Simultaneous multimodal three-photon and optical coherence microscopy of the mouse brain in the 1700 nm optical window .

Multimodal microscopy combining various imaging approaches can provide complementary information about tissue in a single imaging session. Here, we demonstrate a multimodal approach combining three-photon microscopy (3PM) and spectral-domain optical coherence microscopy (SD-OCM). We show that an optical parametric chirped-pulse amplification (OPCPA) laser source, which is the standard source for three-photon fluorescence excitation and third harmonic generation (THG), can be used for simultaneous OCM, 3-photon (3P) fluorescence and THG imaging.

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.

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.

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.

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.

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 mouse brain tissue.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

Volumetric optical coherence microscopy with a high space-bandwidth- 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.

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.

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).

Measurement of dynamic cell-induced 3D displacement fields 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.

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.

Computational high-resolution optical imaging of the living human retina.

High-resolution imaging is of great importance for the fields of biology and medicine. The introduction of hardware-based adaptive optics (HAO) has pushed the limits of optical imaging, enabling high-resolution near diffraction-limited imaging of previously unresolvable structures. In ophthalmology, when combined with optical coherence tomography, HAO has enabled a detailed three-dimensional visualization of photoreceptor distributions and individual nerve fibre bundles in the living human retina.

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.

Computed optical interferometric tomography for high-speed volumetric cellular imaging.

Three-dimensional high-resolution imaging methods are important for cellular-level research. Optical coherence microscopy (OCM) is a low-coherence-based interferometry technology for cellular imaging with both high axial and lateral resolution. Using a high-numerical-aperture objective, OCM normally has a shallow depth of field and requires scanning the focus through the entire region of interest to perform volumetric imaging.

Differentiation of ex vivo human breast tissue using polarization-sensitive optical coherence tomography.

Successful treatment of breast cancer typically requires surgical removal of the tumor. Optical coherence tomography (OCT) has been previously developed for real-time imaging of the surgical margin. However, it can be difficult to distinguish between normal stromal tissue and cancer tissue based on scattering intensity and structure alone.