The importance of polarization-sensitive optical coherence tomography (PS-OCT) has been increasingly recognized in human brain imaging. Despite the recent progress of PS-OCT in revealing white matter architecture and orientation, quantification of fine-scale fiber tracts in the human brain cortex has been a challenging problem, due to a low birefringence in the gray matter. In this study, we investigated the effect of refractive index matching by 2,2’-thiodiethanol (TDE) immersion on the improvement of PS-OCT measurements in ex vivo human brain tissue. We show that we can obtain fiber orientation maps of U-fibers that underlie sulci, as well as cortical fibers in the gray matter, including radial fibers in gyri and distinct layers of fibers exhibiting laminar organization. Further analysis shows that index matching reduces the noise in axis orientation measurements by 56% and 39%, in white and gray matter, respectively. Index matching also enables precise measurements of apparent birefringence, which was underestimated in the white matter by 82% but overestimated in the gray matter by 16% prior to TDE immersion. Mathematical simulations show that the improvements are primarily attributed to the reduction in the tissue scattering coefficient, leading to an enhanced signal-to-noise ratio in deeper tissue regions, which could not be achieved by conventional noise reduction methods.

The mammalian neocortex exhibits a stereotypical laminar organization, with feedforward inputs arriving primarily into layer 4, local computations shaping response selectivity in layers 2/3, and outputs to other brain areas emanating via layers 2/3, 5 and 6. It cannot be assumed a priori that these signatures of laminar differences in neuronal circuitry are reflected in hemodynamic signals that form the basis of functional magnetic resonance imaging (fMRI). Indeed, optical imaging of single-vessel functional responses has highlighted the potential limits of using vascular signals as surrogates for mapping the selectivity of neural responses. Therefore, before fMRI can be employed as an effective tool for studying critical aspects of laminar processing, validation with single-vessel resolution is needed. The primary visual cortex (V1) in cats, with its precise neuronal functional micro-architecture, offers an ideal model system to examine laminar differences in stimulus selectivity across imaging modalities. Here we used cerebral blood volume weighted (wCBV) fMRI to examine if layer-specific orientation-selective responses could be detected in cat V1. We found orientation preference maps organized tangential to the cortical surface that typically extended across depth in a columnar fashion. We then examined arterial dilation and blood velocity responses to identical visual stimuli by using two- and three- photon optical imaging at single-vessel resolution—which provides a measure of the hemodynamic signals with the highest spatial resolution. Both fMRI and optical imaging revealed a consistent laminar response pattern in which orientation selectivity in cortical layer 4 was significantly lower compared to layer 2/3. This systematic change in selectivity across cortical layers has a clear underpinning in neural circuitry, particularly when comparing layer 4 to other cortical layers.

Three-photon excitation microscopy has double-to-triple the penetration depth in biological tissue over two-photon imaging and thus has the potential to revolutionize the visualization of biological processes . However, unlike the plug-and-play operation and performance of lasers used in two-photon imaging, three-photon microscopy presents new technological challenges that require a closer look at the fidelity of laser pulses. We implemented state-of-the-art pulse measurements and developed innovative techniques for examining the performance of lasers used in three-photon microscopy. We then demonstrated how these techniques can be used to provide precise measurements of pulse shape, pulse energy, and pulse-to-pulse intensity variability, all of which ultimately impact imaging. We built inexpensive tools, e.g., a second harmonic generation frequency-resolved optical gating (SHG-FROG) device and a deep-memory diode imaging (DMDI) apparatus to examine laser pulse fidelity. First, SHG-FROG revealed very large third-order dispersion (TOD). This extent of phase distortion prevents the efficient temporal compression of laser pulses to their theoretical limit. Furthermore, TOD cannot be quantified when using a conventional method of obtaining the laser pulse duration, e.g., when using an autocorrelator. Finally, DMDI showed the effectiveness of detecting pulse-to-pulse intensity fluctuations on timescales relevant to three-photon imaging, which were otherwise not captured using conventional instruments and statistics. The distortion of individual laser pulses caused by TOD poses significant challenges to three-photon imaging by preventing effective compression of laser pulses and decreasing the efficiency of nonlinear excitation. Moreover, an acceptably low pulse-to-pulse amplitude variability should not be assumed. Particularly for low repetition rate laser sources used in three-photon microscopy, pulse-to-pulse variability also degrades image quality. If three-photon imaging is to become mainstream, our diagnostics may be used by laser manufacturers to improve system design and by end-users to validate the performance of their current and future imaging systems.

The surface of the human cerebellar cortex is much more tightly folded than the cerebral cortex. Volumetric analysis of cerebellar morphometry in magnetic resonance imaging studies suffers from insufficient resolution, and therefore has had limited impact on disease assessment. Automatic serial polarization-sensitive optical coherence tomography (as-PSOCT) is an emerging technique that offers the advantages of microscopic resolution and volumetric reconstruction of large-scale samples. In this study, we reconstructed multiple cubic centimeters of ex vivo human cerebellum tissue using as-PSOCT. The morphometric and optical properties of the cerebellar cortex across five subjects were quantified. While the molecular and granular layers exhibited similar mean thickness in the five subjects, the thickness varied greatly in the granular layer within subjects. Layer-specific optical property remained homogenous within individual subjects but showed higher cross-subject variability than layer thickness. High-resolution volumetric morphometry and optical property maps of human cerebellar cortex revealed by as-PSOCT have great potential to advance our understanding of cerebellar function and diseases.

Multiphoton microscopy has emerged as the primary imaging tool for studying the structural and functional dynamics of neural circuits in brain tissue, which is highly scattering to light. Recently, three-photon microscopy has enabled high-resolution fluorescence imaging of neurons in deeper brain areas that lie beyond the reach of conventional two-photon microscopy, which is typically limited to ~ 450 µm. Three-photon imaging of neuronal calcium signals, through the genetically-encoded calcium indicator GCaMP6, has been used to successfully record neuronal activity in deeper neocortical layers and parts of the hippocampus in rodents. Bulk-loading cells in deeper cortical layers with synthetic calcium indicators could provide an alternative strategy for labelling that obviates dependence on viral tropism and promoter penetration, particularly in non-rodent species. Here we report a strategy for visualized injection of a calcium dye, Oregon Green BAPTA-1 AM (OGB-1 AM), at 500-600 µm below the surface of the mouse visual cortex in vivo. We demonstrate successful OGB-1 AM loading of cells in cortical layers 5-6 and subsequent three-photon imaging of orientation- and direction- selective visual responses from these cells.

Optical coherence tomography provides volumetric reconstruction of brain structure with micrometer resolution. Gray matter and white matter can be highlighted using conventional and polarization-based contrasts; however, vasculature in fixed brain has not been investigated at large scale due to lack of intrinsic contrast. We present contrast enhancement to visualize the vasculature by perfusing titanium dioxide particles transcardially into the mouse vascular system. The brain, after dissection and fixation, is imaged by a serial optical coherence scanner. Accumulation of particles in blood vessels generates distinguishable optical signals. Among these, the cross-polarization images reveal the vasculature organization remarkably well. The conventional and polarization-based contrasts are still available for probing the gray matter and white matter structures. The segmentation and reconstruction of the vasculature are presented by using a deep learning algorithm. Axonal fiber pathways in the mouse brain are delineated by utilizing the retardance and optic axis orientation contrasts. This is a low-cost method that can be further developed to study neurovascular diseases and brain injury in animal models.

Glioblastoma is a primary malignant brain tumor characterized by highly infiltrative glioma cells. Vasculature and white matter tracts are considered to be the preferred and fastest routes for glioma invasion through brain tissue. In this study, we systematically quantified the routes and motility of the U251 human glioblastoma cell line in mouse brain slices by multimodal imaging. Specifically, we used polarization-sensitive optical coherence tomography to delineate nerve fiber tracts while confocal fluorescence microscopy was used to image cell migration and brain vasculature. Somewhat surprisingly, we found that in mouse brain slices, U251 glioma cells do not follow white matter tracts but rather preferentially migrate along vasculature in both gray and white matter. In addition, U251 cell motility is ∼2-fold higher in gray matter than in white matter (91 vs. 43 μm/h), with a substantial fraction (44%) of cells in both regions invading without close association with vasculature. Interestingly, within both regions, the rates of migration for the perivascular and televascular routes of invasion were indistinguishable. Furthermore, by imaging of local vasculature deformation dynamics during cell migration, we found that U251 cells are capable of exerting traction forces that locally pull on their environment, suggesting the applicability of a "motor-clutch"-based model for migration in vivo. Overall, by quantitatively analyzing the migration dynamics along the diverse pathways followed by invading U251 glioma cells as observed by our multimodal imaging approach, our studies suggest that effective antiinvasive strategies will need to simultaneously limit parallel routes of both perivascular and televascular invasion through both gray and white matter.

We present phase-sensitive measurement of optical rotation using spectral-domain and time-domain low-coherence interferometry. The method utilizes two decorrelated polarization states and simultaneous dual-channel detection provided by polarization-maintaining fiber-based implementation. The sample is placed between polarization optics to control and switch left- and right-handed circular states that experience the sample in forward and backward directions. Phase difference between two interferometric signals yields the optical rotation. Results from glucose and fructose samples are presented for validation.

Spinocerebellar ataxia type 1 (SCA1) is a fatal inherited neurodegenerative disease. In this study, we demonstrate the label-free optical imaging methodology that can detect, with a high degree of sensitivity, discrete areas of degeneration in the cerebellum of the SCA1 mouse models. We used ATXN1[82Q] and ATXN1[30Q]-D776 mice in which the transgene is directed only to Purkinje cells. Molecular layer, granular layer, and white matter regions are analyzed using the intrinsic contrasts provided by polarization-sensitive optical coherence tomography. Cerebellar atrophy in SCA1 mice occurred both in gray matter and white matter. While gray matter atrophy is obvious, indications of white matter atrophy including different birefringence characteristics, and shortened and contorted branches are observed. Imaging results clearly show the loss or atrophy of myelinated axons in ATXN1[82Q] mice. The method provides unbiased contrasts that can facilitate the understanding of the pathological progression in neurodegenerative diseases and other neural disorders.

The lumbar facet capsular ligament (FCL) primarily consists of aligned type I collagen fibers that are mainly oriented across the joint. The aim of this study was to characterize and incorporate in-plane local fiber structure into a multiscale finite element model to predict the mechanical response of the FCL during in vitro mechanical tests, accounting for the heterogeneity in different scales. Characterization was accomplished by using entire-domain polarization-sensitive optical coherence tomography to measure the fiber structure of cadaveric lumbar FCLs ([Formula: see text]). Our imaging results showed that fibers in the lumbar FCL have a highly heterogeneous distribution and are neither isotropic nor completely aligned. The averaged fiber orientation was [Formula: see text] ([Formula: see text] in the inferior region and [Formula: see text] in the middle and superior regions), with respect to lateral-medial direction (superior-medial to inferior-lateral). These imaging data were used to construct heterogeneous structural models, which were then used to predict experimental gross force-strain behavior and the strain distribution during equibiaxial and strip biaxial tests. For equibiaxial loading, the structural model fit the experimental data well but underestimated the lateral-medial forces by [Formula: see text]16% on average. We also observed pronounced heterogeneity in the strain field, with stretch ratios for different elements along the lateral-medial axis of sample typically ranging from about 0.95 to 1.25 during a 12% strip biaxial stretch in the lateral-medial direction. This work highlights the multiscale structural and mechanical heterogeneity of the lumbar FCL, which is significant both in terms of injury prediction and microstructural constituents' (e.g., neurons) behavior.

We present the visualization of the mouse cerebellum and adjacent brainstem using a serial optical coherence scanner, which integrates a vibratome slicer and polarization-sensitive optical coherence tomography for imaging. The scanner provides intrinsic optical contrasts to distinguish the cerebellar cortical layers and white matter. Images from serial scans reveal the large-scale anatomy in detail and map the nerve fiber pathways in the cerebellum and brainstem. By incorporating a water-immersion microscope objective, we also present high-resolution tiled images that delineate fine structures in the cerebellum and brainstem.

The optic axis of birefringent samples indicates the direction of optical anisotropy, which should be described in three-dimensional (3-D) space. We present a method to quantify the complete 3-D optic axis orientation calculated from in-plane optic axis measurements from a polarization-sensitive optical coherence tomography system. The in-plane axis orientations with different illumination angles allow the calculation of the necessary polar angle. The method then provides the information to produce the actual birefringence. The method and results from a biological sample are presented.