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 function of orthopedic tissues such as cartilage and bones are particularly susceptible to aberrant mechanical transformation, which has long been associated with changes to the solid tissue components. There is, however, a growing appreciation for the role of interstitial fluid and tissue water content in degenerative mechanical transformation associated with orthopedic diseases. Yet, the contribution of viscous fluid-like behavior to the overall mechanical integrity of orthopedic tissues remains largely unexplored. We demonstrate wideband laser Speckle rHEologicAl micRoscopy (SHEAR) that harnesses speckle fluctuation induced by natural thermal motion of native light scattering tissue structures for microrheological investigation of orthopedic tissues.
Cells in vivo exist in a 3D porous network of proteins that lend structural support while permitting cellular attachment and migration. Characterization of pore size and microstructural network dynamics is imperative to the biophysical study of cell-ECM interactions and for tissue-engineering applications. We implemented Laser Speckle Microrheology at sub-MHz frequencies to measure mean square displacement (MSD) and its log-log derivative (α) of particles embedded in purified fibrin clots. The power law behavior of the time-dependent MSD provides a measure of pore sizes spanning different particle-to-pore size ratios, relevant for advancing our understanding of cell-ECM interactions.
Biological tissues exhibit distinct viscoelastic behavior across multiple frequency scales. Biophysical interactions between cells and extracellular matrix across this spectrum play an important role in governing many pathophysiological processes. We implemented Laser Speckle Microrheology (LSM) to map and measure frequency-dependent viscous and elastic moduli in tumor specimens and ECM constructs up to the sub-MHz regime. We identified distinct frequency-dependent responses in both elasticity and viscosity across multiple regimes, lending a unique source of micromechanical contrast in tissues. Thus, micromechanical spectroscopy with LSM may provide invaluable biomechanical insights that are inaccessible when solely characterizing elasticity over a limited frequency scale.
We demonstrate applications of photonic-force optical coherence elastography (PF-OCE) on imaging three-dimensional (3D) micro-scale mechanical heterogeneity in collagen phantoms and cell-modified extracellular matrix. In collagen, we observed strong correlations between the measured mechanical heterogeneity and local OCT intensity in the vicinity of beads, which could be related to the presence of fibrous structures. Moreover, we quantitatively imaged micro-scale localized stiffening and increased heterogeneities in fibers surrounding an isolated NIH-3T3 fibroblast enclosed in a 2.5 mg/mL fibrin gel with a volumetric field-of-view (FOV) of ~80μm×350μm×50μm. Our results show that PF-OCE is able to characterize 3D micromechanical properties of biomaterials, and has the potential to observe dynamics of cell-ECM interactions.
We demonstrate resolution-enhanced optical coherent tomography (RE-OCT) via coherent averaging and computational spatial bandwidth expansion. RE-OCT leverages coherent average of the complex OCT signal across multiple acquisitions to suppress noise. The enhanced sensitivity accommodates expansion of spatial bandwidth via multiplication with a spatial-frequency mask. We achieved resolution enhancement of 1.5 times with 40 acquisitions, without sacrificing signal-to-background ratio of single-shot image, in silicone phantom. In collagen, RE-OCT revealed collagen fiber structures that were not resolvable in single-shot or coherent-averaged image. RE-OCT demonstrates an added flexibility to existing image improvement techniques in coherent imaging—resolution, in addition to contrast, enhancement.
Photonic force optical coherence elastography (PF-OCE) is a new approach for volumetric characterization of microscopic mechanical properties of soft materials. PF-OCE utilizes harmonically modulated optical radiation pressure to exert localized mechanical excitation on individual microbeads embedded in viscoelastic media. We present microrheological quantification of complex shear modulus in polyacrylamide gels with PF OCE. Spectroscopic measurements over a frequency range spanning 1 Hz to 7 kHz revealed rich frequency-dependent microstructural dynamics of entangled polymer networks across multiple microrheological regimes. PF-OCE provides an all-optical approach to quantitative three-dimensional mechanical microscopy and broadband spectroscopic microrheological studies of soft materials.
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.
Mechanical properties of cells and tissues play an important role in governing both normal and diseased biological processes. Recent findings in mechanobiology have demonstrated that viscosity, independent of elasticity, of extracellular matrix (ECM) can alter cellular behaviors. To obtain a comprehensive understanding of the mechanical properties of viscoelastic biological tissues for biomedical applications and mechanobiology research, both the elasticity and the viscosity must be characterized. Although optical coherence elastography (OCE) has emerged as a promising tool for probing the mechanical properties of biological tissues, quantitative OCE methods have mostly been limited to elasticity reconstruction or relied on the use of a presumed mechanical model, which may or may not adequately describe the response of a given tissue type. We present the first experimental demonstration of a mechanical model-independent reconstruction of complex shear modulus from direct measurement of surface wave propagation in viscoelastic media with dynamic acoustic radiation force (ARF)-OCE. Our results suggest that elasticity imaging based on shear wave speed alone could overlook potentially significant variations in the viscoelastic properties of biological tissues.
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