics associated with MI as well as aid in the development of therapies that consider biomechanical properties. In this work we utilize noncontact dynamic optical coherence elastography (OCE) to evaluate the changes in cardiac biomechanical properties 6 weeks after MI in a mouse model. We performed complementary analysis based on elastic wave propagation and damping analyses. Our results show that the left ventricle cardiac tissue became more isotropic and softer after 6 weeks in the MI-affected mice as compared to the sham mice based on the elastic wave propagation measurements. The damping measurements also showed that the MI-affected mice had softer left ventricles as compared to the sham mice. Moreover, the damping analysis was able to localize the boundary of the MI-damaged region. These results show that OCE can be a powerful tool for understanding the dynamics in biomechanical changes in murine cardiac tissue and could potentially reveal diseased areas for targeted therapies.

Systemic sclerosis (SSc) is an autoimmune disorder with high mortality due to excessive accumulation of collagen in the skin and internal organs. An accurate and early diagnosis is crucial to ensure effective treatment. Currently, the modified Rodnan skin score (mRSS) is the gold standard for evaluating dermal thickening during SSc onset and progression. However, obtaining the mRSS can be time consuming, and the score has noticeable inter- and intraobserver variabilities. Optical coherence elastography (OCE) is an emerging technique for measuring soft tissue biomechanical properties completely noninvasively. In this work, we demonstrate the first use of OCE combined with analysis of the OCT signal slope (OCTSS) for sclerosis detection in the dorsal forearm of 12 patients. A comparison to clinical diagnoses including dermal thickness assessed by histology, mRSS, and site specific mRSS (SMRSS). Results of both optical assessments demonstrated high correlation (OCE: 0.78 and OCTSS: 0.65) with SMRSS as performed by an experienced physician. Importantly, the correlation of both proposed parameters with the dermal thickness (OCTSS: r=0.78 and OCE: r=0.74) outperformed the SMRSS assessment (r=0.57), demonstrating the effectiveness of using OCT/OCE for monitoring the disease severity of SSc.

Disease alters both the micro-structural and micro-mechanical properties of tissue. These changes in mechanical properties manifest at the macro-scale, enabling clinicians to diagnose disease through manual palpation. This has been a primary motivator for elastography, however, in the development of elastography, manual palpation’s key advantages of dexterity and simplicity are lost. Combining manual palpation and elastography would, potentially, preserve these advantages whilst also providing clinicians with quantitative, high-resolution imaging necessary to overcome the subjective and inherently low spatial resolution of manual palpation. Optical coherence elastography (OCE) is particularly well-suited to imaging subtle changes in mechanical properties owing to its high spatial resolution and sensitivity to nanometer-scale displacement. Additionally, as OCE is an optics-based technique, it is readily implemented in compact probes, such as those already demonstrated in needles and endoscopes. Here, we propose a finger-mounted OCE probe, based on quantitative micro-elastography (QME) in a forward-facing configuration, and using the operator’s finger to apply compressive loading. A compliant silicone layer, with known mechanical properties, is placed on the sample and enables quantification of the sample’s elasticity. This finger-mounted probe is designed to preserve the dexterity of manual palpation, whilst providing quantitative, high-resolution images. In this study, we demonstrate the accuracy of finger-mounted OCE to be >70% in measuring the elasticity of tissue mimicking phantoms, and highlight the ability to delineate materials with different mechanical properties. Further, we present results performed on kangaroo muscle tissue and outline the developments required to translate this into a clinically feasible diagnostic tool.

Introduction: Optical coherence tomography (OCT) is a high-resolution imaging modality which can be used to acquire detailed elastograms of biological tissue. In this investigation, we demonstrate the use of OCT to generate µm-scale strain maps of articular cartilage (AC) under compressive and shear deformations. AC is a dense connective tissue which provides a low-friction surface in synovial joints. The specific alignment of collagen fibrils and proteoglycans (which contribute primarily to shear and compressive stiffness, respectively) give rise to depth-dependent mechanical properties. Methods: Six 6mm diameter samples of articular cartilage were harvested from a calf femur. A custom-designed biaxial loading apparatus applied compressive and shear displacements. Three-dimensional images of the tissue were obtained using a spectral-domain OCT system as the sample was loaded at constant rate of displacement. Both speckle-tracking and phase-shift methods were used to generate strain maps from these images. Results: Under both shear and compressive loading, clear differences in local strain distribution were observed between the superficial, transitional, and radial zones of the cartilage. In shear, the superficial/transitional zones are stiffest while in compression these regions are more compliant than the radial zone. These distributions correspond with existing literature and the known orientation of collagen in the AC. Conclusion: It is feasible to rapidly acquire strain maps in AC using OCT. This technique may be extended to high-throughput screening to nondestructively determine the functionality and failure modes of engineered AC as compared to native tissue.

Pathological change tends to alter tissue mechanical properties, e.g. tissue stiffness. Current elastography technology use tissue stiffness as a signature to diagnose and localize diseases. Our team focus on vibrational optical coherence elastography (OCE) for its capability to increase signal to noise ratio as well as its high resolution comparing other elastography modalities. The result highly relies on the stimulation frequency for vibrational mode might change as frequency varies. A proper frequency range is required however, there hasn’t been a consensus among the research groups. In order to find the proper frequencies, several parameters measured from real experiment are input in transient model of ANSYS to simulate vibrational pattern of the sample with driving frequencies vary from 100Hz to 1000Hz. An upper limit of frequency has been discovered finally.

Demand to reveal fundamental micro-mechanical properties is driven by a growing evidence that altered cellular processes in aging-associated disease environments are caused by a change in the regulating biomechanics. Unlike standard elastography techniques, Brillouin microscopy has shown great capabilities to non-invasively assess the biomechanics in the volume of biological samples, such as the lens cornea, atherosclerotic plaques and cells. Spectral contrast is key in Brillouin microscopy to optically probe biological systems, where the elastic Rayleigh scattering and specular reflection are orders of magnitude greater than the Brillouin signal. Here, we developed a noncontact and label-free imaging method, named background-deflection Brillouin (BDB) microscopy, to investigate the three-dimensional intracellular biomechanics at a sub-micron resolution. Our method exploits diffraction to achieve an unprecedented 10,000-fold enhancement in the spectral contrast of single-stage spectrometers, enabling the first direct biomechanical analysis on intracellular stress granules containing ALS mutant FUS protein in fixed cells. Our results provide insights on an aberrant liquid-to-solid phase transition observed in in-vitro reconstituted droplets of FUS protein, which has been recently proposed as a possible pathogenic mechanism for ALS.

Glaucoma is the leading cause of irreversible blindness in the world, with approximately 75 million patients suffering from the disease. We know that elevated intraocular pressure (IOP) is a key risk factor for the disease, and that sustained lowering of IOP has therapeutic benefit, which point to the importance of biomechanics and mechanobiology in this disease. After providing background information about glaucoma, I will in this talk describe how imaging is helping our understanding of glaucoma. For example, we have used OCT anterior segment imaging to infer mechanical properties of the tissues responsible for controlling IOP, and to relate these properties to the function of these tissues. We have also used photoacoustic imaging to track the location of stem cells delivered into the eye in an attempt to refunctionalize tissues damaged in glaucoma. These efforts are highly collaborative, involving imaging scientists, surgeons, pharmacologists and biomedical engineers.

Laser-induced thermoelastic deformation can be an effective way to induce disturbances in soft biological tissues in elastography and photoacoustcs. A laser pulse results in rapid temperature increase, thermoelastic expansion and generation of compressional and shear waves in the tissue. After the expansion and wave attenuation, a quasi-steady state is reached. For several medical applications of elastography, laser-induced thermoelastic deformation has been proposed as a way to produce strain in the tissue to assess tissue mechanical properties. In combination with measuring tissue response using optical coherence tomography, such an approach could be an effective method for noncontact measurement of tissue mechanical properties. In our previous works we have derived a three-dimensional analytical solution for the quasi-steady state, when the tissue reaches equilibrium after the acoustic and shear waves have decayed. In this work, we consider dynamic tissue response at the moment of time immediately after the laser pulse. In the frequency domain, an analytical expression has been derived for the thermoelastic displacements and stresses caused by absorption of an axially symmetric laser beam. The solution was obtained for the Gaussian radial temperature profile on the upper surface of a viscoelastic half-space. The influence of the shear elastic properties on the elastic wave propagation and displacement profiles was evaluated. The proposed analytical solution could be used to model mechanical and photoacoustic tissue response to laser excitation, as well as to investigate the mechanism of photomechanical laser ablation. This study was supported by NIH grant R01EY022362.

In medicine, both pathological (e.g. cirrhosis) and non-pathological states (e.g. aging) can be characterized by changes in the mechanical properties of biological tissue. The use of optical techniques to measure and map the elastic properties of soft tissue, known as optical elastography, is an emergent field with applications in various clinical disciplines, including ophthalmology and dermatology. In this paper, a brief overview of optical elastography will be provided with a short taxonomy. Categories include appropriate types of tissue models (semi-infinite, single thin layer, composite stacks), clinical tasks (classification or estimation), and excitation modes (transient, continuous, quasi-static, or molecular shift). We will then discuss examples of current advances, including optical coherence elastography using reverberant shear wave fields and Brillouin microscopy. The examples will demonstrate how current and future techniques may address clinical needs. Advantages and disadvantages of these techniques will be presented, augmenting the framework of the categorization system. With emerging applications, the taxonomy may be expanded providing a roadmap to future techniques.

Cellular mechanical properties are known to influence both their cellular and subcellular functions. Whilst methods to assess cellular mechanics such as Atomic Force Microscopy (AFM) are already available, there is an emerging need to measure cellular mechanical properties in a label-free and contactless mode, to allow for long time monitoring of cell behaviour, and to enable measurements of cells embedded in extracellular matrix. In this study, we have employed Digital Holographic Microscopy (DHM) combined with the well-controlled application of hydrostatic pressure to study cellular mechanical properties in real-time and in a noncontact manner. Cyclic stress was applied non-destructively and non-invasively to pancreatic ductal adenocarcinoma Focal Adhesion Kinase (FAK) knockout cells (Panc47-1 -/-null) and their corresponding re-expressing clonal population (Panc47-1 +/+ wild type) within a 25cm² culture flask by a microfluidic pump 24h after seeding. Cyclic stress was successfully applied directly to cells, and corresponding change in volume was recorded in real-time at the nanometre scale for cell, yielding the mechanical properties of the cells. Change in amplitude and/or frequency of the stimuli was translated to corresponding cell response. Differences were observed in relative strain rates between the cell lines under investigation. We have described a novel method to perform optical elastography on live cells at single cell resolution in realtime and non-destructively. This allows for long-term monitoring of mechanical properties during cell proliferation and differentiation, and disease progress. This can be directly related to the biomechanical properties of cells.

Monitoring of the human retina temperature may be useful in the determination of its metabolic state, in the study of environmental damage, and for providing feedback during laser surgery. Due to its location the retina is inaccessible to contact temperature measurements and to standard thermal imaging. Here, we propose the use of a stimulated laser speckle imaging (sLSI) system to monitor thermal deformations in the retina and thus infer retina ground temperature. Our system utilizes laser speckle imaging in combination with a stimulating laser scanning system altering the speckle pattern. Measurements of time dependent speckle cross-correlation along with instantaneous and cumulative speckle shifts are related to the sample temperature. Temperature measurements of optical phantoms and biological media have been achieved and validated through thermocouple measurements of sample temperature.

We present a realization of real-time OCT-based strain mapping by estimating interframe phase-variation gradient using the developed "vector" method. This technique allows for mapping both fairly fast and large, as well as rather small strains, slowly-varying on intervals ~tens of minutes. Optimization of interframe interval for improving signal-to-noise ratio is discussed and experimentally demonstrated. Ultimate stability of strain estimation with the designed OCT setup is experimentally estimated using stable phantoms. Examples of spatially resolved maps of slowly-varying strains are demonstrated. The developed methods can be used in emerging techniques of laser-assisted modification of collagenous tissues (e.g., for such perspective application as fabrication of cartilaginous implants).

Elastography, sometimes referred as seismology of the human body, is an imaging modality recently implemented on medical ultrasound systems. It allows to measure shear waves within soft tissues and gives a tomography reconstruction of the shear elasticity. This elasticity map is useful for early cancer detection. A general overview of this field is given in the first part of the presentation as well as latest developments. The second part, is devoted to the application of time reversal or noise correlation technique in the field of elastography. The idea, as in seismology, is to take advantage of shear waves naturally present in the human body due to muscles activities to construct shear elasticity map of soft tissues. It is thus a passive elastography approach since no shear wave sources are used. In the third part some examples are provided using ultrasounds, MRI or optic to detect shear waves and reconstruct a speed tomography in a human liver, thyroid, brain, in a mouse eye and a single cell.

Incomplete excision of cancerous tissue is a major issue in breast-conserving surgery, with up to 30% of cases requiring re-excision. In vivo quantitative micro-elastography (QME) using a hand-held probe is a promising path towards improved intraoperative margin assessment, potentially improving removal of cancerous tissue during the initial procedure. QME is an OCE technique that requires a modified 3D OCT scan in which each lateral position is acquired in two states, differing by a small compressive axial deformation. Analysis of the axial strain between the two states generates a 3D micro-elastogram that facilitates identification of cancerous tissue. Compressive deformation is typically provided by a piezoelectric actuator. However, this approach presents significant disadvantages for hand-held scanning, most notably: the relatively large size of the actuator; high driving voltages; and the difficulty of hermetically sealing and sterilizing moving parts. Alternatively, deformation may be provided by manual compression, avoiding many of the issues associated with piezoelectric actuation. This approach has yet to be demonstrated in 3D, limiting its utility in surgical applications. Here, we present hand-held 3D QME using a manual compression technique. Our technique requires the user to apply a steadily varying pressure to the tissue in order to generate 3D micro-elastograms. We describe the signal processing developed to enable this approach and present results from both structured phantoms and freshly excised human breast tissue, validated by histology. Furthermore, we analyze repeatability by presenting results from multiple users and benchmark our technique against the piezoelectric-actuated approach.

In OCE, the link between measured displacement and elasticity is non-trivial in complex tissues and a number of simplifying assumptions regarding deformation are made to generate an elastogram. In compression OCE, for instance, elasticity is assumed to be inversely proportional to axial strain (the gradient of axial displacement with depth). However, this assumption relies on the tissue being mechanically uniform. This assumption is typically invalid and limits elastogram resolution. Despite this, few studies have explored OCE resolution in detail. Previously, OCE resolution has been reported laterally as the OCT resolution, and axially as the spatial range of displacement used to estimate axial strain. However, this describes only the ability to resolve axial strain. The ability to resolve features is also dependent on the interplay of mechanical deformation and the model with which it is analyzed. We present a framework for analyzing resolution in OCE, which combines a model of mechanical deformation, using finite-element analysis, with a model of the OCT system and signal processing, based on linear systems theory. We present simulated and experimental elastograms of tissue-mimicking phantoms, showing close correspondence, and demonstrate, for instance, that the resolution of a square 1-mm inclusion can vary, within one image, from 100 μm to 200 μm axially, and from 100 μm to 380 μm laterally. We demonstrate that axial and lateral resolution are directly related to inclusion size and mechanical contrast. Our framework may enable OCE systems to be tailored to specific applications and can be extended to other forms of OCE.

Colon pathologies including colon cancer and ulcerative colitis afflict hundreds of thousands of people in the United States. Clinical detection of colon diseases is generally performed through colonoscopy. However, these methods usually lack the sensitivity or resolution to detect diseased tissue at early stages. Even high resolution optical techniques such as confocal microscopy and optical coherence tomography (OCT) rely on structural features to detect anomalies in tissue, which may not be sufficient for early disease detection. If changes in tissue biomechanical properties precede morphological changes in tissue physiology, then mechanical contrast would enable earlier detection of disease. In this work, we utilized optical coherence elastography (OCE) to assess the biomechanical properties of healthy, cancerous, and colitis tissue. Additionally, the optical properties of each sample were also assessed as a secondary feature to distinguish tissue types. The Young’s modulus, as measured by the propagation of an elastic wave, of the healthy, cancerous, and colitis tissue was 10.8 ± 1.0 kPa, 7.12 ± 1.0 kPa, and 5.1 ± 0.1 kPa, respectively. The variations in the OCT signal intensity over depth, as measured by the slope-removed standard deviation of each A-scan was 5.8 ±.0.3 dB, 5.1 ± 0.4 dB, and 5.5 ± 0.2 dB for healthy, cancerous, and colitis tissue, respectively. This work shows OCT structural imaging combined with OCE can detect minute changes in colon tissue optical scattering and elastic properties, which may be useful for detection various colon diseases, such as colitis and colon cancer.

Coagulopathic bleeding in trauma and surgery increases the perioperative risk. Early dignosis of anticoagulant side-effects could avoid this risk. Rapid and accurate clot diagnosis system is important in assessment of hemodiluted blood coagulation. Routine coagulation analysis usually takes 30 to 60 minutes. Traditional ways for blood coagulation assessment includes thromboelastography and thromboelastometry (TEG/ROTEM). However, neither is ideal for assessing clot mechanical properties due to the poor sensitivity, repeatability and lack of common standardization. In this study, we develop a piezoelectric transducer optical coherence elastography (PZT-OCE) system that enables real-time measurement of viscosity in a drop of blood during coagulation process. During the blood coagulation process, the changes in viscous properties increase the attenuation coefficient. Consequently, coagulation metrics including the initial coagulation time and the clot formation rate can be developed by monitoring the attenuation coefficient in order to assess blood coagulation properties. The system compared blood coagulation metrics between samples with different concentrations of activator kaolin and hemodilution with either NaCl 0.9% or hydroxyethyl starch (HES) 6%. Higher concentration of activator kaolin resulted in a shorter initial coagulation time, and HES 6% caused a more pronounced dilutional hypocoagulation than NaCl 0.9%. The results show that PZT-OCE is sensitive to coagulation abnormalities and able to characterize blood coagulation status based on viscosity-related measurements, which can be used for point-of-care testing for diagnosis of coagulation disorders and monitoring of therapies.

Three-dimensional mapping of refractive index can help understanding several biomedical phenomena such as metastatic potential of tumor cells and alteration of crystalline lens, as well as decouple biomechanical measures in Brillouin microscopy from the refractive index of the sample. Current techniques to measure the distribution of refractive index rely on the determination of the optical phase delay induced by the sample. These approaches require knowing, measuring or assuming the geometrical path of the light, as well as accessing the sample from at least two sides, conditions not trivial for many biological samples. To overcome these limitations, we developed a spectroscopy technique that can measure the local refractive index in an absolute manner, without sampling the optical phase delay; to achieve this goal, we probed two co-localized Brillouin scattering geometries. In both configurations, the photons were phase-matched to the same phonon axis; as a result, the index of refraction is the only physical quantity that affects the ratio of the measured Brillouin frequency shifts, allowing a local, absolute measurement of the refractive index. We performed a proof-of-principle experiment of this method and demonstrated a refractive index accuracy of 0.001, with spatial resolution of ~5μm (lateral) by ~40 μm (axial). The confocal configuration ensures three-dimensional mapping with high resolution, and the epi-detection configuration allows access to the sample from the same side. This technique can potentially constitute a new approach in investigating biological phenomena providing both index of refraction and mechanical information with a single measurement.

Stiffness of the extra cellular matrix (ECM) is recognized as a key regulator of cancer cell proliferation, migration, and invasion. Therefore, technologies that enable non-invasive evaluation of ECM stiffness at cellular scales provide important insights into neoplastic progression. Laser Speckle Microrheology (LSM) is a novel optical tool for measuring tissue stiffness. In LSM, a laser beam illuminates the specimen and fluctuating speckle patterns are captured by a CMOS sensor. Spatio-temporal analysis of speckle intensity yields a map of viscoelastic modulus, G. We validated the accuracy, sensitivity, and dynamic range of LSM by preparing homogeneous gels of assorted viscoelastic properties and comparing LSM measurements with mechanical rheology. To assess the LSM resolution, substrates with micro-scale stiffness patterns were fabricated and tested. Next, we investigated the utility of LSM for mechanical evaluation of the ECM in human breast lesions. Results of phantom studies demonstrated a statistically significant, strong correlation between LSM and rheology (|G|: 30 Pa – 30 kPa, R=0.94, p<5×10-6). Moreover, |G| maps of micro-fabricated phantom, illustrated the capability of LSM in resolving mechanical heterogeneities below 50 µm. Results of tumor tissue measurements, further demonstrated the utility of LSM for micromechanical evaluation of the tumor ECM. To improve the resolution, we developed a novel spatio-temporal analysis of speckle series to obtain multiple sub-pixel shifted maps of the G, and reconstruct a resolution-enhanced |G| map. Studies in micro-fabricated phantoms demonstrated a 5-10 fold resolution enhancement. These results demonstrate competency of the LSM for answering key mechano-biological questions pertinent to caner pathogenies and progression.

Quantitative interferometric microscopy is a power non-invasive technique to extract quantitative cellular and tissue biomechanical and morphological information. On one hand, we will describe several generations of quantitative interferometric microscopic systems with improved spectral contrast, depth resolution, and enhanced sensitivity. In conjunction with these advances in optical imaging techniques, important biomedical applications have become possible. We will focus on the identification of biophysical markers of sickle red blood cells and the study of cancer cell nuclear mechanics in relationship to their metastasis potential.

Surgical correction of ametropias and the identification and management of corneal ectasias are separate but tightly intertwined issues of major significance. Corneal morphologic imaging (topography and tomography) is critical to identify corneal ectatic disorders such as keratoconus and to appropriately screen patients to determine suitability for corneal refractive surgery. A variety of devices and strategies have been used with varying degrees of success, but discrepancy exists in terms of the relative importance of various screening technologies and variables to identify the earliest manifestations of keratoconus. Placido based corneal topography, Scheimpflug imaging, and anterior segment optical coherence tomography, especially epithelial thickness variations, all play a significant role in identifying keratoconic eyes in earlier stages. Despite multiple available technologies, there remains a gap in identifying ectatic corneal disease at its earliest manifestation, and there remains significant controversy and discrepancy in the literature about the relative value of different evaluations in distinguishing keratoconus suspect eyes from normal populations. The latest research shows that combining technologies provides better discriminating capability than using any device in isolation. Early identification of corneal ectasias using current technology is critical, but current tests in the clinic are morphological, not biomechanical, and therefore do not allow a definitive diagnosis at the earliest stages, resulting in some patients incorrectly receiving refractive surgery while others lose vision before cross-linking treatment is initiated. Thus, the need for accurate identification of subclinical ectasia has never been greater. The next step in corneal imaging will address direct biomechanical measurements in an accurate, reproducible way.

Purpose: To measure the spatially resolved compressive stiffness properties under in vivo conditions to evaluate the effects of clinical corneal crosslinking (CXL) on patients with keratoconus. Methods: Patients with keratoconus who were scheduled to undergo CXL were imaged before (<1 week) and after (3 to 6 months) treatment. The study was approved under the Cleveland Clinic Institutional Review Board (IRB #13-213). The imaging procedure consists of a continuous compression with a flat glass plate while imaging with swept-source OCT. The frame-to-frame displacements were measured using speckle tracking. Maps of the first order fit of applied force vs cumulative axial displacement were created. Spatially averaged central anterior and posterior regions were defined to generate a relative stiffness value (k) expressing anterior properties relative to posterior stromal properties. Data previously collected from normal patients were also used for group comparison purposes. Results: Qualitative comparison of the color map representation showed significant differences in the distribution of compressive mechanical properties between all three patient types. Mean k-value were 1.129 ± 0.067 in normal eyes (n=12), 0.988 ± 0.089 in keratoconus eyes (n=8), and 1.27 ± 0.16 in keratoconus eyes after CXL (n=6, p<0.05 for all groups using Mann-Whitney U test). Conclusions: The spatial biomechanical effects of CXL are measurable with in vivo compressive OCE. The normal anterior to posterior stromal force/displacement ratio appears to be reduced in keratoconus and is increased to or even beyond normal levels after CXL due to selective stiffening of the anterior stroma.

The aim of this study was to develop a comprehensive biomechanical model to predict biomechanical properties of all ocular tissues and to compare the simulations with air-puff swept-source OCT data. We developed a novel rheological model of the whole eye. The cornea and the crystalline lens were modelled as a combination of spring and dashpot(s) to describe their viscoelastic properties. In addition, a mass element was included to model lenticular wobbling after air pulse. Finally, the eye retraction (depending on the fatty and muscle tissues behind the eye globe) was modeled by a parallel combination of a spring and dashpot elements, and a mass component described the weight of the eye. We measured deformation profiles of ocular components of the human eyes in-vivo using long-range SS-OCT instrument integrated with air-puff stimulation, which enables to visualize the dynamics of eye through its entire length and to measure the intraocular distances (SS-OCT ocular biometer). The deformations calculated from the model were fitted to the measured profiles data using Levenberg-Marquardt method. The rheological model allowed for predicting the displacements of the cornea and the crystalline lens (1.25mm and 0.115mm, respectively), the eye retraction (0.28mm) and the axial wobbling of the lens within 40ms. The developed model outcomes match well to experimental data of corneal and lenticular hysteresis curves identifying viscoelastic properties of the ocular tissues In conclusion, the proposed rheological model correctly predicts the effects observed with air-puff SS-OCT ocular biometer and it can be used in future modelling of the whole eye biomechanics.

Clinical assessment of corneal biomechanics currently relies on evaluation of the response of a cornea to an air-puff directed at its centre. Despite this method showing potential for identifying the presence of biomechanical abnormalities, it cannot be used to quantify corneal biomechanical properties and it provides limited spatial information. There is currently widespread interest in the development of techniques capable of spatially mapping corneal biomechanics, as access to this information could not only accelerate the diagnosis and enhance the treatment of keratoconus, through enabling customised and individualised treatment protocols; it could also permit technologies such as corneal crosslinking to be optimised to offer a minimally-invasive alternative to refractive surgery, lowering the risk of complications, such as corneal ectasia, associated with current procedures. Here we present a method, using speckle interferometric techniques, to measure the full-field displacement of the cornea in response to intra-ocular pressure changes equivalent to those that occur during the cardiac cycle, over a measurement time of several milliseconds. To demonstrate its effectiveness for biomechanical assessment, ex vivo measurements were performed for 40 porcine corneas and 6 human corneas before and after crosslinking in isolated topographic regions. Prior to crosslinking, all corneas demonstrated significant regional variability in response to loading. Regional reductions in displacement of between 16 % to 80 % were observed after crosslinking dependent upon treatment location. These initial data demonstrate both the necessity for full-field evaluation of biomechanics and the potential for crosslinking to be used to alter the refractive power of the eye.

In corneal collagen cross-linking (CXL), a treatment often used to stall the progression of keratoconus, a degenerative eye disease, corneal stroma is exposed to UV-light to improve mechanical stiffness by inducing covalent bonding. In clinical practice, a photoreactive riboflavin-solution is applied to the cornea and exposed to 3mW/cm2 of 365nm light for 30 minutes to accelerate cross-link formation. While this technique was recently approved for clinical use, time-evolving changes in CXL are not well understood. If the cornea is over-exposed, UV light may penetrate and damage deeper tissues. If underexposed, insufficient cross-linking may occur. Acoustic Micro-Tapping (AuT) with phase-sensitive OCT can non-invasively probe biomechanical changes in porcine and human cornea at multiple time points during UV-illumination using an air-coupled ultrasound transducer to deliver sufficient displacement on the corneal surface to launch a mechanical wave propagating as a guided mode. Here, guided wave propagation was captured at 100 spatial X-locations over 100 Y-planes to generate a 6 x 6 mm map of wave velocity across the corneal surface. The swept-source OCT system operated in BM mode at a functional frame rate of 16 kHz. In this experiment, corneas were scanned every 2 minutes during 30-minute UV exposure to analyze temporal changes in mechanical wave speed, central corneal thickness, and focusing power. Preliminary results suggest that changes in corneal structure and wave speed over time may infer rates of corneal cross-linking to refine UV illumination protocols and improve clinical outcomes.

Optical coherence tomography-based elastography (OCE) can perform localized, quantitative measurements of biomechanical properties. One of the most promising applications of OCE is to measure corneal stiffness, which has been linked to keratoconus, corneal crosslinking, and laser vision correction, and can help improve diagnosis, screening and treatment monitoring. Various techniques have been demonstrated to determine the speed of elastic waves traveling in the cornea and thereby to measure the shear modulus of corneal tissues. Here we present a new approach based on a contact probe with a piezo-electrically vibrating tip. This wave generation approach is robust, provides extensive control over the temporal and spectral profiles of the mechanical stimulus, and allows us to measure traveling wave velocities a frequency range of 1 to 15 kHz. The shorter wavelengths obtained at high frequencies can improve the resolution of traveling wave elastography and enable measurements of stiffer tissues such as the sclera. Direct contact with the corneal surface are routinely performed for intraocular pressure measurements, which suggest that this approach has a path to clinical translation. Interestingly, we found that mechanical stimulation tends to excite a combination of guided and non-guided elastic waves, which must be considered for accurate calculation of the shear modulus and may affect other OCT elastography techniques.

Brillouin spectroscopy provides a non-invasive, label-free method to evaluate the mechanical properties of biological materials. Prior studies have shown that the longitudinal elastic modulus, M, measured by Brillouin spectroscopy correlates with the Young’s modulus, E, of cells and tissues. However, M and E for hydrated materials are both influenced by water content. Using hydrogels as a simple model for hydrated biological materials, we designed experiments to separate the effects of E and water content on M. Polyethylene oxide (PEO) hydrogels were prepared with an average molecular weight of 1, 4 or 8 MDa and water content of 92, 95 or 98.5% (v/v). Polyacrylamide (PA) hydrogels were prepared with 10% acrylamide and 0.03-0.30% N-methylene-bis-acrylamide (w/v). E was measured by rheometry for PEO hydrogels and by unconfined compression for PA hydrogels. M was measured using a custom-built Brillouin spectrometer. For PEO hydrogels, E increased with molecular weight, whilst M was unaffected by molecular weight and decreased with increasing water content. For PA hydrogels, both M and E decreased over time due to swelling, but no single relationship could describe how M changed in terms of E. Regardless of swelling, all values of collapsed onto a single relationship that depended only on water content. After correcting for water content, measurements from Brillouin spectroscopy no longer correlate with Young’s modulus for hydrated materials. This work cautions against the straightforward application of Brillouin spectroscopy for optical elastography, but suggests that Brillouin spectroscopy and microscopy may be useful for investigating mechanisms involving changes in local water content.

Brillouin microscopy is an emerging tool of biomedical imaging [1]. While for a long time Brillouin spectroscopy was considered an exquisite tool of precise spectroscopy, recent advancements in instrumentation, methods of signal and data analysis, and multimodal imaging have paved the way to new applications in the broad area of biomedical research. In my presentation, I will review the recent advances of instrumentation, which result in much higher speed of Brillouin spectroscopy and imaging, while providing better discrimination against the background scattering and better sensitivity of Brillouin microscopy measurements. I will also discuss some new developments which can potentially lead to improved spatial resolution of Brillouin microscopy imaging and which can utilize the quantum nature of light to improve the signal discrimination. The above improvements in instrumentation translated into a number of applications, which allowed tissue assessment using Brillouin microscopy (see, for example, [2]) to be an essential component of broader medical protocols [3-4] to evaluate undergoing modifications of tissues in response to a certain treatment procedure. I will attempt to make sense out of such unusual sensitivity of Brillouin microscopy to the physiologically significant variations in tissue properties and will outline other potential directions of biological and biomedical research, which can be facilitated by continuously improving Brillouin microscopy instruments. [1] Z Meng, AJ Traverso, CW Ballmann, MA Troyanova-Wood, VV Yakovlev, Advances in Optics and Photonics 8 (2), 300-327 (2016). [2] M Troyanova‐Wood, C Gobbell, Z Meng, AA Gashev, VV Yakovlev, Journal of Biophotonics 10(12), 1694-1702 (2017). [3] OY Gasheva, IT Nizamutdinova, L Hargrove, C Gobbell, M Troyanova-Wood, SF Alpini, S Pal, C Du, AR Hitt, VV Yakovlev, MK Newell-Rogers, DC Zawieja, CJ Meininger, GD Alpini, H Francis, AA Gashev, Journal of Experimental Medicine (2018), Under review. [4] D Akilbekova, B Ogay, T Yakupov, M Saesenova, B Umbayev, VV Yakovlev, A Nurakhmetov, K Tazhin, and ZN Utegulov, Journal of Biomedical Optics (2018), Under revisions.

Embryonic development involves the interplay of driving forces that shape the tissue and the mechanical resistance that the tissue offers in response. While increasing evidence has suggested the crucial role of physical mechanisms underlying embryo development, tissue biomechanics is not well understood due to the lack of techniques that can quantify the stiffness of tissue in-situ with 3D high-resolution and in a non-contact manner. In this work, we used two all-optical technique, optical coherence tomography (OCT) and Brillouin microscopy, to map the longitudinal modulus of the neural tube tissue of mouse embryo in-situ. We found the tissue stiffens significantly after the closure of the neural tube at cranial regions by comparing embryos at E 8.5 and E 9.5. In addition, we observed that the region of fusion following neural tube closure is softer than the adjacent neural folds, and the neural folds show a modulus gradient along dorsal-ventral direction. Furthermore, we found the overlaying ectoderm is much softer and more pliable than the closed neural tube, and thus can be distinguished based on its mechanical properties. In conclusion, we demonstrated the capability of OCT and Brillouin microscopy to quantify tissue modulus of mouse embryos in-situ, and observed a distinct change of tissue modulus during the closure of cranial neural tube, suggesting this method could be helpful in investigating the role of tissue biomechanics in the regulation of embryo development.

The presented work shows a novel approach to correct the undesired background in Brillouin spectra. Specifically, we have developed a Brillouin spectroscopy modification suitable for correction of distortions caused by a molecular filter’s absorption, fluorescent background, or ambient room light. Due to the weak intensity of the Brillouin signal, a distortion of the baseline or a partial absorption of Brillouin peak can have strong impact on data analysis. In the worst case, such as investigation of strongly scattering biological material with simultaneous fluorescence measurements, these perturbations can make it impossible to accurately determine the Brillouin shift. A new Sequentially-Shifted Excitation (SSE) Brillouin spectroscopy method has been developed to allow acquisition of quality Brillouin spectra in most challenging of conditions. The idea behind this method is the observation that the Brillouin and elastically scattered light strongly depend on the wavelength of the incident light, and the location of their respective peaks in the final spectrum changes in response to the smallest change in excitation wavelength. On the other hand, the fluorescence background or distortions due to molecular filter absorption remain the same for small, ~1 pm, changes in incident wavelength. SSE Brillouin spectroscopy involves acquiring multiple Brillouin spectra using slightly offset excitation wavelengths, and computationally separating the signal and distortion/background components, thus recovering the Brillouin signal. The application of SSE Brillouin spectroscopy in highly-scattering sample is presented experimentally using sample of cream.

Brillouin spectroscopy is a powerful optical technique for non-contact viscoelastic characterizations of materials. Recently, Brillouin spectroscopy combined with confocal microscopy has found applications in high-resolution three-dimensional mechanical mapping of biological samples. These advances enabled in-vivo biomechanical studies of cells and tissues at sub-cellular resolution, however, Brillouin spectroscopy performances are rapidly degraded by optical aberrations and have therefore been so far limited to homogenous transparent samples. Thus, correcting sample aberrations to enable mechanical characterization within inhomogeneous medium, remains the current barrier on the versatility of this emerging technique. In this work, we developed an adaptive optics (AO) configuration designed for Brillouin scattering spectroscopy to dynamically correct aberrations induced by interrogated samples and optical elements. Our configuration does not require direct wavefront sensing and the injection of a ‘guide-star’; hence, it can be implemented without the need for sample pre-treatment. We used our wavefront corrected Brillouin spectrometer in aberrated phantoms and biological samples and obtained improved precision and resolution of Brillouin spectral analysis; we demonstrated 2.5-fold enhancement in Brillouin signal strength and 1.4-fold improvement in axial resolution as a result of the correction of optical aberrations. We further showed that our correction process is essential when mechanical characterization is performed under low signal-to-noise conditions, as is often the case for low Brillouin gain materials.

Mechanical and optical properties are the main criteria for assessing the health of dental tissue in contemporary dentistry. Dentinal pathological changes can be detected by visuo-tactile and radiographic methods to guide clinicians in establishing a relevant diagnosis and an adapted therapy. However, such approaches cannot give information on the dentinal microstructure. Recently, laser ultrasonic techniques have been deployed to evaluate the mechanical properties of enamel [1,2] However, such techniques lack the resolution to reveal the transitions differences between tissue layers. In this work, we used Brillouin light scattering spectroscopy as a non-contact alternative to probe mechanical changes in dentin and dentin-resin interface at GHz hypersonic frequencies. We obtained maps of the Brillouin frequency shift and linewidth that can be interpreted as maps of sound velocity and viscosity. In addition, we observed the specimens by a homemade nonlinear microscopy setup [3]. A 730 nm wavelength Titanium-sapphire laser was used as an excitation source for two-photon excitation fluorescence microscopy (TPEF), while 1040 nm wavelengthYb:KGW laser was used for second harmonic generation (SHG). Our results show significant changes between healthy tissues and pathological lesions. Such results can help to precisely delineate destructed dentin during clinical procedures, paving the way to minimally invasive strategies. In addition, our simultaneous analysis of Brillouin maps and nonlinear images brings valuable information on structure-related mechanical properties of dentin and dentin-resin adhesive interface. 1. Wang et al, Experimental and numerical studies for nondestructive evaluation of human enamel using laser ultrasonic technique. Appl Opt 52, 6896-6905 (2013). 2. Wang et al., Laser ultrasonic evaluation of human dental enamel during remineralisation treatment. Biomed Opt Express 2, 345-355 (2011). 3. Rabasović M et al. Nonlinear microscopy of chitin and chitinous structures: a case study of two cave-dwelling insects. J Biomed Opt 20, 016010 (2015)

A number of approaches employ optical coherence tomography (OCT) to obtain the mechanical properties of biological tissue. These are generally referred to as optical coherence elastography (OCE), and have demonstrated promising applications with studies in cornea, breast, muscle, skin, and other soft tissues. A particular application of interest is the brain, in which changes in local and global elastic properties may correlate with the onset and progression of degenerative brain diseases. In this preliminary study, mice brains are studied ex vivo and in situ with preservation of the brain/skull anatomical architecture. A small 6 mm diameter portion of the skull is replaced with a glass cap to allow for OCT imaging. Various permutations of source placement for generating shear waves and modes of excitation are evaluated to optimize the experimental setup. The use of reverberant shear wave fields, which takes advantage of inevitable reflections from boundaries and tissue inhomogeneities, allow for estimation of the shear wave speed, which is directly related to the elastic modulus of soft tissues. Preliminary estimates for the shear wave speed in brains of recently deceased mice are obtained. This study demonstrates potential applications in brain OCE ex vivo and in vivo.

Corneal injury is potentially leading to ulceration which remains a major health concern in ocular surface diseases. In vitro studies of new ophthalmic drugs selection are usually performed using excised cornea from slaughtered animals or laboratory animals. However, the outcomes from animal models do not completely reflect the human corneal repair and regeneration process. In vitro human corneal models suit better for the rapid testing of the drug uptake in response to drug’s administration and posology. Therefore, this study aims at establishing an in-vitro 3D corneal model to characterise the corneal wound healing process. Moreover, a functional assessment of corneal morphology and strength change during the healing process is of urgent need. A phase-sensitive optical coherence tomography (OCT) system with a spectral-domain configuration was utilised to probe the structure and mechanical strength of the wounded corneal tissues. In this preliminary study, a human corneal 3D model was successfully established using tissue-engineering techniques and corneal injuries were mimicked with adjustable lesion size and depth. During the healing process, OCT provided an accurate indication of the tissue repair and regeneration. These results will be of great clinical impact to understand the biomechanics of the cornea healing process and the therapeutic effectiveness of regenerative medicine.

Optical coherence tomography (OCT) allows structural and functional imaging of biological tissue with high resolution and high speed. Optical coherence elastography (OCE), a functional extension of OCT, has been used to perform mechanical characterization. A handheld fiber-optic OCE instrument allows high sensitivity virtual palpation of tissue with great convenience and flexibility and can be used in a wide range of clinical settings. Moreover, fiber-optic OCE instruments can be integrated into a needle device to access deep tissue. However, the major challenge in the development of handheld OCE instrument is non-constant motion within the tissue. In this study, a simple and effective method for temporally and spatially adaptive Doppler analysis is investigated. The adaptive Doppler analysis method strategically chooses the time interval (δt) between signals involved in Doppler analysis, to track the motion speed v(z,t) that varies as time (t) and depth (z) in a deformed sample volume under manual compression. The aim is to use an optimal time interval to achieve a large yet artifact free Doppler phase shift for motion tracking.

We describe a real-time 2D motion tracking method based on speckle analysis. We implemented this method in realtime using graphics processing unit (GPU). The capability to track both axial and lateral motions will enable more comprehensive characterization of tissue mechanical properties including Poisson's ratio.

Optical coherence elastography allows the characterization of the mechanical properties of tissues, and can be performed through estimating local displacement maps from subsequent acquisitions of a sample under different loads. This displacement estimation is limited by noise in the images, which can be high in dynamic systems due to the inability to perform long exposures or B-scan averaging. In this work, we propose a framework for simultaneously enhancing both the image quality and displacement map for elastography, by motion compensated denoising with the block-matching and 4D filtering (BM4D) method, followed by a re-estimation of displacement. We adopt the interferometric synthetic aperture microscopy (ISAM) method to enhance the lateral resolution away from the focal plane, and use sub-pixel cross correlation block matching for non-uniform deformation estimation. We validate this approach on data from a commercial spectral domain optical coherence tomography system, whereby we observe an enhancement of both image and displacement accuracy of up to 33% over a standard approach.

There has been a large amount of research focused on studying the biomechanical properties of the lens ex vivo. However, the storage medium of the lenses may affect the biomechanical evaluation during ex vivo measurements, which has been demonstrated with other tissues such as the cornea. In this work, we utilized a focused micro air-pulse and phase-sensitive optical coherence elastography to quantify the changes in lenticular biomechanical properties when incubated in different media, temperatures, and pHs for up to 24 hours. The results show that the lenses became stiffer when incubated at lower temperatures and higher pHs. Meanwhile, lenses incubated in M-199 were more mechanically stable than lenses incubated in PBS and DMEM.

Normal intraocular pressure (IOP) is crucial for proper maintaining of eye-globe geometry, ocular tissue health, and visual acuity. An elevated IOP is associated with diseases such as glaucoma and uveitis. While the effects of an elevated IOP on the delicate tissues of the optic nerve head and retina are well-studied, the changes in lenticular biomechanical properties as a function of IOP are not as clear. Moreover, changes in lenticular biomechanical properties have been implicated in conditions and diseases such as presbyopia and cataract. However, measuring the biomechanical properties of the lens as it sits inside the eye-globe is a challenge, but it is necessary to correctly understand the interplay between lenticular biomechanical properties and IOP. In this work, we utilized optical coherence elastography (OCE) to measure the biomechanical properties of the porcine lens in situ.