We introduce a spectral domain polarization sensitive optical coherence tomography system with balanced detection capability. The design relies on polarization optics to split incoming light into reference and sample paths. Modified light returning from these paths creates the co- and cross-polarization channels, which are respectively coupled into two polarization-maintaining fibers. These fibers carry the light to a custom spectrometer, and their orthogonal axes help form the interference. The spectrometer produces two pairs of highly aligned and focused spectral lines on a camera for optimal balanced detection operation. System performance is characterized and demonstrated for biomedical imaging with improved signal-to-noise ratio.
Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics’ agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, in this article we review an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion article, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed and provide an outlook for the future directions.
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 ex-vivo 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 migratory glioma cells capable of invading into surrounding healthy tissue. The mechanism and the physical paths by which glioma cells are capable of invading healthy brain are not well understood. Using human glioblastoma cell line U251 plated on healthy mouse brain slices, glioma cell migration behavior and dynamics are investigated by multimodality imaging.
Polarization-sensitive optical coherence tomography (PS-OCT) is used to delineate nerve fiber tracts. PS-OCT is capable of generating depth-resolved images of reflectivity, phase retardance and optic axis orientation. Because of the birefringence property of myelin sheath, nerve fiber tracts as small as a few tens of micrometers can be resolved from phase retardance images. Swept field confocal imaging system is used to image U251 cells expressing GFP-actin and brain vasculature stained by Isolectin B4. Cell migration is acquired by time-lapse imaging and then correlated with brain vasculature and nerve fiber tracts after fine registration of the two modalities.
We found out U251 cells preferentially adhere to and migrate along blood vessels. Our data do not suggest a strong correlation between U251 cell migration and white matter tracts distinguished by tissue birefringence. In addition, U251 cell motility is higher in gray matter compared with white matter. Finally, using higher temporal resolution and high magnification, we are able to observe short time-scale dynamic of U251 cells and the ability of U251 cells to exert forces as they deform blood vessels.
Spinocerebellar ataxia type 1 (SCA1) is a fatal inherited neurodegenerative disease. Post-mortem studies showed neurodegeneration involving white matter components in the cerebral lobes, the cerebellar peduncles and the more distal cranial nerves in human patients. However, the progression of SCA1 in the brain remains unclear. We present the study of white matter atrophy of SCA1 mouse models using serial optical coherence scanner (SOCS).
SOCS consists of a polarization sensitive optical coherence tomography and a tissue slicer (vibratome) with associated controls for serial imaging. The optical system has 5.5 µm axial resolution and utilizes a scan lens or a water-immersion microscope objective to provide 10 µm or 4 µm lateral resolution, respectively. Brain imaging with SOCS showed that the reflectivity contrast portrays morphology, and the polarization contrasts primarily probe myelinated nerve fibers in the white matter. In the cerebellum, the cerebellar cortical layers and white matter are distinguished by using intrinsic optical contrasts. We use SOCS to image the cerebellums of SCA1 mouse models. Data have been acquired from multiple sections at different age groups.
The label-free contrasts show the pathological changes in molecular layer in SCA1 mouse models. White matter size in midline section was quantified at different time points to show white matter degeneration. Moment analysis for retardance contrast and distribution of axis orientation contrast reveal white matter atrophy. High-resolution (4 µm) SOCS visualizes the atrophy of fine features in midline sagittal cerebellum sections as well.
KEYWORDS: Brain mapping, Cerebellum, System on a chip, Coherence (optics), Scanners, Optical coherence tomography, Polarization, Vibration control, Control systems, Visualization
We present the serial optical coherence scanner (SOCS), which consists of a polarization sensitive optical coherence tomography and a vibratome with associated controls for serial imaging, to visualize the cerebellum and adjacent brainstem of mouse. The cerebellar cortical layers and white matter are distinguished by using intrinsic optical contrasts. Images from serial scans reveal the large-scale anatomy in detail and map the nerve fiber pathways in the cerebellum and adjacent brainstem. The optical system, which has ~5.5 μm axial resolution, utilizes a scan lens or a water-immersion microscope objective resulting in 10 μm or 4 μm lateral resolution, respectively. The large-scale brain imaging at high resolution requires an efficient way to collect large datasets. It is important to improve the SOCS system to deal with large-scale and large number of samples in a reasonable time. The imaging and slicing procedure for a section took about 4 minutes due to a low speed of the vibratome blade to maintain slicing quality. SOCS has potential to investigate pathological changes and monitor the effects of therapeutic drugs in cerebellar diseases such as spinocerebellar ataxia 1 (SCA1). The SCA1 is a neurodegenerative disease characterized by atrophy and eventual loss of Purkinje cells from the cerebellar cortex, and the optical contrasts provided by SOCS is being evaluated for biomarkers of the disease.
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 ex vivo 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.
Neuroanatomical pathways form the basis for functional activity of brain circuits. In the past, we developed a polarization-sensitive optical coherence tomography with serial scanning to achieve large-scale brain imaging. The system was able to visualize 3D fiber tracts of ~20 um in diameter. To investigate the neuroanatomical pathways at finer scales, we have now built a polarization-maintaining fiber based ultra-high resolution polarization-sensitive optical coherence microscope (PS-OCM) at 1300 nm. The PS-OCM has an axial resolution of 3.5 um in tissue. The detection setup consists of two spectrometers, acquiring spectral interference on orthogonal polarization channels. With a single measurement, the setup generates four contrasts: reflectivity, cross-polarization, retardance and optic axis orientation. To investigate the capability of PS-OCM at different resolutions, we used three microscope objectives that yield lateral resolutions of 6.0 um, 3.4 um and 1.3 um. Blocks of formalin fixed mouse brain and human brain were scanned. The cross-polarization and retardance images clearly depict the neuronal fiber structures, which are comparable with that generated by the maximum projection of volumetric reflectivity data. The optic axis orientation quantifies the in-plane fiber orientation. With the lateral resolution of 1.3 um, the retardance contrast is weak in white matter due to the shallow depth of focus. Overall, the ultra-high resolution PS-OCM provides a new tool to reveal neuroanatomical maps in the brain at cellular resolution.
The optic axis of birefringent tissues indicates the direction of structural anisotropy. Polarization-sensitive Optical Coherence Tomography (PS-OCT) can provide reflectivity contrast as well as retardance and optic axis orientation contrasts that originate from tissue birefringence. We introduce imaging 3D tissue anisotropy by using a single-camera and polarization-maintaining fiber (PMF) based PS-OCT, which utilizes normal and angled illuminations.
Because environmental factors such as the movement of PMF and temperature fluctuations induce arbitrary phase changes, the optic axis orientation measurement has a time-varying offset. In order to measure the absolute axis orientation, we add a calibration path which dynamically provides the arbitrary offset to be subtracted from the relative axis orientation values.
The axis orientation on the normal plane is the 2D projection of the fiber direction in 3D space. We propose to characterize the axis orientation in different planes (xy, xy’ and x’y planes) by using normal and angled illuminations. This allows calculation of the polar angle that completes the orientation information in 3D. Polarization-based optical systems relying on one illumination angle measure the “apparent birefringence” that light encounters rather than the “true birefringence”. Birefringence as a measure of anisotropy is quantified with the orientation information in 3D. The method and validation with a biological tissue are presented. The study can facilitate imaging and mapping the structural connections in anisotropic tissues including the brain.
We report a functional optical coherence tomography cross-sectional scanner to detect neural activity using unmyelinated nerves dissected from squid. The nerves, unstained or stained with a voltage-sensitive dye, were imaged in a nerve chamber. Transient phase changes from backscattered light were detected during action potential propagation. The results show that the scanner can provide high spatiotemporal resolution cross-sectional images of neural activity (15 μs/A-line; 0.25 ms/B-scan; ∼8.5×5.5 μm2 in xz). The advantage of this method compared to monitoring a single depth profile z is a dramatic increase in the number of available sites that can be measured in two spatial dimensions xz with lateral scanning; therefore, the study demonstrates that two-dimensional monitoring of small-scale functional activity would also be feasible.
Quantitative investigations of fiber orientation and structural connectivity at microscopic resolution have led to great challenges for current neuroimaging techniques. Here, we present a structure tensor (ST) analysis of ex vivo rat brain images acquired by a multicontrast (MC) serial optical coherence scanner. The ST considers the gradients of images in local neighbors to generate a matrix whose eigen-decomposition can estimate the local features such as the edges, anisotropy, and orientation of tissue constituents. This computational analysis is applied on the conventional- and polarization-based contrasts of optical coherence tomography. The three-dimensional (3-D) fiber orientation maps are computed from the image stacks of sequential scans both at mesoresolution for a global view and at high-resolution for the details. The computational orientation maps demonstrate a good agreement with the optic axis orientation contrast which measures the in-plane fiber orientation. Moreover, tractography is implemented using the directional information extracted from the 3-D ST. The study provides a unique opportunity to leverage MC high-resolution information to map structural connectivity of the whole brain.
We present a polarization-maintaining (PM) fiber based optical coherence tomography system for polarization-sensitive and phase-sensitive measurements. Using a single detector, a single depth scan accurately yields retardance and polarization-insensitive reflectivity information along an A-line. Interference patterns on the orthogonal polarization channels are frequency multiplexed and extracted by digital band-pass filters. Images are insensitive to sample rotation in plane perpendicular to ranging. The use of PM-fibers allows direct calculation of birefringence without correcting the polarization transformations known in single-mode non-PM fibers. The system can be configured for differential phase measurements with minor modifications. In this case, a dual detector setup in the detection arm is used, and frequency multiplexing is not utilized.
Spectral-Domain Polarization-Sensitive Optical Coherence Tomography (SD-PS-OCT) is a technique developed to measure the thickness and birefringence of the nerve fiber layer in vivo as a tool for the early diagnosis of glaucoma. A clinical SD-PS-OCT system was developed and scans were made around the optic nerve head (ONH) using ten concentric circles of increasing diameter. One healthy volunteer was imaged. Retinal nerve fiber layer thickness and birefringence information was extracted from the data. Polarization-sensitive OCT images were acquired at video rate (29 frames per second (fps), 1000 A-lines / frame) and at 7 fps (1000 A-lines / frame). The last setting improved the signal to noise ratio by approximately 6 dB. Birefringence measurements on the healthy volunteer gave similar results as earlier reported values that were obtained with a time-domain setup. The measurement time was reduced from more than a minute to less than a second.
We have demonstrated non-contact, sub-nanometer optical measurement of neural surface displacement associated with action potential propagation without applying exogenous chemicals or reflection coatings. Signals recorded from crayfish leg nerve using a phase-sensitive optical low coherence reflectometer show that transient neural surface displacement due to action potential propagation is approximately 1 nm in amplitude and 1 ms in duration. Measured optical signals are coincident with electrical action potential arrival to the optical measurement site. Recent experiments indicate signals with similar amplitude and duration are observed in response to repetitive fast stimulation (200 stimuli/s).
We describe a Polarization Sensitive Optical Coherence Tomography (PS-OCT) system with de-correlated horizontal and vertical channels. Construction of PS-OCT depth-resolved images is achieved with a scanning bulk Michelson interferometer and a broadband TiAl2O3 femtosecond laser source. We de-correlate and delay horizontal and vertical channels using a birefringent crystal in the source path and calcite prism pairs in the sample and reference paths. Cross-correlation and phase changes between horizontal and vertical channels are measured at different reference-sample optical delays in correlated and de-correlated PS-OCT. PS-OCT with de-correlated (DPS-OCT) channels can broaden applications to include de-correlated Doppler imaging of blood flow and imaging the retinal nerve fiber layer with delayed channels. We achieve a differential delay of 0-400 microns between vertical and horizontal channels by translating the calcite prisms. DPS-OCT system design and experimental measurements are presented and discussed.
We report results of a study using differential phase optical coherence tomography (DP-OCT) for
measurement of variation of refractive index (n) vs. analyte concentration (C) in translucent solutions and
turbid tissue phantoms. Variation of refractive index with analyte concentration (dn/dC) in aqueous
solutions of glucose, calcium chloride, magnesium chloride, sodium chloride, potassium chloride,
potassium bicarbonate, urea, bovine serum albumin, and bovine globulin was measured. Obtained results
demonstrated: (1) dn/dC for glucose is significantly greater than that of other analytes in the physiological
range; and (2) high sensitivity of DP-OCT method for measurement of analyte concentration.
Osteoarthritis is a heterogeneous disease characterized by progressive loss of cartilage. The earliest biochemical features, which precede gross pathological changes, include non-uniform loss of proteoglycans associated with increase of water content in tissue and finally, fibrillation of the tissue's collagen network. Loss of proteoglycans decreases the ability of cartilage to withstand compressive loading and makes the tissue softer and more susceptible to wear and fibrilation. If the early loss of proteoglycans is detectable by a non-invasive optical technique, progression of the disease may be arrested using, for example, pharmacologic or surgical intervention. When an electric field is applied to cartilage by an electrical stimulator, the current-generated stress gradients are produced and stress deformation occurs. Since differential phase optical coherence tomography is very sensitive to subsurface stress deformation, we propose to stimulate cartilage electrically and detect stress gradients before gross signs of cartilage degeneration appear. Detection of depth-resolved electromechanical stress gradients in cartilage using differential phase optical coherence tomography may be useful to monitor non-invasively cartilage degeneration. Since the streaming potential and other electrokinetic effects in cartilage are directly proportional to proteoglycan density, application of an electric field in cartilage combined with depth-resolved phase sensitive optical measurements may provide a sensitive indicator of cartilage viability on the molecular-level.
We describe a phase sensitive optical low coherence reflectometer using polarization maintaining optical fiber with high lateral, longitudinal and phase resolution for biomedical applications. The system utilizes three sample path configurations (i.e. single beam, lateral beams, and longitudinally delayed channels) and allows investigation of various topics in biomedical research. Applications include measurement of birefringence change, intensity and phase imaging, refractive index measurements, surface analysis, and measurement of solute concentrations.
In many bio-medical optical imaging applications such as monitoring cell and tissue dynamics and imaging of phase objects, ultra-small changes in refractive index must be detected. We describe a fiber-based optical biosensor, which is capable of detecting ultra-small refractive index changes in highly scattering media with high lateral and longitudinal spatial resolution. The system is a dual channel phase-sensitive optical low coherence tomography system that measures relative optical path length differences between the orthogonal modes of the polarization-maintaining fiber.
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