Noninvasive virtual biopsy using micro-registered optical coherence tomography (OCT) in human subjects.

Histological hematoxylin and eosin-stained (H&E) tissue sections are used as the gold standard for pathologic detection of cancer, tumor margin detection, and disease diagnosis. Producing H&E sections, however, is invasive and time-consuming. While deep learning has shown promise in virtual staining of unstained tissue slides, true virtual biopsy requires staining of images taken from intact tissue. In this work, we developed a micron-accuracy coregistration method [micro-registered optical coherence tomography (OCT)] that can take a two-dimensional (2D) H&E slide and find the exact corresponding section in a 3D OCT image taken from the original fresh tissue. We trained a conditional generative adversarial network using the paired dataset and showed high-fidelity conversion of noninvasive OCT images to virtually stained H&E slices in both 2D and 3D. Applying these trained neural networks to in vivo OCT images should enable physicians to readily incorporate OCT imaging into their clinical practice, reducing the number of unnecessary biopsy procedures.

Rapid Cellular-Resolution Skin Imaging with Optical Coherence Tomography Using All-Glass Multifocal Metasurfaces.

Cellular-resolution optical coherence tomography (OCT) is a powerful tool offering noninvasive histology-like imaging. However, like other optical microscopy tools, a high numerical aperture (N.A.) lens is required to generate a tight focus, generating a narrow depth of field, which necessitates dynamic focusing and limiting the imaging speed. To overcome this limitation, we developed a metasurface platform that generates multiple axial foci, which multiplies the volumetric OCT imaging speed by offering several focal planes. This platform offers accurate and flexible control over the number, positions, and intensities of axial foci generated. All-glass metasurface optical elements 8 mm in diameter are fabricated from fused-silica wafers and implemented into our scanning OCT system. With a constant lateral resolution of 1.1 µm over all depths, the multifocal OCT triples the volumetric acquisition speed for dermatological imaging, while still clearly revealing features of stratum corneum, epidermal cells, and dermal-epidermal junctions and offering morphological information as diagnostic criteria for basal cell carcinoma. The imaging speed can be further improved in a sparse sample, e.g., 7-fold with a seven-foci beam. In summary, this work demonstrates the concept of metasurface-based multifocal OCT for rapid virtual biopsy, further providing insights for developing rapid volumetric imaging systems with high resolution and compact volume.

Intravital 3D visualization and segmentation of murine neural networks at micron resolution.

Optical coherence tomography (OCT) allows label-free, micron-scale 3D imaging of biological tissues' fine structures with significant depth and large field-of-view. Here we introduce a novel OCT-based neuroimaging setting, accompanied by a feature segmentation algorithm, which enables rapid, accurate, and high-resolution in vivo imaging of 700 µm depth across the mouse cortex. Using a commercial OCT device, we demonstrate 3D reconstruction of microarchitectural elements through a cortical column. Our system is sensitive to structural and cellular changes at micron-scale resolution in vivo, such as those from injury or disease. Therefore, it can serve as a tool to visualize and quantify spatiotemporal brain elasticity patterns. This highly transformative and versatile platform allows accurate investigation of brain cellular architectural changes by quantifying features such as brain cell bodies' density, volume, and average distance to the nearest cell. Hence, it may assist in longitudinal studies of microstructural tissue alteration in aging, injury, or disease in a living rodent brain.

Flexible method for generating needle-shaped beams and its application in optical coherence tomography.

Needle-shaped beams (NBs) featuring a long depth-of-focus (DOF) can drastically improve the resolution of microscopy systems. However, thus far, the implementation of a specific NB has been onerous due to the lack of a common, flexible generation method. Here we develop a spatially multiplexed phase pattern that creates many axially closely spaced foci as a universal platform for customizing various NBs, allowing flexible manipulations of beam length and diameter, uniform axial intensity, and sub-diffraction-limit beams. NBs designed via this method successfully extended the DOF of our optical coherence tomography (OCT) system. It revealed clear individual epidermal cells of the entire human epidermis, fine structures of human dermal-epidermal junction in a large depth range, and a high-resolution dynamic heartbeat of alive Drosophila larvae.

Angular compounding for speckle reduction in optical coherence tomography using geometric image registration algorithm and digital focusing.

Optical coherence tomography (OCT) suffers from speckle noise due to the high spatial coherence of the utilized light source, leading to significant reductions in image quality and diagnostic capabilities. In the past, angular compounding techniques have been applied to suppress speckle noise. However, existing image registration methods usually guarantee pure angular compounding only within a relatively small field of view in the focal region, but produce spatial averaging in the other regions, resulting in resolution loss and image blur. This work develops an image registration model to correctly localize the real-space location of every pixel in an OCT image, for all depths. The registered images captured at different angles are fused into a speckle-reduced composite image. Digital focusing, based on the convolution of the complex OCT images and the conjugate of the point spread function (PSF), is studied to further enhance lateral resolution and contrast. As demonstrated by experiments, angular compounding with our improved image registration techniques and digital focusing, can effectively suppress speckle noise, enhance resolution and contrast, and reveal fine structures in ex-vivo imaged tissue.

Real-Time Detection of Circulating Tumor Cells in Living Animals Using Functionalized Large Gold Nanorods.

Optical coherence tomography (OCT) can be utilized with significant speckle reduction techniques and highly scattering contrast agents for non-invasive, contrast-enhanced imaging of living tissues at the cellular scale. The advantages of reduced speckle noise and improved targeted contrast can be harnessed to track objects as small as 2 µm in vivo, which enables applications for cell tracking and quantification in living subjects. Here we demonstrate the use of large gold nanorods as contrast agents for detecting individual micron-sized polystyrene beads and single myeloma cells in blood circulation using speckle-modulating OCT. This report marks the first time that OCT has been used to detect individual cells within blood in vivo. This technical capability unlocks exciting opportunities for dynamic detection and quantification of tumor cells circulating in living subjects.

Optimization of the Trade-Off Between Speckle Reduction and Axial Resolution in Frequency Compounding.

We measured the reduction of speckle by frequency compounding using Gaussian pulses, which have the least time-bandwidth product. The experimental results obtained from a tissue mimicking phantom agree quantitatively with numerical simulations of randomly distributed point scatterers. For a fixed axial resolution, the amount of speckle reduction is found to approach a maximum as the number of bands increases while the total spectral range that they cover is kept constant. An analytical solution of the maximal speckle reduction is derived and shows that the maximum improves approximately as the inverse square root of the Gaussian pulse bandwidth. Since the axial resolution is proportional to the inverse of the pulse bandwidth, an optimized trade-off between speckle reduction and axial resolution is obtained. Considerations for the applications of the optimized trade-off are discussed.

Upper limit for angular compounding speckle reduction.

Angular compounding is a technique for reducing speckle noise in optical coherence tomography that is claimed to significantly improve the signal-to-noise ratio (SNR) of images without impairing their spatial resolution. Here, we examine how focal point movements caused by optical aberrations in an angular compounding system may produce unintended spatial averaging and concomitant loss of spatial resolution. Experimentally, we accounted for such aberrations by aligning our system and measuring distortions in images and found that when the distortions were corrected, the speckle reduction by angular compounding was limited. Our theoretical analysis using Monte Carlo simulations indicates that "pure" angular compounding (i.e., with no spatial averaging) over our full numerical aperture (13° in air) can improve the SNR by not more than a factor of 1.3. Illuminating only a partial aperture cannot improve this factor compared to a spatial averaging system with equivalent loss of resolution. We conclude that speckle reduction using angular compounding is equivalent to spatial averaging. Nonetheless, angular compounding may be useful for improving images in applications where the depth of field is important. The distortions tend to be the greatest off the focal plane, and so angular compounding combined with our correction technique can reduce speckle with a minimal loss of resolution across a large depth of field.

Gold Nanoprisms as Optical Coherence Tomography Contrast Agents in the Second Near-Infrared Window for Enhanced Angiography in Live Animals.

Optical coherence tomography angiography (OCTA) is an important tool for investigating vascular networks and microcirculation in living tissue. Traditional OCTA detects blood vessels via intravascular dynamic scattering signals derived from the movements of red blood cells (RBCs). However, the low hematocrit and long latency between RBCs in capillaries make these OCTA signals discontinuous, leading to incomplete mapping of the vascular networks. OCTA imaging of microvascular circulation is particularly challenging in tumors due to the abnormally slow blood flow in angiogenic tumor vessels and strong attenuation of light by tumor tissue. Here, we demonstrate in vivo that gold nanoprisms (GNPRs) can be used as OCT contrast agents working in the second near-infrared window, significantly enhancing the dynamic scattering signals in microvessels and improving the sensitivity of OCTA in skin tissue and melanoma tumors in live mice. With GNPRs as contrast agents, the postinjection OCT angiograms showed 41 and 59% more microvasculature than preinjection angiograms in healthy mouse skin and melanoma tumors, respectively. By enabling better characterization of microvascular circulation in vivo, GNPR-enhanced OCTA could lead to better understanding of vascular functions during pathological conditions, more accurate measurements of therapeutic response, and improved patient prognoses.

The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle.

Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth.

In Vivo Molecular Optical Coherence Tomography of Lymphatic Vessel Endothelial Hyaluronan Receptors.

Optical Coherence Tomography (OCT) imaging of living subjects offers increased depth of penetration while maintaining high spatial resolution when compared to other optical microscopy techniques. However, since most protein biomarkers do not exhibit inherent contrast detectable by OCT, exogenous contrast agents must be employed for imaging specific cellular biomarkers of interest. While a number of OCT contrast agents have been previously studied, demonstrations of molecular targeting with such agents in live animals have been historically challenging and notably limited in success. Here we demonstrate for the first time that microbeads (µBs) can be used as contrast agents to target cellular biomarkers in lymphatic vessels and can be detected by OCT using a phase variance algorithm. This molecular OCT method enables in vivo imaging of the expression profiles of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a biomarker that plays crucial roles in inflammation and tumor metastasis. In vivo OCT imaging of LVYE-1 showed that the biomarker was significantly down-regulated during inflammation induced by acute contact hypersensitivity (CHS). Our work demonstrated a powerful molecular imaging tool that can be used for high resolution studies of lymphatic function and dynamics in models of inflammation, tumor development, and other lymphatic diseases.

Dependency Network Analysis (DEPNA) Reveals Context Related Influence of Brain Network Nodes.

Communication between and within brain regions is essential for information processing within functional networks. The current methods to determine the influence of one region on another are either based on temporal resolution, or require a predefined model for the connectivity direction. However these requirements are not always achieved, especially in fMRI studies, which have poor temporal resolution. We thus propose a new graph theory approach that focuses on the correlation influence between selected brain regions, entitled Dependency Network Analysis (DEPNA). Partial correlations are used to quantify the level of influence of each node during task performance. As a proof of concept, we conducted the DEPNA on simulated datasets and on two empirical motor and working memory fMRI tasks. The simulations revealed that the DEPNA correctly captures the network's hierarchy of influence. Applying DEPNA to the functional tasks reveals the dynamics between specific nodes as would be expected from prior knowledge. To conclude, we demonstrate that DEPNA can capture the most influencing nodes in the network, as they emerge during specific cognitive processes. This ability opens a new horizon for example in delineating critical nodes for specific clinical interventions.

Quantitative contrast-enhanced optical coherence tomography.

We have developed a model to accurately quantify the signals produced by exogenous scattering agents used for contrast-enhanced Optical Coherence Tomography (OCT). This model predicts distinct concentration-dependent signal trends that arise from the underlying physics of OCT detection. Accordingly, we show that real scattering particles can be described as simplified ideal scatterers with modified scattering intensity and concentration. The relation between OCT signal and particle concentration is approximately linear at concentrations lower than 0.8 particle per imaging voxel. However, at higher concentrations, interference effects cause signal to increase with a square root dependence on the number of particles within a voxel. Finally, high particle concentrations cause enough light attenuation to saturate the detected signal. Predictions were validated by comparison with measured OCT signals from gold nanorods (GNRs) prepared in water at concentrations ranging over five orders of magnitude (50 fM to 5 nM). In addition, we validated that our model accurately predicts the signal responses of GNRs in highly heterogeneous scattering environments including whole blood and living animals. By enabling particle quantification, this work provides a valuable tool for current and future contrast-enhanced in vivo OCT studies. More generally, the model described herein may inform the interpretation of detected signals in modalities that rely on coherence-based detection or are susceptible to interference effects.

Cry for her or cry with her: context-dependent dissociation of two modes of cinematic empathy reflected in network cohesion dynamics.

Two empathy-related processes were recently distinguished neuroscientifically: automatic embodied-simulation (ES) based on visceromotor representation of another's affective state via cingulo-insulary circuit, and emotional sharing relying on cognitive 'theory of mind' (ToM) via prefrontal-temporo-parietal circuit. Evidence that these regions are not only activated but also function as networks during empathic experience has yet to been shown. Employing a novel approach by analyzing fMRI fluctuations of network cohesion while viewing films portraying personal loss, this study demonstrates increased connectivity during empathic engagement (probed by behavioral and parasympathetic indices) both within these circuits, and between them and a set of limbic regions. Notably, this effect was context-dependent: when witnessing as a determined-loss presented as a future event, the ToM and ToM-limbic cohesion positively correlated with state- and empathy indices. During the dramatic peak of this condition, the ToM cohesion was positively correlated with the trait-empathy index of personal distress. However, when the loss was presented as a probabilistic real-time occurrence, ToM cohesion negatively correlated with state-empathy index, which positively correlated with ES-limbic cohesion. In this case, it was the ES-limbic cohesion during the emotional peak which was correlated with personal distress scores. The findings indicate a dichotomy between regulated empathy toward determined-loss and vicarious empathy toward a real-time occurrence.

Synchrony and specificity in the maternal and the paternal brain: relations to oxytocin and vasopressin.

OBJECTIVE: Research on the neurobiology of parenting has defined biobehavioral synchrony, the coordination of biological and behavioral responses between parent and child, as a central process underpinning mammalian bond formation. Bi-parental rearing, typically observed in monogamous species, is similarly thought to draw on mechanisms of mother-father synchrony. METHOD: We examined synchrony in mothers' and fathers' brain response to ecologically valid infant cues. Thirty mothers and fathers, comprising 15 couples parenting 4- to 6-month-old infants, were visited at home, and infant play was videotaped. Parents then underwent functional magnetic resonance imaging scanning while observing own-infant compared with standard-infant videos. Coordination in brain response between mothers and fathers was assessed using a voxel-by-voxel algorithm, and gender-specific activations were also tested. Plasma oxytocin and arginine vasopressin, neuropeptides implicated in female and male bonding, were examined as correlates. RESULTS: Online coordination in maternal and paternal brain activations emerged in social-cognitive networks implicated in empathy and social cognition. Mothers showed higher amygdala activations and correlations between amygdala response and oxytocin. Fathers showed greater activations in social-cognitive circuits, which correlated with vasopressin. CONCLUSIONS: Parents coordinate online activity in social-cognitive networks that support intuitive understanding of infant signals and planning of adequate caregiving, whereas motivational-limbic activations may be gender specific. Although preliminary, these findings demonstrate synchrony in the brain response of two individuals within an attachment relationship, and may suggest that human attachment develops within the matrix of biological attunement and brain-to-brain synchrony between attachment partners.

Portraying emotions at their unfolding: a multilayered approach for probing dynamics of neural networks.

Dynamic functional integration of distinct neural systems plays a pivotal role in emotional experience. We introduce a novel approach for studying emotion-related changes in the interactions within and between networks using fMRI. It is based on continuous computation of a network cohesion index (NCI), which is sensitive to both strength and variability of signal correlations between pre-defined regions. The regions encompass three clusters (namely limbic, medial prefrontal cortex (mPFC) and cognitive), each previously was shown to be involved in emotional processing. Two sadness-inducing film excerpts were viewed passively, and comparisons between viewer's rated sadness, parasympathetic, and inter-NCI and intra-NCI were obtained. Limbic intra-NCI was associated with reported sadness in both movies. However, the correlation between the parasympathetic-index, the rated sadness and the limbic-NCI occurred in only one movie, possibly related to a "deactivated" pattern of sadness. In this film, rated sadness intensity also correlated with the mPFC intra-NCI, possibly reflecting temporal correspondence between sadness and sympathy. Further, only for this movie, we found an association between sadness rating and the mPFC-limbic inter-NCI time courses. To the contrary, in the other film in which sadness was reported to commingle with horror and anger, dramatic events coincided with disintegration of these networks. Together, this may point to a difference between the cinematic experiences with regard to inter-network dynamics related to emotional regulation. These findings demonstrate the advantage of a multi-layered dynamic analysis for elucidating the uniqueness of emotional experiences with regard to an unguided processing of continuous and complex stimulation.