Laser speckle rheological microscopy reveals wideband viscoelastic spectra of biological tissues.

Viscoelastic transformation of tissue drives aberrant cellular functions and is an early biomarker of disease pathogenesis. Tissues scale a range of viscoelastic moduli, from biofluids to bone. Moreover, viscoelastic behavior is governed by the frequency at which tissue is probed, yielding distinct viscous and elastic responses modulated over a wide frequency band. Existing tools do not quantify wideband viscoelastic spectra in tissues, leaving a vast knowledge gap. We present wideband laser speckle rheological microscopy (WB-SHEAR) that reveals elastic and viscous response over sub-megahertz frequencies previously not investigated in tissue. WB-SHEAR uses an optical, noncontact approach to quantify wideband viscoelastic spectra in specimens spanning a range of moduli from low-viscosity fibrin to highly elastic bone. Via laser scanning, micromechanical imaging is enabled to access wideband viscoelastic spectra in heterogeneous tumor specimens with high spatial resolution (25 micrometers). The ability to interrogate the viscoelastic landscape of diverse biospecimens could transform our understanding of mechanobiological processes in various diseases.

Regeneration of collecting lymphatic vessels following injury.

Secondary lymphedema is a debilitating condition driven by impaired regeneration of lymphatic vasculature following lymphatic injury, surgical removal of lymph nodes in cancer patients or infection. However, the extent to which collecting lymphatic vessels regenerate following injury remains unclear. Here, we employed a novel mouse model of lymphatic injury in combination with state-of-the-art lymphatic imaging to demonstrate that the implantation of an optimized fibrin gel following lymphatic vessel injury leads to the growth and reconnection of the injured lymphatic vessel network, resulting in the restoration of lymph flow to the draining node. Intriguingly, we found that fibrin implantation elevates the tissue levels of CCL5, a potent macrophage-recruiting chemokine. Notably, CCL5-KO mice displayed a reduced ability to reconnect injured vessels following fibrin gel implantation. These novel findings shed light on the mechanisms underlying lymphatic regeneration and suggest that enhancing CCL5 signaling may be a promising therapeutic strategy for enhancing lymphatic regeneration.

Speckle rheological spectroscopy reveals wideband viscoelastic spectra of biological tissues.

Mechanical transformation of tissue is not merely a symptom but a decisive driver in pathological processes. Comprising intricate network of cells, fibrillar proteins, and interstitial fluid, tissues exhibit distinct solid-(elastic) and liquid-like (viscous) behaviours that span a wide band of frequencies. Yet, characterization of wideband viscoelastic behaviour in whole tissue has not been investigated, leaving a vast knowledge gap in the higher frequency range that is linked to fundamental intracellular processes and microstructural dynamics. Here, we present wideband Speckle rHEologicAl spectRoScopy (SHEARS) to address this need. We demonstrate, for the first time, analysis of frequency-dependent elastic and viscous moduli up to the sub-MHz regime in biomimetic scaffolds and tissue specimens of blood clots, breast tumours, and bone. By capturing previously inaccessible viscoelastic behaviour across the wide frequency spectrum, our approach provides distinct and comprehensive mechanical signatures of tissues that may provide new mechanobiological insights and inform novel disease prognostication.

Recent advances in optical elastography and emerging opportunities in the basic sciences and translational medicine [Invited].

Optical elastography offers a rich body of imaging capabilities that can serve as a bridge between organ-level medical elastography and single-molecule biophysics. We review the methodologies and recent developments in optical coherence elastography, Brillouin microscopy, optical microrheology, and photoacoustic elastography. With an outlook toward maximizing the basic science and translational clinical impact of optical elastography technologies, we discuss potential ways that these techniques can integrate not only with each other, but also with supporting technologies and capabilities in other biomedical fields. By embracing cross-modality and cross-disciplinary interactions with these parallel fields, optical elastography can greatly increase its potential to drive new discoveries in the biomedical sciences as well as the development of novel biomechanics-based clinical diagnostics and therapeutics.

Light-sheet photonic force optical coherence elastography for high-throughput quantitative 3D micromechanical imaging.

Quantitative characterisation of micro-scale mechanical properties of the extracellular matrix (ECM) and dynamic cell-ECM interactions can significantly enhance fundamental discoveries and their translational potential in the rapidly growing field of mechanobiology. However, quantitative 3D imaging of ECM mechanics with cellular-scale resolution and dynamic monitoring of cell-mediated changes to pericellular viscoelasticity remain a challenge for existing mechanical characterisation methods. Here, we present light-sheet photonic force optical coherence elastography (LS-pfOCE) to address this need by leveraging a light-sheet for parallelised, non-invasive, and localised mechanical loading. We demonstrate the capabilities of LS-pfOCE by imaging the micromechanical heterogeneity of fibrous collagen matrices and perform live-cell imaging of cell-mediated ECM micromechanical dynamics. By providing access to 4D spatiotemporal variations in the micromechanical properties of 3D biopolymer constructs and engineered cellular systems, LS-pfOCE has the potential to drive new discoveries in mechanobiology and contribute to the development of novel biomechanics-based clinical diagnostics and therapies.

Resolution-enhanced OCT and expanded framework of information capacity and resolution in coherent imaging.

Spatial resolution in conventional optical microscopy has traditionally been treated as a fixed parameter of the optical system. Here, we present an approach to enhance transverse resolution in beam-scanned optical coherence tomography (OCT) beyond its aberration-free resolution limit, without any modification to the optical system. Based on the theorem of invariance of information capacity, resolution-enhanced (RE)-OCT navigates the exchange of information between resolution and signal-to-noise ratio (SNR) by exploiting efficient noise suppression via coherent averaging and a simple computational bandwidth expansion procedure. We demonstrate a resolution enhancement of 1.5 × relative to the aberration-free limit while maintaining comparable SNR in silicone phantom. We show that RE-OCT can significantly enhance the visualization of fine microstructural features in collagen gel and ex vivo mouse brain. Beyond RE-OCT, our analysis in the spatial-frequency domain leads to an expanded framework of information capacity and resolution in coherent imaging that contributes new implications to the theory of coherent imaging. RE-OCT can be readily implemented on most OCT systems worldwide, immediately unlocking information that is beyond their current imaging capabilities, and so has the potential for widespread impact in the numerous areas in which OCT is utilized, including the basic sciences and translational medicine.

Computational 4D-OCM for label-free imaging of collective cell invasion and force-mediated deformations in collagen.

Traction force microscopy (TFM) is an important family of techniques used to measure and study the role of cellular traction forces (CTFs) associated with many biological processes. However, current standard TFM methods rely on imaging techniques that do not provide the experimental capabilities necessary to study CTFs within 3D collective and dynamic systems embedded within optically scattering media. Traction force optical coherence microscopy (TF-OCM) was developed to address these needs, but has only been demonstrated for the study of isolated cells embedded within optically clear media. Here, we present computational 4D-OCM methods that enable the study of dynamic invasion behavior of large tumor spheroids embedded in collagen. Our multi-day, time-lapse imaging data provided detailed visualizations of evolving spheroid morphology, collagen degradation, and collagen deformation, all using label-free scattering contrast. These capabilities, which provided insights into how stromal cells affect cancer progression, significantly expand access to critical data about biophysical interactions of cells with their environment, and lay the foundation for future efforts toward volumetric, time-lapse reconstructions of collective CTFs with TF-OCM.

Investigation of multiple scattering in space and spatial-frequency domains: with application to the analysis of aberration-diverse optical coherence tomography.

Optical microscopy suffers from multiple scattering (MS), which limits the optical imaging depth into scattering media. We previously demonstrated aberration-diverse optical coherence tomography (AD-OCT) for MS suppression, based on the principle that for datasets acquired with different aberration states of the imaging beam, MS backgrounds become decorrelated while single scattering (SS) signals remain correlated, so that a simple coherent average can be used to enhance the SS signal over the MS background. Here, we propose a space/spatial-frequency domain analysis framework for the investigation of MS in OCT, and apply the framework to compare AD-OCT (using astigmatic beams) to standard Gaussian-beam OCT via experiments in scattering tissue phantoms. Utilizing this framework, we found that increasing the astigmatic magnitude produced a large drop in both MS background and SS signal, but the decay experienced by the MS background was larger than the SS signal. Accounting for the decay in both SS signal and MS background, the overall signal-to-background ratio (SBR) of AD-OCT was similar to the Gaussian control after about 10 coherent averages, when deeper line foci was positioned at the plane-of-interest and the line foci spacing was smaller than or equal to 80 µm. For an even larger line foci spacing of 160 µm, AD-OCT resulted in a lower SBR than the Gaussian-beam control. This work provides an analysis framework to gain deeper levels of understanding and insights for the future study of MS and MS suppression in both the space and spatial-frequency domains.

Spatial localization of mechanical excitation affects spatial resolution, contrast, and contrast-to-noise ratio in acoustic radiation force optical coherence elastography.

The notion that a spatially confined mechanical excitation would produce an elastogram with high spatial resolution has motivated the development of various elastography techniques with localized mechanical excitation. However, a quantitative investigation of the effects of spatial localization of mechanical excitation on the spatial resolution of elastograms is still lacking in optical coherence elastography (OCE). Here, we experimentally investigated the effect of spatial localization of acoustic radiation force (ARF) excitation on spatial resolution, contrast, and contrast-to-noise ratio (CNR) of dynamic uniaxial strain elastograms in dynamic ARF-OCE, based on a framework for analyzing the factors that influence the quality of the elastogram at different stages of the elastography workflow. Our results show that localized ARF excitation with a smaller acoustic focal spot size produced a strain elastogram with superior spatial resolution, contrast, and CNR. Our results also suggest that the spatial extent spanned by the displacement response in the sample may connect between the spatial localization of the mechanical excitation and the resulting elastogram quality. The elastography framework and experimental approach presented here may provide a basis for the quantitative analysis of elastogram quality in OCE that can be adapted and applied to different OCE systems and applications.

Spectroscopic photonic force optical coherence elastography.

We demonstrate spectroscopic photonic force optical coherence elastography (PF-OCE). Oscillations of microparticles embedded in viscoelastic hydrogels were induced by harmonically modulated optical radiation pressure and measured by phase-sensitive spectral-domain optical coherence tomography. PF-OCE can detect microparticle displacements with pico- to nano-meter sensitivity and millimeter-scale volumetric coverage. With spectroscopic PF-OCE, we quantified viscoelasticity over a broad frequency range from 1 Hz to 7 kHz, revealing rich microstructural dynamics of polymer networks across multiple microrheological regimes. Reconstructed frequency-dependent loss moduli of polyacrylamide hydrogels were observed to follow a general power scaling law G''∼ω0.75, consistent with that of semiflexible polymer networks. Spectroscopic PF-OCE provides an all-optical approach to microrheological studies with high sensitivity and high spatiotemporal resolution, and could be especially beneficial for time-lapse and volumetric mechanical characterization of viscoelastic materials.

Microrheological quantification of viscoelastic properties with photonic force optical coherence elastography.

Photonic force optical coherence elastography (PF-OCE) is a new approach for volumetric characterization of microscopic mechanical properties of three-dimensional viscoelastic medium. It is based on measurements of the complex mechanical response of embedded micro-beads to harmonically modulated radiation-pressure force from a weakly-focused beam. Here, we utilize the Generalized Stokes-Einstein relation to reconstruct local complex shear modulus in polyacrylamide gels by combining PF-OCE measurements of bead mechanical responses and experimentally measured depth-resolved radiation-pressure force profile of our forcing beam. Data exclusion criteria for quantitative PF-OCE based on three noise-related parameters were identified from the analysis of measurement noise at key processing steps. Shear storage modulus measured by quantitative PF-OCE was found to be in good agreement with standard shear rheometry, whereas shear loss modulus was in agreement with previously published atomic force microscopy results. The analysis and results presented here may serve to inform practical, application-specific implementations of PF-OCE, and establish the technique as a viable tool for quantitative mechanical microscopy.

Photonic force optical coherence elastography for three-dimensional mechanical microscopy.

Optical tweezers are an invaluable tool for non-contact trapping and micro-manipulation, but their ability to facilitate high-throughput volumetric microrheology of biological samples for mechanobiology research is limited by the precise alignment associated with the excitation and detection of individual bead oscillations. In contrast, radiation pressure from a low-numerical aperture optical beam can apply transversely localized force over an extended depth range. Here we present photonic force optical coherence elastography (PF-OCE), leveraging phase-sensitive interferometric detection to track sub-nanometer oscillations of beads, embedded in viscoelastic hydrogels, induced by modulated radiation pressure. Since the displacements caused by ultra-low radiation-pressure force are typically obscured by absorption-mediated thermal effects, mechanical responses of the beads were isolated after independent measurement and decoupling of the photothermal response of the hydrogels. Volumetric imaging of bead mechanical responses in hydrogels with different agarose concentrations by PF-OCE was consistent with bulk mechanical characterization of the hydrogels by shear rheometry.

Depth-resolved measurement of optical radiation-pressure forces with optical coherence tomography.

A weakly focused laser beam can exert sufficient radiation pressure to manipulate microscopic particles over a large depth range. However, depth-resolved continuous measurement of radiation-pressure force profiles over an extended range about the focal plane has not been demonstrated despite decades of research on optical manipulation. Here, we present a method for continuous measurement of axial radiation-pressure forces from a weakly focused beam on polystyrene micro-beads suspended in viscous fluids over a depth range of 400 µm, based on real-time monitoring of particle dynamics using optical coherence tomography (OCT). Measurements of radiation-pressure forces as a function of beam power, wavelength, bead size, and refractive index are consistent with theoretical trends. However, our continuous measurements also reveal localized depth-dependent features in the radiation-pressure force profiles that deviate from theoretical predictions based on an aberration-free Gaussian beam. The combination of long-range radiation pressure and OCT offers a new mode of quantitative optical manipulation and detection with extended spatial coverage. This may find applications in the characterization of optical tractor beams, or volumetric optical manipulation and interrogation of beads in viscoelastic media.