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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Localization of cortical tissue optical changes during seizure activity in vivo with optical coherence tomography.

Optical coherence tomography (OCT) is a high resolution, minimally invasive imaging technique, which can produce depth-resolved cross-sectional images. In this study, OCT was used to detect changes in the optical properties of cortical tissue in vivo in mice during the induction of global (pentylenetetrazol) and focal (4-aminopyridine) seizures. Through the use of a confidence interval statistical method on depth-resolved volumes of attenuation coefficient, we demonstrated localization of regions exhibiting both significant positive and negative changes in attenuation coefficient, as well as differentiating between global and focal seizure propagation.

In vivo optical microscopy of peripheral nerve myelination with polarization sensitive-optical coherence tomography.

Assessing nerve integrity and myelination after injury is necessary to provide insight for treatment strategies aimed at restoring neuromuscular function. Currently, this is largely done with electrical analysis, which lacks direct quantitative information. In vivo optical imaging with sufficient imaging depth and resolution could be used to assess the nerve microarchitecture. In this study, we examine the use of polarization sensitive-optical coherence tomography (PS-OCT) to quantitatively assess the sciatic nerve microenvironment through measurements of birefringence after applying a nerve crush injury in a rat model. Initial loss of function and subsequent recovery were demonstrated by calculating the sciatic function index (SFI). We found that the PS-OCT phase retardation slope, which is proportional to birefringence, increased monotonically with the SFI. Additionally, histomorphometric analysis of the myelin thickness and g-ratio shows that the PS-OCT slope is a good indicator of myelin health and recovery after injury. These results demonstrate that PS-OCT is capable of providing nondestructive and quantitative assessment of nerve health after injury and shows promise for continued use both clinically and experimentally in neuroscience.

Decreased light attenuation in cerebral cortex during cerebral edema detected using optical coherence tomography.

Cerebral edema develops in response to a variety of conditions, including traumatic brain injury and stroke, and contributes to the poor prognosis associated with these injuries. This study examines the use of optical coherence tomography (OCT) for detecting cerebral edema in vivo. Three-dimensional imaging of an in vivo water intoxication model in mice was performed using a spectral-domain OCT system centered at 1300 nm. The change in attenuation coefficient was calculated and cerebral blood flow was analyzed using Doppler OCT techniques. We found that the average attenuation coefficient in the cerebral cortex decreased over time as edema progressed. The initial decrease began within minutes of inducing cerebral edema and a maximum decrease of 8% was observed by the end of the experiment. Additionally, cerebral blood flow slowed during late-stage edema. Analysis of local regions revealed the same trend at various locations in the brain, consistent with the global nature of the cerebral edema model used in this study. These results demonstrate that OCT is capable of detecting in vivo optical changes occurring due to cerebral edema and highlights the potential of OCT for precise spatiotemporal detection of cerebral edema.

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 vivo detection of cortical optical changes associated with seizure activity with optical coherence tomography.

The most common technology for seizure detection is with electroencephalography (EEG), which has low spatial resolution and minimal depth discrimination. Optical techniques using near-infrared (NIR) light have been used to improve upon EEG technology and previous research has suggested that optical changes, specifically changes in near-infrared optical scattering, may precede EEG seizure onset in in vivo models. Optical coherence tomography (OCT) is a high resolution, minimally invasive imaging technique, which can produce depth resolved cross-sectional images. In this study, OCT was used to detect changes in optical properties of cortical tissue in vivo in mice before and during the induction of generalized seizure activity. We demonstrated that a significant decrease (P < 0.001) in backscattered intensity during seizure progression can be detected before the onset of observable manifestations of generalized (stage-5) seizures. These results indicate the feasibility of minimally-invasive optical detection of seizures with OCT.

Thinned-skull cortical window technique for in vivo optical coherence tomography imaging.

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

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.

In vivo imaging of human burn injuries with polarization-sensitive optical coherence tomography.

The accurate determination of burn depth is critical in the clinical management of burn wounds. Polarization-sensitive optical coherence tomography (PS-OCT) has been proposed as a potentially non-invasive method for determining burn depth by measuring thermally induced changes in the structure and birefringence of skin, and has been investigated in pre-clinical burn studies with animal models and ex vivo human skin. In this study, we applied PS-OCT to the in-vivo imaging of two pediatric burn patients. Deep and superficial burned skins along with contralateral controls were imaged in 3D. The imaging size was 8 mm × 6 mm × 2 mm in width, length, and depth in the air respectively, and the imaging time was approximately 6 s per volume. Superficially burned skins exhibited the same layered structure as the contralateral controls, but more visible vasculature and reduced birefringence compared to the contralateral controls. In contrast, a deeply burned skin showed loss of the layered structure, almost absent vasculature, and smaller birefringence compared to superficial burns. This study suggested the vasculature and birefringence as parameters for characterizing burn wounds.