This PDF file contains the front matter associated with SPIE Proceedings Volume 10496 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Optical coherence elastography (OCE) holds great promise for quantitative characterization of corneal elasticity including robust measurements of the intraocular pressure (IOP) independent of corneal mechanical properties. To translate this method into a viable clinical tool, however, requires wideband, highly accurate mechanical wave measurements using mechanical stimulation requiring no physical contact with the cornea. We have developed a method of non-contact mechanical stimulation of soft media with precise spatial and temporal shaping. We call it acoustic micro-tapping (AuT) because it employs focused, air-coupled ultrasound (US) to induce significant mechanical displacement at the boundary of a soft material using reflection-based radiation force. Combining it with high-speed, four-dimensional (three space dimensions plus time) phase-sensitive optical coherence tomography (PhS-OCT) creates a non-contact tool for high-resolution and quantitative dynamic elastography of soft tissue at near real-time imaging rates. To demonstrate this approach, we present OCE results on a porcine cornea using a homemade, focused 1 MHz air-coupled piezoelectric transducer with a matching layer to launch an US wave through air onto the sample surface. To provide an acoustic line source approximating a 1-D excitation, the transducer was made from a cylindrical segment of a piezoelectric tube. A high-speed (1.6 MHz A-Scan rate) PhS-OCT system was utilized to measure acoustic wave propagation in the cornea at different intraocular pressures (IOPs). Results from this OCE study demonstrate that an air-coupled US wave reflected from an air/tissue interface provides significant radiation force to generate displacement for elasticity imaging for full mechanical characterization of the cornea.

Brillouin microspectroscopy is an emerging technique in optical elastography. It allows measuring local high-frequency viscoelastic modulus in cells and tissues in a matter of seconds or hundreds of milliseconds. BISTRO (Brillouin Imaging and Sensing via Time-Resolved Optical) measurements relies on impulsive stimulated Brillouin scattering to increase the signal strength and, hence, the speed of imaging, which can be as high as 1,000,000 pixels per second. However, there are several other untapped advantages of BISTRO measurements, and a particular intriguing one is the accuracy of Brillouin shift and linewidth assessments, which are substantially improved through BISTRO assessment. This allows high quality measurements not only to be done fast and more accurately, but also allows highly reproducible measurements over an extended period of time.

Wave-based optical elastography is a rapidly emerging technique for viscoelastic assessment of tissues due to its high displacement sensitivity and simple implementation. This method does not require prior knowledge of mechanical load characteristics, such as the applied preload and applied stress on the sample. However, current truly noncontact excitation methods are limited by their inability to produce broadband waves with high frequency content. Lower frequency wave content is constrained by boundary conditions, and thus, requires specifically tailored mechanical models that consider the sample geometry. In this work, we demonstrate that rapid vaporization of perfluorocarbon inside dye nanoparticles (NP) with a pulsed laser can produce high frequency and broadband elastic waves in tissue mimicking agar phantoms. As a comparison, a focused air-pulse was used as an alternative excitation method. The elastic waves were imaged by an ultra-fast low-coherence line-field holography system. Our results show that the NPs produced elastic waves with frequencies up to ~9 kHz, while the air-pulse was only able to produce waves with frequency content up to ~2 kHz. The elastic wave dispersion curves were fitted to the analytical solution of a Rayleigh wave model to quantify viscoelasticity. Analysis of the broadband high-frequency waves produced by the NPs yielded more accurate quantification of the sample viscoelasticity, demonstrating the benefits of optically excited elastic waves.

Traditionally, chemical and molecular markers have been the predominate method in diagnostics. Recently, alternate methods of determining tissue and disease characteristics have been proposed based on testing the mechanical behavior of biomaterials. Existing methods for performing elastography measurements, such as atomic force microscopy, compression testing, and ultrasound elastography, require either extensive sample processing or have poor resolution. In the present work, we demonstrate an optical polarimetric elastography device to characterize the mechanical properties of salmon skeletal muscle. A fiber-coupled 1550nm laser paired with an optical polarizer is used to create a fiber optic sensing region. By measuring the change in polarization from the initial state to the final state within the fiber sensing region with a polarimeter, the loading-unloading curves can be determined for the biomaterial. The device is used to characterize the difference between samples with a range of collagen membranes. The loading-unloading curves are used to determine the change in polarization phase and energy loss of the samples at 10%, 20% and 30% strain. As expected, the energy loss is a better metric for measuring the mechanical properties of the tissues because it incorporates the entire loading-unloading curve rather than a single point. Using this metric, it is demonstrated the device can repeatedly differentiate between the different membrane configurations.

Optical elastography emerged in the late 1990s as a way to non-invasively assess tissue biomechanical properties (i.e. optical palpation to sense tissue stiffness). Advances in optical engineering, such as optical coherence tomography, were essential to the growth of this field and provided the high-speed, high-resolution imaging required to quantify microscopic tissue deformation dynamics, which can be the basis for distinguishing normal and diseased tissue. This review will cover the development and evolution of optical elastography applications for ocular tissues and discuss the challenges to deploying this technology for clinical use.

Age-related macular degeneration is the leading cause of blindness in the elderly population, with a high demand for early diagnosis since the symptoms are irreversible. Current structural and functional imaging modalities include fundus autofluorescence, optical coherence tomography, and angiography, and are often not sufficient for early stage detection, which is mostly characterized by changes in tissue composition. A technology that enables the in-vivo imaging of the posterior ocular globe is essential for gaining insight into the natural mechanical anatomy of the eye, as well as the changes that take place with ocular diseases. However, in-vivo mechanical imaging of the retina remains a challenge and is currently not available. In this study, we report on the development of acoustic radiation force optical coherence elastography (ARF-OCE) to visualize and quantify the stiffness map of in-vivo retinal tissues based on the Voigt model. We demonstrate the elasticity mapping of an in-vivo rabbit retina, showing the stiffness variations across 5 different layers, ranging from 3 kPa to 16 kPa on the ganglion to the sclera sides. In addition, we introduce a diseased rabbit model based primarily on blue light exposure, and have found a difference in the layered stiffness where inflammation occurred. The results show that the ARF-OCE system has the capability to noninvasively detect tissue abnormalities in-vivo, and represents a significant step toward the development of the ARF-OCE system for clinical use.

In vision correction surgeries, the corneal stroma is subject to limbal-relaxing incisions which change the focusing power of the cornea, but can damage tissue and put the patient at risk of complication. A non-invasive method to launch a mechanical wave in tissue, referred to as Acoustic Micro-Tapping (AuT), is demonstrated with phase-sensitive spectral domain OCT (SD-OCT) to probe for biomechanical changes in porcine and human cornea samples following arcuate keratotomy (AK). This method uses an air-coupled ultrasound transducer to deliver sufficient displacement on the corneal surface to launch a mechanical wave propagating as a guided mode. Rayleigh-Lamb wave propagation is captured at 100 spatial locations 6 mm across the corneal surface, resulting in a high resolution elastogram. The SD-OCT system operates in the MB mode at a functional frame rate of 47 kHz to detect local wave behavior for analysis of the group velocity, group displacement amplitude, displacement attenuation, phase velocity over the bandwidth of the excitation, mean frequency, and bandwidth. An analysis of mechanical wave behavior shows reduced wave speed up to 20% following an incision through 3/4th of the cornea in porcine tissue samples, indicating a potential reduction in elastic modulus. This technique was performed on porcine and human corneas following PRK incision to demonstrate progress toward clinical translation.

Non-surgical thermo-mechanical reshaping of avascular collagenous tissues (cartilages and cornea) using moderate heating by IR-laser irradiation is an emerging technology that can find important applications in visioncorrection problems and preparation of cartilaginous implants in otolaryngology. To estimate both transient interframe strains and cumulative resultant strains produced by the laser irradiation of the tissue we use and improved version of strain mapping developed in our previous work related to compressional phase-sensitive optical coherence tomography. To reveal microstructural changes in the tissue regions where irradiation-produced strains do not disappear after temperature equilibration, we apply compressional optical coherence elastography in order to visualize the resultant variations in the tissue stiffness. The so-found regions of the stiffness reduction are attributed to formation of microscopic pores, existence of which agree with independent data obtained using methods of high-resolution microscopy.

Biomechanical properties of the cornea play key role in accurate measurement of the intraocular pressure (IOP). The aim of this study is to assess the impact of IOP on corneal hysteresis in porcine (ex vivo) and human (in vivo) eyes using swept source optical coherence tomography combined with the air-puff system (air-puff SS-OCT). We developed air-puff SS-OCT to assess rapid dynamics of porcine corneas during the air pulse application. Both tissue displacement x(t) and air stimulus F(t) are acquired simultaneously that enables generation of corneal hysteresis F(x), which is a direct signature of viscoelastic properties of the cornea. The hysteresis loop can be quantified by calculation the parameters including maximum apex displacement, central corneal thickness, hysteresis area, elastic moduli etc. Firstly, the corneal response of 35 ex-vivo porcine eyes to the air puff is determined for IOP ranging from 5 to 35 mmHg. The IOP level is set by a custom pressure control system. The IOP causes highly correlated changes in the proposed parameters of the hysteresis curve. Secondly, we investigate the modification of corneal hysteresis in 30 human corneas in vivo. The IOP is modulated by installation of 0,2% brimonidine eye drops (Alphagan) decreasing the IOP. The IOP is measured with air-puff non-contact tonometer (Topcon) and Goldmann tonometer and compared with hystereses generated by air-puff SS-OCT. To conclude, IOP generates changes of corneal viscoelasticity in ex vivo animal model and in vivo human eyes. Non-invasive character, micrometer resolution and fast acquisition make our approach attractive for in vivo studies.

We report on a multimodal imaging system comprising optical coherence tomography (OCT), pulse echo ultrasound imaging (USI), and acoustic radiation force optical coherence elastography (ARF OCE), capable of volumetric structural and mechanical imaging with micrometer-to-centimeter- scale spatial coverage. A spectral-domain OCT setup (1300 nm central wavelength, with transverse and axial resolutions of 6-8 μm and 3.5 μm, respectively) imaged the sample from above, and a 10 MHz immersion ultrasound transducer provided a counter-propagating co-aligned beam for both USI and ARF excitation from below. Although typical ARF elastography systems report acoustic focal spot sizes greater than 300 μm, studies indicate that reducing the region of excitation (ROE) improves mechanical contrast. To decrease the ROE diameter, we designed and fabricated acoustic lenses made of silicone and agar of various curvatures to increase the numerical aperture of the acoustic beam. We achieved a spot size of 240 μm – a 28% decrease from an initial spot size of 330 μm. We characterized mechanical resolution of the ARF-OCE elastograms using a gelatin-agar co-gel phantom exhibiting a sharp ‘step’ in mechanical properties. Differentiating the mechanical step response, we obtained the mechanical impulse response with FWHM of 165±2 μm using ARF excitation with ROE diameter of 700 μm at the sample surface. Our results suggest that mechanical resolution (width of the impulse response function) cannot be described by just the ROE or OCT resolution alone. Future work will aim to further reduce the ROE, and will further investigate the effects of ROE and ARF excitation frequency on mechanical resolution.

Laser induced elastic waves in soft media have great potential to characterize tissue biomechanical properties. The instantaneous increase in local temperature caused by absorption of laser energy leads to a mechanical perturbation in the sample, which can then propagate as a pressure (or an elastic) wave. The generation of the elastic wave can be via thermoelastic or ablative processes depending on the absorption coefficient of the sample and incident laser fluence. It is critical to differentiate between these regimes because only the thermoelastic regime is useful for nondestructive analysis of tissues. To investigate the transition point between these two different regimes, we induced elastic waves in tissue mimicking agar phantoms mixed with different concentrations of graphite powder. The elastic waves were excited by a 532nm pulsed laser with a pulse duration of 6 ns. The fluence of the pulsed laser was tuned from 0.08 J/cm² to 3.19 J/cm² , and the elastic wave was captured by ultra-fast line-field low coherent holography system capable of single-shot elastic wave imaging with nanometer-scale displacement sensitivity. Different concentrations of graphite powder enabled excitation in sample with controlled and variable attenuation coefficient, enabling measurement of the transition between the thermoelastic and ablative regimes. The results show that the transition from thermoelastic to ablative generated waves was 0.75 J/cm² and 1.84 J/cm² for phantoms with optical attenuation coefficients of 6.64±0.32 mm-1 and 26.19±1.70 mm^-1, respectively. Our results show promise for all optical biomechanical characterization of tissues.

Ablative fractional skin laser is widely applied for various skin conditions, especially for cosmetic repairing and promoting the located drug delivery. Although the influence of laser treatment over the skin has been explored before in means of excision and biopsy with microscopy, these approaches are invasive, only morphological and capable of distorting the skin. In this paper the authors use fresh porcine skin samples irradiated by the lasers, followed by detected by using Optical Coherence Tomography (OCT). This advanced optical technique has the ability to present the high resolution structure image of treated sample. The results shows that laser beams can produce holes left on the surface after the irradiation. The depth of holes can be affected by changes of laser energy while the diameter of holes have no corresponding relation. Plus, OCT, as a valuable imaging technology, is capable of monitoring the clinical therapy procedure and assisting the calibration.

Surface acoustic wave (SAW) elastography has been proven to be a non-invasive, non-destructive method for accurately characterizing tissue elastic properties. Current SAW elastography technique tracks generated surface acoustic wave impulse point by point which are a few millimeters away. Thus, reconstructed elastography has low lateral resolution. To improve the lateral resolution of current SAW elastography, a new method was proposed in this research. A M-B scan mode, high spatial resolution phase sensitive optical coherence tomography (PhS-OCT) system was employed to track the ultrasonically induced SAW impulse. Ex-vivo porcine skin specimen was tested using this proposed method. A 2D fast Fourier transform based algorithm was applied to process the acquired data for estimating the surface acoustic wave dispersion curve and its corresponding penetration depth. Then, the ex-vivo porcine skin elastogram was established by relating the surface acoustic wave dispersion curve and its corresponding penetration depth. The result from the proposed method shows higher lateral resolution than that from current SAW elastography technique, and the approximated skin elastogram could also distinguish the different layers in the skin specimen, i.e. epidermis, dermis and fat layer. This proposed SAW elastography technique may have a large potential to be widely applied in clinical use for skin disease diagnosis and treatment monitoring.

Recently we used ultrasound from an air-coupled transducer for non-contact excitation of broadband mechanical waves in soft tissue such as cornea. The transient displacement, generated by “Acoustic Micro-Tapping” (AuT), was then measured using phase-sensitive spectral domain OCT (SD-OCT). In addition traditional surface wave speed measurement, we investigated complementary methods to characterize the mechanical properties of the target material. We note that the maximum frequency, as well as the group velocity, of the surface wave is related to both the phase velocity of the material and the spatial width of the acoustic pulse. If the spatial and temporal profile of the excitation is well defined, it may be possible to infer elastic modulus from the frequency profile of a propagating mechanical wave. To assess the effect of the spatial profiles of the AuT excitation on frequency profiles of resulting mechanical waves, acoustic pulses with different spatial width (from 0.1 to 1 mm) were applied to agar phantoms with different shear modulus (from 1 to 100 kPa) to generate mechanical waves, and a SD-OCT system with a functional frame rate of 47 kHz was used to track wave propagation. For validation, simulations with the same acoustic and mechanical properties were performed using a finite element method (FEM) to analyze induced wave propagation. The phantom experiment and simulation exhibited similar increase in the maximum frequency with decreasing excitation width. Both estimates also agreed well with previous theoretical results.

Living cells sense and respond to their microenvironment through chemical and physical signals. In vivo, their interactions are determined both by the adjacent cells and by the surrounding extracellular matrix (ECM) network. Tumor development is regulated by complex interactions of both normal and cancerous cells with their ECM. Such interactions are not well understood due mainly to the lack of appropriate characterization techniques covering length scales ranging from the molecular to the matrix/cellular level. Our goal is to study the optical/structural and mechanical properties of ECM fibrillar structures in physiological and tumor environments, from the single protein to the cellular/tissue level and to investigate tumor vascularization mechanisms. Combining fluorescence resonance energy transfer and multiple beam interferometry through surface forces apparatus characterization, we measured the molecular conformation, Young’s modulus and viscosity of the ECM deposited by cancer-associated fibroblasts preconditioned with tumor soluble factors derived from an aggressive breast cancer cells line. Our results reveal that tumor factors promote (i) single ECM protein unfolding, (ii) overall ECM stiffening, and (iii) increased ECM viscosity with respect to control. We next quantified the effect of this altered tumor-associated ECM on cell proangiogenic capability. Our findings indicate that the unfolded and stiff tumor-associated ECM significantly enhances the secretion of vascular growth factors by surrounding stromal cells. Collectively, our multi-scale analysis suggests that conformation and mechanics of ECM proteins at both the molecular and the matrix/tissue levels significantly dysregulate the downstream proangiogenic behavior of surrounding cells, which likely contributes to tumor vascularization and development.

Mechanical properties are critical in regulating pathophysiological cell behavior via mechano-transduction. Although biological tissues are generally regarded as viscoelastic, mechanobiology studies mainly focused on characterizing tissue elasticity and investigating cell behavior as a function of substrate stiffness. Moreover, mechanical properties are often derived using bulk testing techniques, likely being poorly representative of the local biomechanical environment felt by cells. Aiming at characterizing micro-mechanical viscoelastic properties at typical cell length-scales in physiological-like conditions, we present here a new testing approach to perform dynamic nano-indentation measurements in liquid (e.g. PBS, culture medium) at controlled temperature (e.g. 37 °C). Our method involves a customized version of Optics11’s PIUMA Nanoindenter, which is based on a unique ferrule-top opto-mechanical cantilever force transducer operated by a z-axis piezoelectric motor, and which has been modified to enable user-defined load- and displacement-controlled measurements (e.g. creep, stress-relaxation, DMA and constant strain rate tests). Moreover, a temperature (T) sensor has been integrated with that of the PIUMA sample stage to control the actual sample T via a new master-slave control loop. In this study, we characterized the viscoelastic properties of PDMS samples, gelatin hydrogels at different temperatures and concentrations, single smooth muscle cells, and healthy and infarcted myocardial tissue samples. Experimental data within the linear viscoelastic region were fitted to Generalised Maxwell models, deriving instantaneous and equilibrium elastic moduli, and characteristic relaxation times. This method could be beneficial for better investigating soft tissues/(bio)materials mechanics and for designing new mechano-mimetic substrates for tissue engineering, disease modelling and cell mechanobiology studies.

Determining the mechanical properties of tissue such as elasticity and viscosity is fundamental for better understanding and assessment of pathological and physiological processes. Dynamic optical coherence elastography uses shear/surface wave propagation to estimate frequency-dependent wave speed and Young’s modulus. However, for dispersive tissues, the displacement pulse is highly damped and distorted during propagation, diminishing the effectiveness of peak tracking approaches. The majority of methods used to determine mechanical properties assume a rheological model of tissue for the calculation of viscoelastic parameters. Further, plane wave propagation is sometimes assumed which contributes to estimation errors. To overcome these limitations, we invert a general wave propagation model which incorporates (1) the initial force shape of the excitation pulse in the space-time field, (2) wave speed dispersion, (3) wave attenuation caused by the material properties of the sample, (4) wave spreading caused by the outward cylindrical propagation of the wavefronts, and (5) the rheological-independent estimation of the dispersive medium. Experiments were conducted in elastic and viscous tissue-mimicking phantoms by producing a Gaussian push using acoustic radiation force excitation, and measuring the wave propagation using a swept-source frequency domain optical coherence tomography system. Results confirm the effectiveness of the inversion method in estimating viscoelasticity in both the viscous and elastic phantoms when compared to mechanical measurements. Finally, the viscoelastic characterization of collagen hydrogels was conducted. Preliminary results indicate a relationship between collagen concentration and viscoelastic parameters which is important for tissue engineering applications.

Brillouin scattering microscopy is the only potential tool to realize microscopic mapping of mechanical property in multicellular system (i.e. colony, tissue) with sub-cell resolution. We built a laser-scanning Brillouin microscope system for our biological study on spatial heterogeneity of stiffness in multicellular system. High-numerical-aperture (NA) objective lens (NA ≥ 0.7) and a dual-VIPA based spectrometer were employed to achieve high spatial resolution and high sensitivity, respectively. Addition of a spatial filter at a Fourier plane of the EMCCD detector surface effectively rejected strongly reflected excitation light without loss of Brillouin scattering signal, which accordingly allowed us to observe cells just above glass substrate surface. We performed three-dimensional imaging of Brillouin scattering to see three-dimensional stiffness distribution within a multicellular system. It was found that cells in relatively central region or in close vicinity of glass substrate have higher stiffness, which agrees to biological prediction. We also found presence of anomaly cells with much higher elasticity than surrounding cells. The stiffness imaging was applied to various kinds of multicellular systems including ES cell colonies upon differentiation and artificial tissue grown from iPS cells. The results certified the effectiveness of Brillouin scattering microscope in mechanobiology, developmental biology and regenerative medicine. Several practical issues for biomedical application will also be discussed.

The nucleus is the largest and stiffest organelle of eukaryotic cells, and as such, its mechanical properties are tightly related to various cell functions. Many efforts have been devoted to characterize the mechanical properties of nucleus, but the current techniques generally need physical contact of the cell and staining of the nucleus and thus cannot acquire the mechanical information directly. Brillouin microscope is an integration of a confocal microscope and a Brillouin spectrometer, which measures the spectral shift due to the spontaneous Brillouin light scattering, and from that the longitudinal modulus of the sample can be quantified. In this work, by combining the standard Brillouin microscope with the microfluidic technique, we developed a Brillouin flow cytometry that can quantify the mechanical properties of the intact cellular nucleus in a non-contact and label-free manner. As cell flows through a microfluidic channel, its mechanical property at different regions will be sampled by a sub-micron beam spot of the Brillouin microscope. The mechanical information of the nucleus from the cell population can then be identified and extracted via data post-processing, which is further confirmed by co-registering Brillouin data with fluorescence data from the same cell. Currently, the overall throughput of this technique is about 200 cells per hour, mainly relies on the acquisition speed of the spectrometer, which could be readily improved with available technology. We verified the capability of this all-optical technique by distinguishing the stiffness changes of the nucleus that are relevant to physiological and pathological phenomena.

The mechanical properties of biological tissues are increasingly recognized as crucial parts of signaling cascades involved in developmental and pathological processes. While most techniques measuring intrinsic mechanical properties necessitate invasive sample preparations or are currently applicable only to large sample dimensions, confocal Brillouin microscopy provides means to quantify the mechanical properties of single cells and tissues in a contact- and label-free manner. Here, we show for the first time a systematic application of confocal Brillouin microscopy to quantify physical properties of tissues in vivo. By using our custom-built Brillouin microscope, zebrafish larvae were probed in all anatomical planes, at different time points during development and after spinal cord injury. These experiments revealed that confocal Brillouin microscopy is capable of detecting the mechanical properties of distinct anatomical structures without interfering with the animal’s natural development. We furthermore detected an increasing Brillouin shift of spinal cord tissue during development and a transiently decreasing Brillouin shift after spinal cord injury. The presented work constitutes the first step towards an in vivo assessment of spinal cord tissue mechanics during regeneration, provides a basis to identify key determinants of mechanical tissue properties and allows to test their importance in combination with biochemical and genetic factors.

Spontaneous Brillouin scattering is an inelastic scattering process arising from inherent thermal density fluctuations, or acoustic phonons, propagating in a medium. The recent development of high throughput efficiency Virtually Imaged Phased Array (VIPA) etalons and high sensitivity CCD cameras has dramatically reduced the data acquisition time, in turn enabling the extension of Brillouin spectroscopy from a point sampling technique to an imaging modality. Hitherto 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 to optically probe biological systems, where the elastic Rayleigh scattering and specular reflection are orders of magnitude greater than the Brillouin signal. In VIPA spectrometers, the elastic background light introduces crosstalk signals that overwhelms the weak Brillouin peaks, thus impeding the acquisition of biomechanical images. One method to increase the contrast is to add more etalons in tandem and crossed with respect to each other. Nevertheless, this comes at the cost of a reduced throughput efficiency and a significantly increase system complexity. Here we demonstrate a method to increase the contrast by more than 30dB respect to standard VIPA spectrometers without the requirement of any additional optical or dispersive components. Our method was demonstrated by acquiring Brillouin images of single cells at a sub-micron spatial resolution, where the biomechanical properties of individual cellular structures were investigated.

We propose that antihistamine desloratadine affects lipid content of tissues, a change that can be accessed with Raman and Brillouin spectroscopies. Antihistamines are commonly prescribed to alleviate allergy symptoms. However, reports indicate increase in appetite and weight gain among their possible side effects. This study examines the relationship between the antihistamines’ use and obesity. Four groups of rats consumed regular or high-lipid diets while daily receiving either desloratadine or placebo. We analyzed changes in the chemical composition and local elasticity of skin and adipose tissue samples using Raman and Brillouin spectroscopy respectively. Both the medicated regular-diet group and the non-medicated high-lipid-diet group showed an increase in samples’ elasticity and lipid content compared to the control group that received placebo. Interestingly, the adipose tissues’ elasticity was significantly lower in the high-lipid-diet group receiving daily desloratadine compared to other groups. Raman and Brillouin spectroscopy demonstrated that desloratadine does affect tissues’ lipid content. Antihistamines may contribute to weight gain as shown by an increased lipid content in the medicated regular-diet group. However, it remains unclear why a combination of antihistamines and a high-lipid diet decreased the elasticity of adipose. This observation may indicate a change in the adipose tissue’s density or lipid absorption.

We discuss the use of Brillouin Light Scattering Microspectroscopy (BLSM) on Liquid Biopsies for obtaining complementary information in the diagnosis of different diseases. The viscoelastic properties of blood are known to play an important role for many processes necessary for survival including tissue perfusion, oxygen delivery and general circulation. They are understood to be dominated by the dense red blood cell suspension, with the plasma often modelled as a Newtonian fluid serving as an extracellular matrix. Much effort has been devoted to studying the mechanical properties of red blood cells, variations of which have been linked to numerous hereditary and metabolic disorders. Recent studies have shown also a non-Newtonian viscoelastic behavior of plasma. Though the biochemical composition of plasma can change at the onset of diseases, it is unclear if and how the structural-mechanical properties are affected. Here we discuss the measurement of the high-frequency viscoelastic properties of plasma from diseased blood using BLSM. BLSM utilizes the inelastic scattering of light from inherent thermal density fluctuations (acoustic phonons) to derive mechanical parameters such as the Longitudinal Storage and Loss Moduli. Since BLSM probes very fast relaxation processes it can be sensitive to structural/conformational changes of macromolecules. By mapping the variation and scaling of the storage and loss parameters also as a function of dilution and temperature we observe subtle differences between plasma from healthy and diseased samples. We discuss possible origins of these differences, and their potential for complementing liquid biopsies.

Circulating tumor cells form metastases by reaching a distant microcirculation, undergoing transendothelial migration, entering into the remote tissue and proliferating. Microfluidic assays have recently been developed that enable the visualization and quantification of this process within vascular networks that recapitulate many aspects of the in vivo microcirculation. The assays are created by seeding endothelial cells in co-culture with fibroblasts or pericytes within a fibrin gel. In 1 day, the networks form, and in 4 to 7 days they are perfusable with medium. At that point, tumor cells, with or without accompanying immune cells, are streamed into the network, some fraction of which will arrest and extravasate into the surrounding matrix. Models of this type have been used to study several aspects of this process. These studies have provided detailed data on the ability of different tumor cell types to extravasate, the adhesion molecules they use to pull themselves through the endothelial monolayer, and the effects of various other cell types in the intravascular space (neutrophils and platelets), and the extracellular matrix (fibroblasts, pericytes, myoblasts, and osteoblasts). Some studies have been carried out for over one week in order to observe the initial stages of growth of the metastatic tumor. Other vascular network models have also been developed that can be used for longer-term studies, with more realistic network morphologies and remodeled matrix composition.

Fiber optic microindentation sensors that have the potential to be integrated into arthroscopic instruments and to allow localizing degraded articular cartilage are presented in this paper. The indenters consist of optical fibers with integrated Bragg gratings as force sensors. In a basic configuration, the tip of the fiber optic indenter consists of a cleaved fiber end, forming a cylindrical flat punch indenter geometry. When using this indenter geometry, high stresses at the edges of the cylinder are present, which can disrupt the tissue structure. This is avoided with an improved version of the indenter. A spherical indenter tip that is formed by melting the end of the glass fiber. The spherical fiber tip shows the additional advantage of strongly reducing reflections from the fiber end. This allows a reduction of the length of the fiber optic sensor element from 65 mm of the flat punch type to 27 mm of the spherical punch. In order to compare the performance of both indenter types, in vitro stress-relaxation indentation experiments were performed on bovine articular cartilage with both indenter types, to assess biomechanical properties of bovine articular cartilage. For indentation depths between 60 μm and 300 μm, the measurements with both indenter types agreed very well with each other. This shows that both indenter geometries are suitable for microindentation measuremnts . The spherical indenter however has the additional advantage that it minimizes the risk to damage the surface of the tissue and has less than half dimensions than the flat indenter.

A characteristic sign of aging skin is loss of firmness. Current bioinstruments to measure the influence of formulations on skin mechanics are limited in sensitivity and have high operator variability. To address these issues an optical elastography system was developed. Regions of the upper inner bicep were treated with various commercial formulations including wrinkle reducers, firming films, and moisturizers. These regions along with adjacent untreated areas that served as internal controls were imaged with a custom designed optical elastography system. The elastography system employed a polarized 50 mW 532 nm cw laser as an illumination source, a CCD camera imaging the skin at 160 Hz frame rate and a polarization analyzer aligned parallel to the incident beam. The skin was mechanically loaded using compressed air reduced in pressure and modulated using a proportional valve to provide a 1 Hz sinusoidally varying pressure to the skin with a peak force of 0.15 N. Subjects ranging in ages from 19 to 60 years old were recruited with IRB approval. Displacement and strain encoded elastograms were generated simultaneously for the treated and untreated areas. The ratio of the strain response in the two regions was calculated to quantify the relative effect of the skin agents. Significant differences were found in the strain response to the imposed loads between treated and control areas in all age groups and genders. Optical elastography systems such as the one prototyped in this study may prove to be useful for the cosmetics industry for assessing product efficacy.

Elevated intraocular pressure (IOP) deforms the lamina cribrosa (LC), a structure within the optic nerve head (ONH) in the back of the eye. Evidence suggests that these deformations trigger events that eventually cause irreversible blindness, and have therefore been studied in-vivo using optical coherence tomography (OCT), and ex-vivo using OCT and a diversity of techniques. To the best of our knowledge, there have been no in-situ ex-vivo studies of LC mechanics. Our goal was two-fold: to introduce a technique for measuring 3D LC deformations from OCT, and to determine whether deformations of the LC induced by elevated IOP differ between in-vivo and in-situ ex-vivo conditions. A healthy adult rhesus macaque monkey was anesthetized and IOP was controlled by inserting a 27- gauge needle into the anterior chamber of the eye. Spectral domain OCT was used to obtain volumetric scans of the ONH at normal and elevated IOPs. To improve the visibility of the LC microstructure the scans were first processed using a novel denoising technique. Zero-normalized cross-correlation was used to find paired corresponding locations between images. For each location pair, the components of the 3D strain tensor were determined using non-rigid image registration. A mild IOP elevation from 10 to 15mmHg caused LC effective strains as large as 3%, and about 50% larger in-vivo than in-situ ex-vivo. The deformations were highly heterogeneous, with substantial 3D components, suggesting that accurate measurement of LC microstructure deformation requires high-resolution volumes. This technique will help improve understanding of LC biomechanics and how IOP contributes to glaucoma.

The change of tissue elasticity has been recognized as a biomarker of many disease conditions. Elastography has been investigated by observing the strain and stress correlation or shear wave propagation in tissue. The strain measurement can be achieved via speckle tracking in ultrasound (US) and optical modalities whereas the stress in deep tissue cannot be directly measured. Assuming that the collapsing of the vasculature could reflect the stress exerted on a tissue volume, the photoacoustic (PA) signals of the hemoglobin content within the vasculature could be an alternative measurement of the stress. This study investigates the strain-PA correlation with phantoms and a rat model in vivo. Parallel PA-US imaging was achieved by combining a compact linear US array and fiber optics delivering 720nm illumination. The phantom study simulated the vasculature with a piece of sponge soaked in ink, and the surrounding tissue with porcine gel with varied elasticity. The strain was generated by pushing the PA-US probe against the phantom surface. A correlation of 0.9 was found between the strain within the sponge and the PA signal changes. The rat model possesses inflammatory and fibrotic intestinal strictures comparable to those in the Crohn’s disease patients. The strain within the strictures was achieved by pushing the PA-US probe against the rats’ abdominal walls. Approximately twice more significant PA signal changes were observed in the fibrotic strictures than those in inflammatory ones under the same strain (p<0.001). All the results support that strain-PA imaging is capable of estimating the tissue elasticity.

There is a significant interest in characterizing mechanical properties of the brain tissue due to the role of mechanics in neurodevelopment and neurological disorders. Previous Scanning Force Microscopy studies have reported that brain tissue has highly heterogeneous mechanical properties. Yet, it is not known how the structural components of the brain such as neurons, glial cells, their axons and dendrites, and extracellular matrix contribute to these stiffness variations. To investigate the structure-stiffness relation in brain tissue and thus solve this issue, we have employed dynamic indentation-controlled mapping with a spatial resolution of ~50 µm to measure viscoelastic properties of hippocampus and cortex on isolated horizontal mouse brain sections. Nonlinear-viscoelastic nature of the brain tissue was observed by oscillatory ramp testing, where stiffness increased linearly with the strain (strain < 10 %); frequency sweeps revealed frequency-stiffening (1-10 Hz) with the phase delay in the range of 15-30˚. Viscoelasticity maps showed that regions with distinct mechanical properties correspond to morphological layers with the mean storage modulus varying from 779±77 Pa for granular layer to 3260±74 Pa for stratum lacunosum-moleculare (mean ± SE). Density of the nuclei was estimated for the measured regions and was found to negatively correlate with the stiffness, except for alveus, mostly composed of axonal fibers, being significantly softer than all other high-stiffness low-cell-density regions. Taken together, our study shows that our novel indentation method is able to map mechanical differences of the brain at the cellular level, leading to a better understanding of the relation between tissue composition and stiffness.

The majority of tissue related diseases are known to alter tissue structure. During the last 10 years, increasing efforts have been put into the development of new techniques that could provide in vivo information on tissue morphology. Optical coherence tomography (OCT) is known to provide structural information in situ. However, there is also a strong demand to evaluate the mechanical properties of biological tissues in vivo. To address this need, we combined microindentation with non-invasive OCT imaging to determine spatiotemporal distributions of mechanical properties of in vivo and formaldehyde fixed chicken embryos. The use of OCT allows us to quantify changes in tissue morphology and to localize indentation at specific regions. To measure viscoelastic properties of living tissue, indentation tests are simultaneously performed on in vivo HH8-HH12 chicken embryos using a cantilever based force transducer. After performing live tissue indentation, we probed the properties of formaldehyde fixed embryo. The same general contrasts of elasticity between different histological regions were found, but the average value was found to be higher for the fixed sample. Furthermore, with this technique it is possible to follow the remodeling of tissue elastic properties during embryonic development, measure viscoelastic properties of living tissue, and investigate correlations between local mechanical properties during cell migration and differentiation. This method is applicable to a wide variety of biological samples and can provide new insight to better understand the link between the mechanical response of tissue and its biological structure, and to compare diseased tissues with healthy one.

Thermo-mechanical effect of laser radiation is a basis of new method of normalization of intraocular pressure in glaucomatous eyes due to laser-assisted pore formation in eye sclera. Laser-induced creation of pores in sclera increases hydraulic permeability. Stability of laser-induced pore system is achieved via gas nano-bubbles arisen in the sclera under laser radiation as a result of temperature dependency of gas solubility. The stabilization of laser-induced gas and pore systems in the tissue is an important mechanism for a long lasting healing of glaucoma observed in clinical trials with one year follow-up.

Polarization sensitive optical coherence tomography (PS-OCT) measures tissue birefringence, while optical coherence elastography (OCE) reveals the mechanical property of the tissue. Since both birefringence and mechanical properties are associated with tissue microstructures such as collagen, simultaneous PS-OCT and OCE measurement will provide useful insight for the tissue microstructures. In this paper, we present a combined PS-OCT and OCE technique. The PS-OCT is based on Jones matrix OCT theory. It measures a tomography of Jones matrix. Birefringence tomography is then deduced from the Jones matrix. The OCE is obtained with active tissue compression. The tissue compression was performed by a ring piezoelectric (PZT) actuator installed in front of an objective. A glass slip is attached at the fore-end of the PZT to push a tissue. Multiple cross sections were synchronously measured through the glass slip with gradual tissue compression (0 – 12 µm at maximum). The raw Jones matrix tomography consists of 4 complex-valued OCT images. Two of the 4 images (two orthogonal detection polarization images of a single input polarization) are processed by a correlation based tissue displacement-and-deformation analysis method [Kurokawa et al., Opt. Lett., 2153-, 2015]. It provides polarization artifact free cross-sectional maps of (1) axial displacement, (2) lateral displacement, and (3) microstructural deformation. The method was evaluated by measuring porcine muscle tissues. It was observed that the muscle has subdomains with different deformation properties. In addition, the cross-sectional deformation map distinctively visualized that the muscle and adipose has significantly different deformation properties. Different muscle samples shows different birefringence strength.

Many complex diseases such as diastolic dysfunction and some types of cardiomyopathy are often characterized by an increased stiffness of heart muscles which can potentially cause heart failure. While changes of heart muscle’s geometry could be detected by various imaging methods, non-invasive measurements of stiffness of the heart muscle are desired to assess such areas of the heart tissues without invasive surgery. A novel minimally-invasive method of stiffness assessment of heart muscle – optical coherent elastography (OCE) – is based on a combination of applied acoustic radiation force for mechanical excitation of tissue with subsequent phase-sensitive optical coherence tomography (psOCT) measurements of spatio-temporal response of tissue. A minimally invasive probe comprising a small, 2x2 mm size, low-frequency (<5MHz) ultrasound transducer and a clinically approved psOCT imaging fiber was incorporated into a single housing such that psOCT beam and acoustic excitation beam were parallel. Acoustic radiation pressure pulse was applied to initiate tissue displacement and propagation of shear waves that were detected by psOCT. Given the known offset between ultrasound and psOCT beams, the speed of shear waves was measured and shear elastic modulus of the heart tissues can be reconstructed. The initial results demonstrate that our OCE probe can produce and measure the displacements on the order of several ten nanometers in heart tissue-mimicking phantoms. The results indicate that translate-rotate scanning of OCE probe can simultaneously image the tissue and map its shear elastic modulus.

HIFU is a truly noninvasive, acoustic therapeutic technique that utilizes high intensity acoustic field in the focus to kill the targeted tissue for disease treatment purpose. The mechanical properties of targeted tissue changes before and after treatment, and this change can be accurately detected by shear wave elastography. Hence, shear wave elastography is usually used for monitoring HIFU treatment asynchronously. To improve the low spatial resolution in ultrasound shear wave elastography, and to perform diseases diagnosis, treatment and monitoring in the same system, a new setup that combines HIFU and PhS-OCT system was proposed in this study. This proposed setup could do 1) HIFU treatment when the transducer works at high energy level, 2) ultrasound induced shear wave optical coherence elastography for HIFU treatment asynchronous monitoring when the transducer works at low energy level. Ex-vivo bovine liver tissue was treated at the same energy level for different time (0s, 1s, 5s, 9s) in this research. Elastography was performed on the lesion area of the sample after HIFU treatment, and the elastogram was reconstructed by the time of flight time method. The elastogram results clearly show the boundary of HIFU lesion area and surrounding normal tissue, even for 1s treatment time. And the average elasticity of the lesion grows linearly as the treatment time increases. Combined with OCT needle probe, the proposed method has a large potential not only to be used for superficial diseases treatment, but also to be used for high-precision-demanded diseases treatment, e.g. nervous disease treatment.

Biomechanical properties of mammalian bones, such as strength, toughness and plasticity, are essential for understanding how microscopic scale mechanical features can link to macroscale bones’ strength and fracture resistance. We employ Brillouin light scattering (BLS) micro-spectroscopy for local assessment of elastic properties of bones under compression and the efficacy of the tissue engineering approach based on heparin-conjugated fibrin (HCF) hydrogels, bone morphogenic proteins (BMPs) and osteogenic stem cells in the regeneration of the bone tissues. BLS is noninvasive and label-free imaging modality for probing mechanical properties of hard tissues that can give information on structure-function properties of normal and pathological tissues. Results showed that HCF gels containing combination of all factors had the best effect with complete defect regeneration at week 9 and that the bones with fully consolidated fractures have higher values of elastic moduli compared to the bones with defects.