Enhanced spectral-domain optical coherence tomography (SD-OCT) using in situ ultrasonic virtual tunable optical waveguides.

A conventional optical lens can enhance lateral resolution in optical coherence tomography (OCT) by focusing the input light onto the sample. However, the typical Gaussian beam profile of such a lens will impose a tradeoff between the depth of focus (DOF) and the lateral resolution. The lateral resolution is often compromised to achieve a mm-scale DOF. We have experimentally shown that using a cascade system of an ultrasonic virtual tunable optical waveguide (UVTOW) and a short focal-length lens can provide a large DOF without severely compromising the lateral resolution compared to an external lens with the same effective focal length. In addition, leveraging the reconfigurability of UVTOW, we show that the focal length of the cascade system can be tuned without the need for mechanical translation of the optical lens. We compare the performance of the cascade system with a conventional optical lens to demonstrate enhanced DOF without compromising the lateral resolution as well as reconfigurability of UVTOW for OCT imaging.

Interferometric mapping of material properties using thermal perturbation.

Optical phase changes induced by transient perturbations provide a sensitive measure of material properties. We demonstrate the high sensitivity and speed of such methods, using two interferometric techniques: quantitative phase imaging (QPI) in transmission and phase-resolved optical coherence tomography (OCT) in reflection. Shot-noise-limited QPI can resolve energy deposition of about 3.4 mJ/cm2 in a single pulse, which corresponds to 0.8 °C temperature rise in a single cell. OCT can detect deposition of 24 mJ/cm2 energy between two scattering interfaces producing signals with about 30-dB signal-to-noise ratio (SNR), and 4.7 mJ/cm2 when SNR is 45 dB. Both techniques can image thermal changes within the thermal confinement time, which enables accurate single-shot mapping of absorption coefficients even in highly scattering samples, as well as electrical conductivity and many other material properties in biological samples at cellular scale. Integration of the phase changes along the beam path helps increase sensitivity, and the signal relaxation time reveals the size of hidden objects. These methods may enable multiple applications, ranging from temperature-controlled retinal laser therapy or gene expression to mapping electric current density and characterization of semiconductor devices with rapid pump-probe measurements.

Transparent nanocrystalline yttria-stabilized-zirconia calvarium prosthesis.

Laser-based diagnostics and therapeutics show promise for many neurological disorders. However, the poor transparency of cranial bone (calvaria) limits the spatial resolution and interaction depth that can be achieved, thus constraining opportunity in this regard. Herein, we report preliminary results from efforts seeking to address this limitation through use of novel transparent cranial implants made from nanocrystalline yttria-stabilized zirconia (nc-YSZ). Using optical coherence tomography (OCT) imaging of underlying brain in an acute murine model, we show that signal strength is improved when imaging through nc-YSZ implants relative to native cranium. As such, this provides initial evidence supporting the feasibility of nc-YSZ as a transparent cranial implant material. Furthermore, it represents a crucial first step towards realization of an innovative new concept we are developing, which seeks to eventually provide a clinically-viable means for optically accessing the brain, on-demand, over large areas, and on a chronically-recurring basis, without need for repeated craniectomies. FROM THE CLINICAL EDITOR: In this study, transparent nanocrystalline yttria-stabilized-zirconia is used as an experimental "cranium prosthesis" material, enabling the replacement of segments of cranial bone with a material that allows for optical access to the brain on a recurrent basis using optical imaging methods such as OCT.

In vitro formation and extended culture of highly metabolically active and contractile tissues.

3D cell culture models have gained popularity in recent years as an alternative to animal and 2D cell culture models for pharmaceutical testing and disease modeling. Polydimethylsiloxane (PDMS) is a cost-effective and accessible molding material for 3D cultures; however, routine PDMS molding may not be appropriate for extended culture of contractile and metabolically active tissues. Failures can include loss of culture adhesion to the PDMS mold and limited culture surfaces for nutrient and waste diffusion. In this study, we evaluated PDMS molding materials and surface treatments for highly contractile and metabolically active 3D cell cultures. PDMS functionalized with polydopamine allowed for extended culture duration (14.8 ± 3.97 days) when compared to polyethylamine/glutaraldehyde functionalization (6.94 ± 2.74 days); Additionally, porous PDMS extended culture duration (16.7 ± 3.51 days) compared to smooth PDMS (6.33 ± 2.05 days) after treatment with TGF-ß2 to increase culture contraction. Porous PDMS additionally allowed for large (13 mm tall × 8 mm diameter) constructs to be fed by diffusion through the mold, resulting in increased cell density (0.0210 ± 0.0049 mean nuclear fraction) compared to controls (0.0045 ± 0.0016 mean nuclear fraction). As a practical demonstration of the flexibility of porous PDMS, we engineered a vascular bioartificial muscle model (VBAM) and demonstrated extended culture of VBAMs anchored with porous PDMS posts. Using this model, we assessed the effect of feeding frequency on VBAM cellularity. Feeding 3×/week significantly increased nuclear fraction at multiple tissue depths relative to 2×/day. VBAM maturation was similarly improved in 3×/week feeding as measured by nuclear alignment (23.49° ± 3.644) and nuclear aspect ratio (2.274 ± 0.0643) relative to 2x/day (35.93° ± 2.942) and (1.371 ± 0.1127), respectively. The described techniques are designed to be simple and easy to implement with minimal training or expense, improving access to dense and/or metabolically active 3D cell culture models.

Enhanced peripheral nerve regeneration by mechano-electrical stimulation.

To address limitations in current approaches for treating large peripheral nerve defects, the presented study evaluated the feasibility of functional material-mediated physical stimuli on peripheral nerve regeneration. Electrospun piezoelectric poly(vinylidene fluoride-trifluoroethylene) nanofibers were utilized to deliver mechanical actuation-activated electrical stimulation to nerve cells/tissues in a non-invasive manner. Using morphologically and piezoelectrically optimized nanofibers for neurite extension and Schwann cell maturation based on in vitro experiments, piezoelectric nerve conduits were synthesized and implanted in a rat sciatic nerve transection model to bridge a critical-sized sciatic nerve defect (15 mm). A therapeutic shockwave system was utilized to periodically activate the piezoelectric effect of the implanted nerve conduit on demand. The piezoelectric nerve conduit-mediated mechano-electrical stimulation (MES) induced enhanced peripheral nerve regeneration, resulting in full axon reconnection with myelin regeneration from the proximal to the distal ends over the critical-sized nerve gap. In comparison, a control group, in which the implanted piezoelectric conduits were not activated in vivo, failed to exhibit such nerve regeneration. In addition, at both proximal and distal ends of the implanted conduits, a decreased number of damaged myelination (ovoids), an increased number of myelinated nerves, and a larger axonal diameter were observed under the MES condition as compared to the control condition. Furthermore, unlike the control group, the MES condition exhibited a superior functional nerve recovery, assessed by walking track analysis and polarization-sensitive optical coherence tomography, demonstrating the significant potential of the piezoelectric conduit-based physical stimulation approach for the treatment of peripheral nerve injury.

GPU accelerated real-time multi-functional spectral-domain optical coherence tomography system at 1300 nm.

We present a GPU accelerated multi-functional spectral domain optical coherence tomography system at 1300 nm. The system is capable of real-time processing and display of every intensity image, comprised of 512 pixels by 2048 A-lines acquired at 20 frames per second. The update rate for all four images with size of 512 pixels by 2048 A-lines simultaneously (intensity, phase retardation, flow and en face view) is approximately 10 frames per second. Additionally, we report for the first time the characterization of phase retardation and diattenuation by a sample comprised of a stacked set of polarizing film and wave plate. The calculated optic axis orientation, phase retardation and diattenuation match well with expected values. The speed of each facet of the multi-functional OCT CPU-GPU hybrid acquisition system, intensity, phase retardation, and flow, were separately demonstrated by imaging a horseshoe crab lateral compound eye, a non-uniformly heated chicken muscle, and a microfluidic device. A mouse brain with thin skull preparation was imaged in vivo and demonstrated the capability of the system for live multi-functional OCT visualization.

Sound Induced Vibrations Deform the Organ of Corti Complex in the Low-Frequency Apical Region of the Gerbil Cochlea for Normal Hearing : Sound Induced Vibrations Deform the Organ of Corti Complex.

Human speech primarily contains low frequencies. It is well established that such frequencies maximally excite the cochlea near its apex. But, the micromechanics that precede and are involved in this transduction are not well understood. We measured vibrations from the low-frequency, second turn in intact gerbil cochleae using optical coherence tomography (OCT). The data were used to create spatial maps that detail the sound-evoked motions across the sensory organ of Corti complex (OCC). These maps were remarkably similar across animals and showed little variation with frequency or level. We identify four, anatomically distinct, response regions within the OCC: the basilar membrane (BM), the outer hair cells (OHC), the lateral compartment (lc), and the tectorial membrane (TM). Results provide evidence that active processes in the OHC play an important role in the mechanical interplay between different OCC structures which increases the amplitude and tuning sharpness of the traveling wave. The angle between the OCT beam and the OCC makes that we captured radial motions thought to be the effective stimulus to the mechano-sensitive hair bundles. We found that TM responses were relatively weak, arguing against a role in enhancing mechanical hair bundle deflection. Rather, BM responses were found to closely resemble the frequency selectivity and sensitivity found in auditory nerve fibers (ANF) that innervate the low-frequency cochlea.

Automated epidermal thickness quantification of in vitro human skin equivalents using optical coherence tomography.

Human skin equivalents (HSEs) are in vitro models of human skin. They are used to study skin development, diseases, wound healing and toxicity. The gold standard of analysis is histological sectioning, which both limits three-dimensional assessment of the tissue and prevents live culture monitoring. Optical coherence tomography (OCT) has previously been used to visualize in vivo human skin and in vitro models. OCT is noninvasive and enables real-time volumetric analysis of HSEs. The techniques presented here demonstrate the use of OCT imaging to track HSE epidermal thickness over 8 weeks of culture and improve upon previous processing of OCT images by presenting algorithms that automatically quantify epidermal thickness. Through volumetric automated analysis, HSE morphology can be accurately tracked in real time.

Magnetic Field-Modulated Plasmonic Scattering of Hybrid Nanorods for FFT-Weighted OCT Imaging in NIR-II.

We report a method for fast Fourier transform (FFT)-weighted optical coherence tomography (OCT) in the second biological tissue transparency window by actively modulating the plasmonic scattering of Fe3O4@Au hybrid nanorods using magnetic fields. Instead of tracking the nanoparticles' lateral displacement in conventional magnetomotive OCT imaging, we monitor the nanorod rotation and optical signal changes under an alternating magnetic field in real time. The coherent rotation of the nanorods with the field produces periodic OCT signals, and the FFT is then used to convert the periodic OCT signals in the time domain to a single peak in the frequency domain. This allows automatic screening of nanorod signals from the random biological noises and reconstruction of FFT-weighted images using a computer program based on a time-sequence image set. Compared with conventional magnetomotive OCT, the FFT-weighted imaging technique creates enhanced OCT images with dB-scale contrast over an order of magnitude higher than the original images.

Retinal imaging using adaptive optics optical coherence tomography with fast and accurate real-time tracking.

One of the main obstacles in high-resolution 3-D retinal imaging is eye motion, which causes blur and distortion artifacts that require extensive post-processing to be corrected. Here, an adaptive optics optical coherence tomography (AOOCT) system with real-time active eye motion correction is presented. Correction of ocular aberrations and of retinal motion is provided by an adaptive optics scanning laser ophthalmoscope (AOSLO) that is optically and electronically combined with the AOOCT system. We describe the system design and quantify its performance. The AOOCT system features an independent focus adjustment that allows focusing on different retinal layers while maintaining the AOSLO focus on the photoreceptor mosaic for high fidelity active motion correction. The use of a high-quality reference frame for eye tracking increases revisitation accuracy between successive imaging sessions, allowing to collect several volumes from the same area. This system enables spatially targeted retinal imaging as well as volume averaging over multiple imaging sessions with minimal correction of motion in post processing.

Development of a Vascularized Human Skin Equivalent with Hypodermis for Photoaging Studies.

Photoaging is an important extrinsic aging factor leading to altered skin morphology and reduced function. Prior work has revealed a connection between photoaging and loss of subcutaneous fat. Currently, primary models for studying this are in vivo (human samples or animal models) or in vitro models, including human skin equivalents (HSEs). In vivo models are limited by accessibility and cost, while HSEs typically do not include a subcutaneous adipose component. To address this, we developed an "adipose-vascular" HSE (AVHSE) culture method, which includes both hypodermal adipose and vascular cells. Furthermore, we tested AVHSE as a potential model for hypodermal adipose aging via exposure to 0.45 ± 0.15 mW/cm2 385 nm light (UVA). One week of 2 h daily UVA exposure had limited impact on epidermal and vascular components of the AVHSE, but significantly reduced adiposity by approximately 50%. Overall, we have developed a novel method for generating HSE that include vascular and adipose components and demonstrated potential as an aging model using photoaging as an example.

Polarization-sensitive optical frequency domain imaging based on unpolarized light.

Polarization-sensitive optical coherence tomography (PS-OCT) is an augmented form of OCT, providing 3D images of both tissue structure and polarization properties. We developed a new method of polarization-sensitive optical frequency domain imaging (PS-OFDI), which is based on a wavelength-swept source. In this method the sample was illuminated with unpolarized light, which was composed of two orthogonal polarization states (i.e., separated by 180° in the Poincaré sphere) that are uncorrelated to each other. Reflection of these polarization states from within the sample was detected simultaneously and independently using a frequency multiplexing scheme. This simultaneous sample probing with two polarization states enabled determination of the depth-resolved Jones matrices of the sample. Polarization properties of the sample were obtained by analyzing the sample Jones matrices through eigenvector decomposition. The new PS-OFDI system ran at 31K wavelength-scans/s with 3072 pixels per wavelength-scan, and was tested by imaging a polarizer and several birefringent tissues such as chicken muscle and human skin. Lastly the new PS-OFDI was applied to imaging two cancer animal models: a mouse model by injecting cancer cells and a hamster cheek pouch model. These animal model studies demonstrated the significant differences in tissue polarization properties between cancer and normal tissues in vivo.

Formation of 3D Self-Organized Neuron-Glial Interface Derived from Neural Stem Cells via Mechano-Electrical Stimulation.

Due to dissimilarities in genetics and metabolism, current animal models cannot accurately depict human neurological diseases. To develop patient-specific in vitro neural models, a functional material-based technology that offers multi-potent stimuli for enhanced neural tissue development is devised. An electrospun piezoelectric poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)) nanofibrous scaffold is systematically optimized to maximize its piezoelectric properties while accommodating the cellular behaviors of neural stem cells. Hydro-acoustic actuation is elegantly utilized to remotely activate the piezoelectric effect of P(VDF-TrFE) scaffolds in a physiologically-safe manner for the generation of cell-relevant electric potentials. This mechano-electrical stimulation, which arose from the deflection of the scaffold and its consequent generation of electric charges on the scaffold surface under hydro-acoustic actuation, induces the multi-phenotypic differentiation of neural stem cells simultaneously toward neuronal, oligodendrocytic, and astrocytic phenotypes. As compared to the traditional biochemically-mediated differentiation, the 3D neuron-glial interface induced by the mechano-electrical stimulation results in enhanced interactions among cellular components, leading to superior neural connectivity and functionality. These results demonstrate the potential of piezoelectric material-based technology for developing functional neural tissues in vitro via effective neural stem cell modulation with multi-faceted regenerative stimuli.

In vivo 3D human vocal fold imaging with polarization sensitive optical coherence tomography and a MEMS scanning catheter.

We present in-vivo 3D human vocal fold images with polarization sensitive optical coherence tomography (PS-OCT). Characterizing the extent and location of vocal fold lesions provides useful information in guiding surgeons during phonomicrosurgery. Previous studies showed that PS-OCT imaging can distinguish vocal fold lesions from normal tissue, but these studies were limited to 2D cross-sectional imaging and were susceptible to sampling error. In-vivo 3D endoscopic imaging was performed by using a recently developed 2-axis MEMS scanning catheter and a spectral domain OCT (SD-OCT), running at 18.5 frames/s. Imaging was performed in the operating room with patients under general anesthesia and 3D images were acquired either by 2D scanning of the scanner on the sites of interest or by combining 1D scanning and manual sliding to capture whole length of the vocal fold. Vocal fold scar, polyps, nodules, papilloma and malignant lesions were imaged and characteristics of individual lesions were analyzed in terms of spatial distribution and variation of tissue structure and birefringence. The 3D large sectional PS-OCT imaging showed that the spatial extent of vocal fold lesions can be found non-invasively with good contrast from normal tissue.

The optoretinogram reveals the primary steps of phototransduction in the living human eye.

Photoreceptors initiate vision by converting photons to electrical activity. The onset of the phototransduction cascade is marked by the isomerization of photopigments upon light capture. We revealed that the onset of phototransduction is accompanied by a rapid (<5 ms), nanometer-scale electromechanical deformation in individual human cone photoreceptors. Characterizing this biophysical phenomenon associated with phototransduction in vivo was enabled by high-speed phase-resolved optical coherence tomography in a line-field configuration that allowed sufficient spatiotemporal resolution to visualize the nanometer/millisecond-scale light-induced shape change in photoreceptors. The deformation was explained as the optical manifestation of electrical activity, caused due to rapid charge displacement following isomerization, resulting in changes of electrical potential and surface tension within the photoreceptor disc membranes. These all-optical recordings of light-induced activity in the human retina constitute an optoretinogram and hold remarkable potential to reveal the biophysical correlates of neural activity in health and disease.

Retinal imaging with polarization-sensitive optical coherence tomography and adaptive optics.

Various layers of the retina are well known to alter the polarization state of light. Such changes in polarization may be a sensitive indicator of tissue structure and function, and as such have gained increased clinical attention. Here we demonstrate a polarization-sensitive optical coherence tomography (PS-OCT) system that incorporates adaptive optics (AO) in the sample arm and a single line scan camera in the detection arm. We quantify the benefit of AO for PS-OCT in terms of signal-to-noise, lateral resolution, and speckle size. Double pass phase retardation per unit depth values ranging from 0.25 degrees/microm to 0.65 degrees/microm were found in the birefringent nerve fiber layer at 6 degrees eccentricity, superior to the fovea, with the highest values being noticeably higher than previously reported with PS-OCT around the optic nerve head. Moreover, fast axis orientation and degree of polarization uniformity measurements made with AO-PS-OCT demonstrate polarization scrambling in the retinal pigment epithelium at the highest resolution reported to date.

Two-axis magnetically-driven MEMS scanning catheter for endoscopic high-speed optical coherence tomography.

A two-axis scanning catheter was developed for 3D endoscopic imaging with spectral domain optical coherence tomography (SD-OCT). The catheter incorporates a micro-mirror scanner implemented with microelectromechanical systems (MEMS) technology: the micro-mirror is mounted on a two-axis gimbal comprised of folded flexure hinges and is actuated by magnetic field. The scanner can run either statically in both axes or at the resonant frequency (>= 350Hz) for the fast axis. The assembled catheter has an outer diameter of 2.8 mm and a rigid part of 12 mm in length. Its scanning range is +/- 20 in optical angle in both axes with low voltages (1 approximately 3V), resulting in a scannable length of approximately 1 mm at the surface in both axes, even with the small catheter size. The catheter was incorporated with a multi-functional SD-OCT system for 3D endoscopic imaging. Both intensity and polarization-sensitive images could be acquired simultaneously at 18.5K axial scans/s. In vivo 3D images of human fingertips and oral cavity tissue are presented as a demonstration.

Autocalibration of spectral-domain optical coherence tomography spectrometers for in vivo quantitative retinal nerve fiber layer birefringence determination.

Accurate wavelength assignment of each spectral element for spectral-domain optical coherence tomography (SD-OCT) and optical frequency domain imaging (OFDI) is required for proper construction of biological tissue cross-sectional images. This becomes more critical for functional extensions of these techniques, especially in polarization-sensitive optical coherence tomography (PS-OCT), where incorrect wavelength assignment between the two orthogonal polarization channels leads to polarization artifacts. We present an autocalibration method for wavelength assignment that does not require separate calibration measurements and that can be applied directly on actual data. Removal of the birefringence artifact is demonstrated in a PS-OCT system with picometer accuracy in the relative wavelength assignment, resulting in a residual phase error of 0.25 deg/100 microm. We also demonstrate, for the first time, a quantitative birefringence map of an in vivo human retinal nerve fiber layer.

OCT intensity and phase fluctuations correlated with activity-dependent neuronal calcium dynamics in the Drosophila CNS [Invited].

Phase-resolved OCT and fluorescence microscopy were used simultaneously to examine stereotypic patterns of neural activity in the isolated Drosophila central nervous system. Both imaging modalities were focused on individually identified bursicon neurons known to be involved in a fixed action pattern initiated by ecdysis-triggering hormone. We observed clear correspondence of OCT intensity, phase fluctuations, and activity-dependent calcium-induced fluorescence.

Pulsed illumination spectral-domain optical coherence tomography for human retinal imaging.

We present pulsed illumination spectral-domain optical coherence tomography (SD-OCT) for in vivo human retinal imaging. We analyze the signal-to-noise (SNR) for continuous wave (CW) and pulsed illumination SD-OCT. The lateral beam scan motion is responsible for a SNR drop due to lateral scanning induced interference fringe washout. Pulsed illumination can reduce the SNR drop by shorter sample illumination time during the integration time of a camera. First, we demonstrate the SNR benefit of pulsed illumination over CW as function of lateral scan speed for a paper sample. Finally, we show better SNR in retinal images of a normal subject with pulsed illumination SD-OCT over CW at high lateral scanning speed.