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.

Closed-loop wavefront sensing and correction in the mouse brain with computed optical coherence microscopy.

Optical coherence microscopy (OCM) uses interferometric detection to capture the complex optical field with high sensitivity, which enables computational wavefront retrieval using back-scattered light from the sample. Compared to a conventional wavefront sensor, aberration sensing with OCM via computational adaptive optics (CAO) leverages coherence and confocal gating to obtain signals from the focus with less cross-talk from other depths or transverse locations within the field-of-view. Here, we present an investigation of the performance of CAO-based aberration sensing in simulation, bead phantoms, and ex vivo mouse brain tissue. We demonstrate that, due to the influence of the double-pass confocal OCM imaging geometry on the shape of computed pupil functions, computational sensing of high-order aberrations can suffer from signal attenuation in certain spatial-frequency bands and shape similarity with lower order counterparts. However, by sensing and correcting only low-order aberrations (astigmatism, coma, and trefoil), we still successfully corrected tissue-induced aberrations, leading to 3× increase in OCM signal intensity at a depth of ∼0.9 mm in a freshly dissected ex vivo mouse brain.

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.

Obesity-associated Adipose Stromal Cells Promote Breast Cancer Invasion Through Direct Cell Contact and ECM Remodeling.

Obesity increases the risk and worsens the prognosis for breast cancer due, in part, to altered adipose stromal cell (ASC) behavior. Whether ASCs from obese individuals increase migration of breast cancer cells relative to their lean counterparts, however, remains unclear. To test this connection, multicellular spheroids composed of MCF10A-derived tumor cell lines of varying malignant potential and lean or obese ASCs were embedded into collagen scaffolds mimicking the elastic moduli of interstitial breast adipose tissue. Confocal image analysis suggests that tumor cells alone migrate insignificantly under these conditions. However, direct cell-cell contact with either lean or obese ASCs enables them to migrate collectively, whereby obese ASCs activate tumor cell migration more effectively than their lean counterparts. Time-resolved optical coherence tomography (OCT) imaging suggests that obese ASCs facilitate tumor cell migration by mediating contraction of local collagen fibers. Matrix metalloproteinase (MMP)-dependent proteolytic activity significantly contributes to ASC-mediated tumor cell invasion and collagen deformation. However, ASC contractility is also important, as co-inhibition of both MMPs and contractility is necessary to completely abrogate ASC-mediated tumor cell migration. These findings imply that obesity-mediated changes of ASC phenotype may impact tumor cell migration and invasion with potential implications for breast cancer malignancy in obese patients.

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.

Computed optical coherence microscopy of mouse brain ex vivo.

The compromise between lateral resolution and usable imaging depth range is a bottleneck for optical coherence tomography (OCT). Existing solutions for optical coherence microscopy (OCM) suffer from either large data size and long acquisition time or a nonideal point spread function. We present volumetric OCM of mouse brain ex vivo with a large depth coverage by leveraging computational adaptive optics (CAO) to significantly reduce the number of OCM volumes that need to be acquired with a Gaussian beam focused at different depths. We demonstrate volumetric reconstruction of ex-vivo mouse brain with lateral resolution of 2.2 µm, axial resolution of 4.7 µm, and depth range of ∼1.2 mm optical path length, using only 11 OCT data volumes acquired on a spectral-domain OCM system. Compared to focus scanning with step size equal to the Rayleigh length of the beam, this is a factor of 4 fewer datasets required for volumetric imaging. Coregistered two-photon microscopy confirmed that CAO-OCM reconstructions can visualize various tissue microstructures in the brain. Our results also highlight the limitations of CAO in highly scattering media, particularly when attempting to reconstruct far from the focal plane or when imaging deep within the sample.

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.

Quantitative reconstruction of time-varying 3D cell forces with traction force optical coherence microscopy.

Cellular traction forces (CTFs) play an integral role in both physiological processes and disease, and are a topic of interest in mechanobiology. Traction force microscopy (TFM) is a family of methods used to quantify CTFs in a variety of settings. State-of-the-art 3D TFM methods typically rely on confocal fluorescence microscopy, which can impose limitations on acquisition speed, volumetric coverage, and temporal sampling or coverage. In this report, we present the first quantitative implementation of a new TFM technique: traction force optical coherence microscopy (TF-OCM). TF-OCM leverages the capabilities of optical coherence microscopy and computational adaptive optics (CAO) to enable the quantitative reconstruction of 3D CTFs in scattering media with minute-scale temporal sampling. We applied TF-OCM to quantify CTFs exerted by isolated NIH-3T3 fibroblasts embedded in Matrigel, with five-minute temporal sampling, using images spanning a 500 × 500 × 500 µm3 field-of-view. Due to the reliance of TF-OCM on computational imaging methods, we have provided extensive discussion of the equations, assumptions, and failure modes of these methods. By providing high-throughput, label-free, volumetric imaging in scattering media, TF-OCM is well-suited to the study of 3D CTF dynamics, and may prove advantageous for the study of large cell collectives, such as the spheroid models prevalent in mechanobiology.

Aberration-diverse optical coherence tomography for suppression of multiple scattering and speckle.

Multiple scattering is a major barrier that limits the optical imaging depth in scattering media. In order to alleviate this effect, we demonstrate aberration-diverse optical coherence tomography (AD-OCT), which exploits the phase correlation between the deterministic signals from single-scattered photons to suppress the random background caused by multiple scattering and speckle. AD-OCT illuminates the sample volume with diverse aberrated point spread functions, and computationally removes these intentionally applied aberrations. After accumulating 12 astigmatism-diverse OCT volumes, we show a 10 dB enhancement in signal-to-background ratio via a coherent average of reconstructed signals from a USAF target located 7.2 scattering mean free paths below a thick scattering layer, and a 3× speckle contrast reduction from an incoherent average of reconstructed signals inside the scattering layer. This AD-OCT method, when implemented using astigmatic illumination, is a promising approach for ultra-deep volumetric optical coherence microscopy.

Traction Force Microscopy for Noninvasive Imaging of Cell Forces.

The forces exerted by cells on their surroundings play an integral role in both physiological processes and disease progression. Traction force microscopy is a noninvasive technique that enables the in vitro imaging and quantification of cell forces. Utilizing expertise from a variety of disciplines, recent developments in traction force microscopy are enhancing the study of cell forces in physiologically relevant model systems, and hold promise for further advancing knowledge in mechanobiology. In this chapter, we discuss the methods, capabilities, and limitations of modern approaches for traction force microscopy, and highlight ongoing efforts and challenges underlying future innovations.

Volumetric optical coherence microscopy with a high space-bandwidth-time product enabled by hybrid adaptive optics.

Optical coherence microscopy (OCM) is a promising modality for high resolution imaging, but has limited ability to capture large-scale volumetric information about dynamic biological processes with cellular resolution. To enhance the throughput of OCM, we implemented a hybrid adaptive optics (hyAO) approach that combines computational adaptive optics with an intentionally aberrated imaging beam generated via hardware adaptive optics. Using hyAO, we demonstrate the depth-equalized illumination and collection ability of an astigmatic beam compared to a Gaussian beam for cellular-resolution imaging. With this advantage, we achieved volumetric OCM with a higher space-bandwidth-time product compared to Gaussian-beam acquisition that employed focus-scanning across depth. HyAO was also used to perform volumetric time-lapse OCM imaging of cellular dynamics over a 1mm × 1mm × 1mm field-of-view with 2 µm isotropic spatial resolution and 3-minute temporal resolution. As hyAO is compatible with both spectral-domain and swept-source beam-scanning OCM systems, significant further improvements in absolute volumetric throughput are possible by use of ultrahigh-speed swept sources.

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.

Measurement of dynamic cell-induced 3D displacement fields in vitro for traction force optical coherence microscopy.

Traction force microscopy (TFM) is a method used to study the forces exerted by cells as they sense and interact with their environment. Cell forces play a role in processes that take place over a wide range of spatiotemporal scales, and so it is desirable that TFM makes use of imaging modalities that can effectively capture the dynamics associated with these processes. To date, confocal microscopy has been the imaging modality of choice to perform TFM in 3D settings, although multiple factors limit its spatiotemporal coverage. We propose traction force optical coherence microscopy (TF-OCM) as a novel technique that may offer enhanced spatial coverage and temporal sampling compared to current methods used for volumetric TFM studies. Reconstructed volumetric OCM data sets were used to compute time-lapse extracellular matrix deformations resulting from cell forces in 3D culture. These matrix deformations revealed clear differences that can be attributed to the dynamic forces exerted by normal versus contractility-inhibited NIH-3T3 fibroblasts embedded within 3D Matrigel matrices. Our results are the first step toward the realization of 3D TF-OCM, and they highlight the potential use of OCM as a platform for advancing cell mechanics research.

Intraoperative optical coherence tomography for assessing human lymph nodes for metastatic cancer.

BACKGROUND: Evaluation of lymph node (LN) status is an important factor for detecting metastasis and thereby staging breast cancer. Currently utilized clinical techniques involve the surgical disruption and resection of lymphatic structure, whether nodes or axillary contents, for histological examination. While reasonably effective at detection of macrometastasis, the majority of the resected lymph nodes are histologically negative. Improvements need to be made to better detect micrometastasis, minimize or eliminate lymphatic disruption complications, and provide immediate and accurate intraoperative feedback for in vivo cancer staging to better guide surgery. METHODS: We evaluated the use of optical coherence tomography (OCT), a high-resolution, real-time, label-free imaging modality for the intraoperative assessment of human LNs for metastatic disease in patients with breast cancer. We assessed the sensitivity and specificity of double-blinded trained readers who analyzed intraoperative OCT LN images for presence of metastatic disease, using co-registered post-operative histopathology as the gold standard. RESULTS: Our results suggest that intraoperative OCT examination of LNs is an appropriate real-time, label-free, non-destructive alternative to frozen-section analysis, potentially offering faster interpretation and results to empower superior intraoperative decision-making. CONCLUSIONS: Intraoperative OCT has strong potential to supplement current post-operative histopathology with real-time in situ assessment of LNs to preserve both non-cancerous nodes and their lymphatic vessels, and thus reduce the associated risks and complications from surgical disruption of lymphoid structures following biopsy.

Real-time Imaging of the Resection Bed Using a Handheld Probe to Reduce Incidence of Microscopic Positive Margins in Cancer Surgery.

Wide local excision (WLE) is a common surgical intervention for solid tumors such as those in melanoma, breast, pancreatic, and gastrointestinal cancer. However, adequate margin assessment during WLE remains a significant challenge, resulting in surgical reinterventions to achieve adequate local control. Currently, no label-free imaging method is available for surgeons to examine the resection bed in vivo for microscopic residual cancer. Optical coherence tomography (OCT) enables real-time high-resolution imaging of tissue microstructure. Previous studies have demonstrated that OCT analysis of excised tissue specimens can distinguish between normal and cancerous tissues by identifying the heterogeneous and disorganized microscopic tissue structures indicative of malignancy. In this translational study involving 35 patients, a handheld surgical OCT imaging probe was developed for in vivo use to assess margins both in the resection bed and on excised specimens for the microscopic presence of cancer. The image results from OCT showed structural differences between normal and cancerous tissue within the resection bed following WLE of the human breast. The ex vivo images were compared with standard postoperative histopathology to yield sensitivity of 91.7% [95% confidence interval (CI), 62.5%-100%] and specificity of 92.1% (95% CI, 78.4%-98%). This study demonstrates in vivo OCT imaging of the resection bed during WLE with the potential for real-time microscopic image-guided surgery.