Glioma Cell Migration Dynamics in Brain Tissue Assessed by Multimodal Optical Imaging.

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 µm2/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.

Polarization-sensitive optical coherence tomography reveals gray matter and white matter atrophy in SCA1 mouse models.

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.

Low-coherence interferometry for phase-sensitive measurement of optical rotation.

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.

Polarization sensitive optical coherence microscopy for brain imaging.

Optical coherence tomography (OCT) and optical coherence microscopy (OCM) have demonstrated the ability to investigate cyto- and myelo-architecture in the brain. Polarization-sensitive OCT provides sensitivity to additional contrast mechanisms, specifically the birefringence of myelination and, therefore, is advantageous for investigating white matter fiber tracts. In this Letter, we developed a polarization-sensitive optical coherence microscope (PS-OCM) with a 3.5 µm axial and 1.3 µm transverse resolution to investigate fiber organization and orientation at a finer scale than previously demonstrated with PS-OCT. In a reconstructed mouse brain section, we showed that at the focal depths of 20-70 µm, the PS-OCM reliably identifies the neuronal fibers and quantifies the in-plane orientation.

Polarization-based balanced detection for spectral-domain optical coherence tomography.

We present a new design for spectral-domain optical coherence tomography that allows balanced detection using a single camera. The design uses polarization optics to encode the light in reference and sample arms. Two parallel and highly aligned spectra, which carry out-of-phase interference signals, in-phase common noise, and auto-interference terms, are focused on the camera, which performs the digital balanced detection for each wavelength. The optical system is characterized and tested for tissue imaging. Results demonstrate consistent signal gains in depth and suppression of DC and sample auto-interference. The design could be further amended for polarization-sensitive imaging and might demonstrate a market for manufacturing dual-line cameras with analog-balanced detection capability.

Serial optical coherence scanner for large-scale brain imaging at microscopic resolution.

We describe a serial optical coherence scanner (SOCS) for high resolution imaging of ex-vivo brain. SOCS integrates a multi-contrast optical coherence tomography and a vibratome slicer to establish comprehensive brain anatomy and fiber pathways in three-dimensional space. Rat brain images are demonstrated by utilizing intrinsic optical contrasts including back-scattering, birefringence and optic axis orientation, which are simultaneously generated from the same dataset. Volumetric images from serial scans are combined to realize large scale brain maps. Nerve fiber tracts are globally described in 3D by retardance, and delicately delineated by cross-polarization at the resolution of 15×15×5.5µm(3). In-plane orientations of the tracts are quantified by optic axis orientation. SOCS offers a new solution for complete reconstructions of macroscopic tissues such as primate and human brains at microscopic resolution. The technique also opens up varieties of opportunities for connectome studies and systematic investigations on neurological diseases and brain disorders.

Blockface histology with optical coherence tomography: a comparison with Nissl staining.

Spectral domain optical coherence tomography (SD-OCT) is a high resolution imaging technique that generates excellent contrast based on intrinsic optical properties of the tissue, such as neurons and fibers. The SD-OCT data acquisition is performed directly on the tissue block, diminishing the need for cutting, mounting and staining. We utilized SD-OCT to visualize the laminar structure of the isocortex and compared cortical cytoarchitecture with the gold standard Nissl staining, both qualitatively and quantitatively. In histological processing, distortions routinely affect registration to the blockface image and prevent accurate 3D reconstruction of regions of tissue. We compared blockface registration to SD-OCT and Nissl, respectively, and found that SD-OCT-blockface registration was significantly more accurate than Nissl-blockface registration. Two independent observers manually labeled cortical laminae (e.g. III, IV and V) in SD-OCT images and Nissl stained sections. Our results show that OCT images exhibit sufficient contrast in the cortex to reliably differentiate the cortical layers. Furthermore, the modalities were compared with regard to cortical laminar organization and showed good agreement. Taken together, these SD-OCT results suggest that SD-OCT contains information comparable to standard histological stains such as Nissl in terms of distinguishing cortical layers and architectonic areas. Given these data, we propose that SD-OCT can be used to reliably generate 3D reconstructions of multiple cubic centimeters of cortex that can be used to accurately and semi-automatically perform standard histological analyses.

Cross-validation of serial optical coherence scanning and diffusion tensor imaging: a study on neural fiber maps in human medulla oblongata.

We established a strategy to perform cross-validation of serial optical coherence scanner imaging (SOCS) and diffusion tensor imaging (DTI) on a postmortem human medulla. Following DTI, the sample was serially scanned by SOCS, which integrates a vibratome slicer and a multi-contrast optical coherence tomography rig for large-scale three-dimensional imaging at microscopic resolution. The DTI dataset was registered to the SOCS space. An average correlation coefficient of 0.9 was found between the co-registered fiber maps constructed by fractional anisotropy and retardance contrasts. Pixelwise comparison of fiber orientations demonstrated good agreement between the DTI and SOCS measures. Details of the comparison were studied in regions exhibiting a variety of fiber organizations. DTI estimated the preferential orientation of small fiber tracts; however, it didn't capture their complex patterns as SOCS did. In terms of resolution and imaging depth, SOCS and DTI complement each other, and open new avenues for cross-modality investigations of the brain.

Spectral-domain low-coherence interferometry for phase-sensitive measurement of Faraday rotation at multiple depths.

We describe a method for differential phase measurement of Faraday rotation from multiple depth locations simultaneously. A polarization-maintaining fiber-based spectral-domain interferometer that utilizes a low-coherent light source and a single camera is developed. Light decorrelated by the orthogonal channels of the fiber is launched on a sample as two oppositely polarized circular states. These states reflect from sample surfaces and interfere with the corresponding states of the reference arm. A custom spectrometer, which is designed to simplify camera alignment, separates the orthogonal channels and records the interference-related oscillations on both spectra. Inverse Fourier transform of the spectral oscillations in k-space yields complex depth profiles, whose amplitudes and phase difference are related to reflectivity and Faraday rotation within the sample, respectively. Information along a full depth profile is produced at the camera speed without performing an axial scan for a multisurface sample. System sensitivity for the Faraday rotation measurement is 0.86 min of arc. Verdet constants of clear liquids and turbid media are measured at 687 nm.

Visualizing the complex 3D geometry of the perfusion border zone in isolated rabbit heart.

Myocardial infarction, caused by a major blockage of a coronary artery, creates a border zone (BZ) between perfused and nonperfused tissue, which is believed to be the origin of fatal cardiac arrhythmias. We used a combination of optical clearing and polarization-sensitive optical coherence tomography to visualize a three-dimensional organization of the BZ in isolated rabbit hearts (n=5) at the microscopic level with a high spatial resolution. We found that the BZ has a complex three-dimensional structure with nonperfused areas penetrating into perfused tissue with finger-like projections. These "fingers" may play an important role in the initiation and maintenance of ventricular arrhythmias.

Potassium titanyl phosphate laser tissue ablation: development and experimental validation of a new numerical model.

The photoselective vaporization of prostate (PVP) green light (532 nm) laser is increasingly being used as an alternative to the transurethral resection of prostate (TURP) for treatment of benign prostatic hyperplasia (BPH) in older patients and those who are poor surgical candidates. In order to achieve the goals of increased tissue removal volume (i.e., "ablation" in the engineering sense) and reduced collateral thermal damage during the PVP green light treatment, a two dimensional computational model for laser tissue ablation based on available parameters in the literature has been developed and compared to experiments. The model is based on the control volume finite difference and the enthalpy method with a mechanistically defined energy necessary to ablate (i.e., physically remove) a volume of tissue (i.e., energy of ablation E(ab)). The model was able to capture the general trends experimentally observed in terms of ablation and coagulation areas, their ratio (therapeutic index (TI)), and the ablation rate (AR) (mm(3)/s). The model and experiment were in good agreement at a smaller working distance (WD) (distance from the tissue in mm) and a larger scanning speed (SS) (laser scan speed in mm/s). However, the model and experiment deviated somewhat with a larger WD and a smaller SS; this is most likely due to optical shielding and heat diffusion in the laser scanning direction, which are neglected in the model. This model is a useful first step in the mechanistic prediction of PVP based BPH laser tissue ablation. Future modeling efforts should focus on optical shielding, heat diffusion in the laser scanning direction (i.e., including 3D effects), convective heat losses at the tissue boundary, and the dynamic optical, thermal, and coagulation properties of BPH tissue.

Neurophotonic tools for microscopic measurements and manipulation: status report.

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, this status report reviews 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 report, 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.

Reconstructing micrometer-scale fiber pathways in the brain: multi-contrast optical coherence tomography based tractography.

Comprehensive understanding of connective neural pathways in the brain has put great challenges on the current imaging techniques, for which three-dimensional (3D) visualization of fiber tracts with high spatiotemporal resolution is desirable. Here we present optical imaging and tractography of rat brain ex-vivo using multi-contrast optical coherence tomography (MC-OCT), which is capable of simultaneously generating depth-resolved images of reflectivity, phase retardance, optic axis orientation and, for in-vivo studies, blood flow images. Using the birefringence property of myelin sheath, nerve fiber tracts as small as a few tens of micrometers can be resolved and neighboring fiber tracts with different orientations can be distinguished in cross-sectional optical slices, 2D en-face images and 3D volumetric images. Combinational contrast of MC-OCT images enables visualization of the spatial architecture and nerve fiber orientations in the brain with unprecedented detail. The results suggest that optical tractography, by virtue of its direct accessibility to nerve fibers, has the potential to validate diffusion magnetic resonance images and investigate structural connections in normal brain and neurological disorders. In addition, an endoscopic MC-OCT may be useful in neurosurgical interventions to aid in placement of deep brain stimulating electrodes.

Aggregation affects optical properties and photothermal heating of gold nanospheres.

Laser heating of gold nanospheres (GNS) is increasingly prevalent in biomedical applications due to tunable optical properties that determine heating efficiency. Although many geometric parameters (i.e. size, morphology) can affect optical properties of individual GNS and their heating, no specific studies of how GNS aggregation affects heating have been carried out. We posit here that aggregation, which can occur within some biological systems, will significantly impact the optical and therefore heating properties of GNS. To address this, we employed discrete dipole approximation (DDA) simulations, Ultraviolet-Visible spectroscopy (UV-Vis) and laser calorimetry on GNS primary particles with diameters (5, 16, 30 nm) and their aggregates that contain 2 to 30 GNS particles. DDA shows that aggregation can reduce the extinction cross-section on a per particle basis by 17-28%. Experimental measurement by UV-Vis and laser calorimetry on aggregates also show up to a 25% reduction in extinction coefficient and significantly lower heating (~ 10%) compared to dispersed GNS. In addition, comparison of select aggregates shows even larger extinction cross section drops in sparse vs. dense aggregates. This work shows that GNS aggregation can change optical properties and reduce heating and provides a new framework for exploring this effect during laser heating of nanomaterial solutions.

Swept-source polarization-sensitive optical coherence tomography based on polarization-maintaining fiber.

We present a swept-source polarization-sensitive optical coherence tomography system based on a polarization-maintaining fiber interferometer. The system produces reflectivity and birefringence information along a depth profile with a single sweep of the optical spectrum. Unlike single-mode fiber systems, retardance and relative optical axis orientation images are calculated without compensation. The source is a 45 mW polygon-based swept-source centered at 1290 nm and tuned at a rate of 28 kHz. The interferometer consists of a single polarization-maintaining coupler that utilizes balanced detection for improved performance. Characterization data shows that this system yields accurate measurements with high sensitivity (106.2 dB) comparable to conventional setups. Images of biological tissues with high dynamic range are demonstrated.

Polarization-maintaining fiber based polarization-sensitive optical coherence tomography in spectral domain.

We demonstrate a polarization-sensitive spectral-domain optical coherence tomography system based on polarization-maintaining fiber technology. Using a single-line-scan camera, the system produces reflectivity and retardance information along a depth profile with a single measurement. The relative axis orientation is available as well. System design and characterization and images of a biological tissue are presented.

Optical coherence tomography phase measurement of transient changes in squid giant axons during activity.

Noncontact optical measurements reveal that transient changes in squid giant axons are associated with action potential propagation and altered under different environmental (i.e., temperature) and physiological (i.e., ionic concentrations) conditions. Using a spectral-domain optical coherence tomography system, which produces real-time cross-sectional images of the axon in a nerve chamber, axonal surfaces along a depth profile are monitored. Differential phase analyses show transient changes around the membrane on a millisecond timescale, and the response is coincident with the arrival of the action potential at the optical measurement area. Cooling the axon slows the electrical and optical responses and increases the magnitude of the transient signals. Increasing the NaCl concentration bathing the axon, whose diameter is decreased in the hypertonic solution, results in significantly larger transient signals during action potential propagation. While monophasic and biphasic behaviors are observed, biphasic behavior dominates the results. The initial phase detected was constant for a single location but alternated for different locations; therefore, these transient signals acquired around the membrane appear to have local characteristics.

Measurement of Faraday rotation using phase-sensitive low-coherence interferometry.

We report a differential phase sensor capable of measuring Faraday rotation in reflection mode with a single measurement and a small field-depth factor. Based on a polarization-maintaining-fiber low-coherence interferometer, the sensor measures phase difference between two decorrelated and oppositely polarized circular states incident on a sample. Sensitivity of the sensor for the Faraday rotation is 0.31 arcmin, allowing applications on small volumes of liquids. The Verdet constants of various liquids, including clear and turbid samples, are measured at 857 nm.

Contrast-enhanced serial optical coherence scanner with deep learning network reveals vasculature and white matter organization of mouse brain.

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.

Polarization-sensitive optical coherence tomography based on polarization-maintaining fibers and frequency multiplexing.

We report a novel polarization-maintaining fiber based optical coherence tomography for single detector imaging of tissue reflectivity and birefringence. A single depth scan yields quantitative birefringence information along the A-line accurately. Since the orthogonal polarization channels are frequency multiplexed, the polarization information is extracted by using digital band-pass filters. Here, we introduce the optical system and present the reflectivity and birefringence images of biological tissues with an axial resolution of 7.9 microm and SNR of 30 dB.