Corneal collagen crosslinking (CXL) is a surgical procedure to treat corneal ectasia. Ultraviolet (UV) light and riboflavin are combined to modify corneal microstructure by forming additional chemical bounds between stromal collagen fibers. Because of limited in-depth penetration of riboflavin and UV-light attenuation, a demarcation between treated and untreated regions can be observed, suggesting a two-layer structure after treatment. Here, we present a method of elastic moduli reconstruction in both cornea layers using OCE-tracked guided waves and an analytical model of a 2-layer nearly incompressible transversely isotropic (NITI) medium. An example of reconstruction is demonstrated in an ex vivo human cornea.

Quantifying the mechanical properties in thin layered structures poses a challenge due to the limited spatial resolution of the available mechanical analysis tools. Here, we propose a broadband (0.1 – 10 kHz) Rayleigh-wave optical coherence elastography technique and a bilayer-based inverse model to determine the elastic moduli of all three skin layers in vivo with high reliability. Our results show that 4 – 10 kHz, a previously unexplored frequency range, is critical to resolve the thin epidermis. Our proposed method is valuable for depth-resolved mechanical characterization of various layered materials and tissues.

Evaluating biomechanical properties is crucial to manage skin care and help guide skin cosmetic and reconstruction procedures. Wave-based OCE tracks mechanical waves propagating along the skin surface, where wave speed is used to reconstruct skin elasticity. The first obstacle to quantifying skin elasticity is anisotropy, i.e. 3 shear moduli must be determined. Second, the surface wave group velocity, usually measured experimentally, is very different from the phase velocity needed for elasticity reconstruction. Here we propose methods to overcome these challenges, test the methods using numerical simulations and use them to reconstruct all shear moduli in human skin in vivo.

Assessing the biomechanical properties of tissues can have important applications for disease diagnosis and treatment monitoring. Optical coherence elastography is an established technology for measuring the biomechanical properties of tissues, and has been implemented ex vivo, in vivo, and clinically. Various steps are required for effective translation of this technology, including making improvements in data analysis and processing. OCE data can be inherently noisy, a single data acquisition can consist of gigabytes of data, and data processing can be lengthy. In this work, we examine how convolutional neural networks can be implemented to efficiently process OCE data.

Ovarian cancer remains the deadliest of the gynecological cancers, where this arises from poor screening and imaging tools that can detect early disease. To demonstrate underlying differences in fiber morphology and density which are related to tissue stiffness, in a spectrum of normal and diseases, we use a combination of wavelength dependent Second Harmonic Generation (SHG) and optical scattering measurements. To gain insight into how these structural details affect resulting underlying cellular dynamics, we have used multiphoton excited fabrication to create SHG image-based orthogonal models from collagen/GelMA that represent both the collagen matrix morphology and stiffness (~2-8 kPa) of normal ovarian stroma and high grade serous ovarian cancers (HGSOC). This overall approach of SHG microscopy and image-based tissue engineered scaffolds affords decoupling the roles of matrix morphology, stiffness and cell genotype and affords hypothesis testing of the factors giving rise to disease progression and metastasis. Further, more established fabrication techniques cannot simultaneously reproduce both the 3D collagen fiber morphology and stiffness.

Solid tumours are typically first diagnosed by palpation, revealing increased stiffness, while cancer cells are usually reported to be softer. The mechanical characteristics are not universal and depend on the cell type and the stage of development. Current techniques are usually point or 2D measurement techniques that lack depth penetration in 3D samples. We are developing optical coherence mechano-microscopy integrated with a confocal fluorescence microscope as a platform to investigate the mechanical phenotypes of 3D cancer models, mimicking the conditions in the native tumour microenvironment. This platform enables unique measurement of the 3D elasticity (i.e. Young’s modulus) of metastatic and non-metastatic breast cancer cell spheroids embedded in GelMAl, presenting the extracellular matrix, co-registered with fluorescence images. Our findings show that cells at the centre of non-metastatic cancer cell spheroids are softer (5.8 kPa) than the cells at the periphery (12.7 kPa). In contrast, migrating cells at the periphery of the metastatic cancer cell spheroids are softer (5.7 kPa) than the less motile cells at the centre of these spheroids (8.0 kPa).

Cancer remains one of the most important contributors to premature mortality at the global level. The elastic properties of cells and tissue have been shown to correlate with normal, dysplastic, and cancerous states. In this work, we rely on time-resolved Brillouin scattering to characterise cancerous and normal cells with contrast provided by their elastic properties. In doing so, we achieved proof of concept that artificial intelligence can be used to differentiate between cancerous and normal cell lines with a low number of highly localised measurements. A differentiation accuracy of 93%, was obtained probing in a volume of a few microns corresponding to a single phonon measurement. Our findings suggest the possibility of potential applications for diagnostics.

Mechanical alterations that accompany neoplasia are believed to drive the hallmarks of cancer, including immune modulation. Here, we exploit laser Speckle rHEologicAl micRoscopy (SHEAR), to evaluate the shear viscoelastic modulus, G*(x,y,ω), of breast carcinoma specimens and to investigate the associations between G* and the presence of tumor-infiltrating lymphocytes(TILs). Results demonstrate that in high-TILs tumors, the spatially averaged |G*| is significantly reduced and the gradient of |G*| is significantly increased. Moreover, stroma-to-epithelium contrast of |G*| trends higher, whereas entropy trends lower. These outcomes suggest that the micro-mechanical properties of the tumor microenvironment likely correlate with the immune response.

Since its development over three decades ago, ultrasound elasticity imaging has been established for delineating tissue mechanical property as a relevant biomarker for numerous pathologies including those in liver, kidney, cardiovascular, and musculoskeletal diseases and cancer, among many other applications. While early implementations qualitatively assessed tissue stiffness by monitoring strain induced by external mechanical actuators, the field rapidly progressed to exploit acoustic radiation force and physiological motion as excitation sources, and mathematical relationships between the propagation of induced shear waves and underlying tissue viscoelasticity were derived to support quantitative evaluation of elastic and viscous moduli, albeit with constraints on the geometric complexity of the tissue medium. The field of ultrasound elasticity imaging is once again accelerating rapidly as advancements in transducer design and fabrication, high-performance computing, and artificial intelligence enable hand-held point-of-care systems and wearable sensors, manipulation and integration of data from more than a thousand simultaneous channels for highly-focused volumetric and multi-modal imaging, quantitative elastic and viscous modulus estimation in complex media, and extension to transcranial applications in brain. This presentation will include a review of these emerging technology development areas, and their impact on the future of ultrasound elasticity imaging will be posited in the context of clinical medicine.

Neural tube closure (NTC) is a highly synchronized morphological process driven by mechanical forces, and any disruptions during this process can lead to neural tube defects (NTDs). However, mechanical properties associated with NTDs are largely unknown. To understand the correlation between NTDs and biomechanical properties, we imaged NTC using multimodal Brillouin microscopy and optical coherence tomography in two mutant mice lines, where the genes Mthfd1l and Fuz were inactivated. We also imaged cerebral organoids cultured in dolutegravir for 10 and 14 days. Our results showed a clear link between NTDs and neural tube biomechanical properties.

Tissue elasticity regulates the reproductive function of the mammalian ovary. However, characterizing micro-scale elasticity of entire intact ovaries in three-dimensions (3-D) remains challenging. This limits our ability to study how the elasticity of different ovarian structures, such a follicles, oocytes, and corpus luteum, as well as the entire ovary, affect ovarian function. In this work, for the first time, we extend quantitative micro-elastography to characterize 3-D micro-scale elasticity of entire murine ovaries. We demonstrate distinct elasticity patterns of key ovarian structures as well as changes in tissue elasticity with age.

The viscoelastic properties and microstructure of the fibrin clot network are compromised in patients with coagulation abnormalities. In this study, we harness two novel optical approaches, iCoagLab and Spectrally Encoded Confocal Microscopy (SECM), to investigate how modifications in viscoelastic properties of blood clots are reflected in the microstructural features of the fibrin network in coagulopathic patients. We observe that blood clot viscoelasticity is directly related to fibrin density, fiber length, and fiber straightness of a fully polymerized fibrin network.

Mechanical properties of cells and tissues have been shown to play a crucial role in development and disease, but standard techniques for probing them are usually invasive and limited to the sample’s surface. In contrast, Brillouin microscopy is an emerging optical technique that enables non-contact measurement of viscoelastic properties of a material with diffraction-limited resolution in 3D. In my talk, I will describe our ongoing technology developments in this emerging field and present our current efforts to study the role of mechanical properties in developing organisms such as zebrafish, Drosophila and Platynereis embryos.

In the life sciences, mechanical properties of cells can be used as a diagnostic tool for breast cancer or liver fibrosis, but current techniques require long integration times. The promising impulsive stimulated Brillouin scattering microscopy is an all-optical approach allowing for both a non-invasive and high-resolution measurement. The study reveals that maximizing pulse energy and pulse width achieves high SNR and fast image acquisition, but caution is necessary to stay below the phototoxicity threshold. Imaging at a 10 kHz repetition rate with 20 averages yields good SNR and an imaging speed of 2 ms/pixel, with potential for improvement. A new technique using asymmetric pump focus shows promise in increasing the lateral resolution.

Brillouin microscopy is a label-free and noncontact optical method to measure fundamental viscoelastic properties of biological systems. One of the main challenges in Brillouin microscopy is given by the strong Rayleigh background light overcoming the Brillouin peaks that are spectrally located at a few GHz from the laser frequency. Here, we demonstrate a common-path and broadband notch filter providing an unprecedented 65 dB extinction ratio. We demonstrate the filter by acquiring Brillouin spectra of highly turbid samples, such as pure milk and unprocessed vertebra bone tissues.

In order to realise the clinical potential of Brillouin scattering-based techniques, it is critical to develop an endoscopic probe for measuring elasticity in future in-vivo environments. We have developed a phonon probe which actively injects high amplitude GHz strain pulses into specimens and have demonstrated proof of concept this technique can be used for high resolution 3D imaging. In this talk we show that this new technology is highly applicable to the 3D elasticity imaging of biological tissue from the single-cell scale to multi-cellular organisms and provides a future pathway for the clinical application of in-vivo Brillouin spectroscopy of tissue.

In this work, we developed a new multimodal technology that can simultaneously measure a subset of mechanical, optical, and acoustical properties of the sample, which is based on the integration of Brillouin and photoacoustic (PA) microscopy. Notably, this integration offers a novel approach to probing the refractive index of the sample, which is inaccessible through each technique alone. As a proof-of-concept, we demonstrated the feasibility of the combined setup to obtain the co-registered Brillouin and time-resolved PA signals in a synthetic phantom made of kerosene and CuSO4 aqueous solution. In addition, we measured the refractive index of saline solutions and validated the result in comparison with previously reported data.

The impact of LASIK surgery on corneal biomechanics remains unclear. To address this, we used air-coupled ultrasonic optical coherence elastography (ACUS-OCE) system to examine the full spatial-dependent elasticity of corneas before and after LASIK. We measured Lamb wave propagation speed and average corneal thickness in 16 semi-meridians to calculate the Shear Modulus (G) in 30 healthy control subjects (n=60 corneas) and 8 patients (n= 16 corneas) pre- and 1-month post-LASIK. G decreased from 52±4 kPa to 43±5 kPa after LASIK intervention. In summary, LASIK decreased corneal stiffness, which is consistent with the loss of anterior stroma containing high lamellae interweaving.

Glaucoma is a leading cause of blindness. Previously, we quantified the deformations in scleral tissue components through a PAM-FEA methodology. This study furthers our examination of perilimbal sclera (PLS) and aqueous veins in intraocular pressure (IOP) regulation using ex vivo porcine eyes. Our results reveal that the cross-sectional area change of the aqueous veins and strain of PLS are strongly and positively correlated with the steady-state IOP (n=8, R2=0.90, R2=0.57, respectively) and the IOP elevation rate at a constantly increasing flow rate (n=8, R2=0.89, R2=0.58, respectively). These findings support our hypothesis that the PLS and aqueous veins with less deformation are correlated with elevated steady state IOP and less adaptive behavior of eyes to increased aqueous outflow.

Irregularities of ocular pulsatility are associated with some of the most severe eye diseases. Here, we present a high-speed visible light OCT for simultaneous high-resolution structural imaging and measuring local heartbeat-induced micro-displacements in corneas of rodents. The analysis of complex OCT signal dynamics across repeated frame acquisitions enables tissue motion measurements with high spatial and temporal resolution. As a proof of concept, we demonstrate in vivo imaging in corneas of wildtype mice and reveal subtle structural deformations related to ocular pulsation. Our data in mouse eyes hold promise for a passive, noncontact elastography technique that may enable a new paradigm of OCT based measurements and image contrast.

Biomechanical testing of human skin in vivo is important for studying the aging process and pathological conditions such as skin cancer. Brillouin microscopy allows non-contact measurements of the bulk elastic modulus of the skin, providing information on the mechanical properties of the tissue. Here, we use the combination of Brillouin microscopy and OCT imaging for motion-corrected, depth-resolved biomechanical testing of human skin in vivo. We found that the experimentally measured Brillouin frequency shift of the epidermis is higher than that of the dermis, indicating the higher stiffness of the upper skin layer.

The nanoscale ultrastructure of cells can be analyzed using quantitative phase imaging to examine variations in refractive index. By studying cell response to mechanical stimuli, these refractive index variations can be related to viscoelastic properties such as shear modulus as determined by atomic force microscopy. In contrast, the spatial organization of these variations is characterized using fractal dimension which is seen to increase with cancer progression. Our recent work has linked these two measurements using multiscale measurements of optical phase variance to calculate disorder strength and in turn to determine the fractal dimension of the structures. Disorder strength is seen to vary with imaging resolution with fractal dimension revealed in the observed trends over length scale. Studies will be presented which enable distinction of cell phenotype using these two parameters while their combined use presents a new approach for better understanding cellular restructuring. Finally, modulation of these parameters is examined as a means to study early carcinogenic due to heavy metal exposure.

Alterations in tissue mechanical properties have emerged as not merely a symptom of cancer but a decisive regulator of various oncogenic processes. The tumor microenvironment exhibits a combination of elastic and viscous traits spanning a wide frequency spectrum, leading to a perceived paradox of both matrix stiffening and ‘liquidization’ that coexist during malignant transformation and metastasis. Yet, seminal mechanobiological studies have largely relied on a single mechanical descriptor of elasticity. We present wideband laser Speckle rHEologicAl micRoscopy (SHEAR) to investigate the elastic and viscous spectral signatures that exist over >5 decades of frequencies in various types of clinical tumor specimens.

The stiffness of a biological tissue is a great indicator of its health state. Thus, adding quantitative stiffness to medical imaging systems could be a strong aid for diagnosis, notably in cases of small lesions or inaccessible tissues. In our team, we developed noise correlation elastography for full field coherent imaging technics such as digital holography or FFOCT). In the present study, we demonstrate the advantages of this method for the non-invasive quantification of mechanical anisotropy in fibrous biological tissues, both when validating it on finite-difference simulated data, in anisotropic tissue-mimicking polymer fantoms, and ex-vivo and in-vivo biological samples.

We propose a novel optical microrheology technique that assesses tissue viscoelasticity by tracking the stochastic motion of optical vortices in speckles from turbid tissues. We validate the optical vortex tracking microrheology by illustrating the universal inverse relation between vortex displacements and the viscoelastic modulus. Moreover, we notice the phase angle of the viscoelastic sample is directly related to the vortices subdiffusion exponent. We also observe that the subdiffusion of optical vortices exhibits diverse characteristics for homogeneous and inhomogeneous samples. We believe the optical vortex tracking microrheology may find wide applications in different clinical scenarios like localizing tumors and fabricating biomaterials.

The last few decades have seen the emergence of a huge number of optical imaging techniques. Among them, full-field optical coherence tomography (FF-OCT) has become valuable for many biomedical applications. Indeed, FF-OCT is a noninvasive and label-free imaging technique that produces high-resolution 3D microscopic images of scattering biological samples. However, FF-OCT images alone lack of specificity for accurate diagnosis. That is why it is necessary to add new contrast modalities to FF-OCT technique such as elastography. Indeed, coupling FF-OCT with shear wave elastography allows quantitative estimation of the stiffness at a resolution of a few micrometers. We present here our first results on coupling single-shot off-axis FF-OCT (SO-FF-OCT) method with transient shear wave elastography method.

Systemic sclerosis (SSc-scleroderma) is an autoimmune disorder resulting in excessive collagen accumulation in the skin and internal organs. Currently, the modified Rodnan Skin Score (mRSS) is the gold standard for evaluating dermal thickening due to SSc. However, mRSS has noticeable inter- and intra-observer variabilities as quantified by the interclass correlation coefficient ICC: 0.6-0.75. In this work, optical coherence elastography (OCE) combined with structural and functional optical coherence tomography (OCT) image analysis was used to assess skin thickness in 57 SSc patients and 19 healthy volunteers. The data also demonstrate that OCT/OCE could outperform semi-quantitative clinical assessments such as mRSS. Overall, these results demonstrate that OCT/OCE could be useful for rapid, noninvasive, and objective assessments of SSc onset and monitoring skin disease progression and treatment response.

The analysis of the biomechanical properties is a key factor in disease diagnosis at an earlier stage. It has found several applications in fields such as systemic sclerosis. Optical coherence elastography (OCE) is one of the techniques which has been used to characterize the mechanical properties of the skin. We designed a two-beam OCE system with better phase stability (50 times) than a single-point measurement. This system allows for minimizing bulk tissue movement. We used a piezo transducer to excite acoustic waves in the tissue and two beams to measure the acoustic wave velocity. We measured the elasticity of different ager phantoms and skin.

This study explores Atomic Force Microscopy's (AFM) applicability for 3D analysis by determining its "mechanical focal plane" in soft composites. The investigators determined the AFM's ability to measure mechanics from a distance when target components are immersed in a secondary medium. Using the Kelvin-Voigt model under quasi-static and dynamic conditions, a sample material with polystyrene beads embedded in agarose gel is analyzed at varying scanning parameters. The results include a model of the effective depth and the effect that a secondary medium has on the ability to measure an embedded component's properties.