Advanced analysis of domain walls in Mg doped LiNbO3 crystals with high resolution OCT.

The structure of domain walls (DW) in ferroelectric media is of great interest as this material is used for frequency doublers and other applications. We show that the structure of the DWs can nicely be visualized by high resolution optical coherence tomography (OCT). While the high group refractive index of lithium niobate allows a resolution much better than 1 µm, the large dispersion can blur the image and has to be compensated. Therefore, we developed an adaptive dispersion compensation algorithm based on maximizing the intensity of the DWs. By measuring a group of DWs, the mean period of the DWs could be measured with an accuracy of less than 10 nm differentiating samples with only 30 nm distinct periods. By analyzing the peak position, amplitude and phase shift within a DW, we were able to determine steps in the DW of only 50 nm. Furthermore, the inclined course of the DWs in a fan-shaped frequency doubler could be displayed. Therefore, we conclude that OCT is able to provide valuable information about the structure of domain walls in periodically poled lithium niobate (PPLN).

NADPH oxidase 4 protects against development of endothelial dysfunction and atherosclerosis in LDL receptor deficient mice.

AIMS: Endothelial dysfunction is an early step in the development of atherosclerosis. Increased formation of superoxide anions by NADPH oxidase Nox1, 2, and 5 reduces nitric oxide availability and can promote endothelial dysfunction. In contrast, recent evidence supports a vasoprotective role of H2O2 produced by main endothelial isoform Nox4. Therefore, we analysed the impact of genetic deletion of Nox4 on endothelial dysfunction and atherosclerosis in the low-density lipoprotein receptor (Ldlr) knockout model. METHODS AND RESULTS: Ex vivo analysis of endothelial function by Mulvany myograph showed impaired endothelial function in thoracic aorta of Nox4(-/-)/Ldlr(-/-) mice. Further progression of endothelial dysfunction due to high-fat diet increased atherosclerotic plaque burden and galectin-3 staining in Nox4(-/-)/Ldlr(-/-) mice compared with Ldlr(-/-) mice. Under physiological conditions, loss of Nox4 does not influence aortic vascular function. In this setting, loss of Nox4-derived H2O2 production could be partially compensated for by nNOS upregulation. Using an innovative optical coherence tomography approach, we were able to analyse endothelial function by flow-mediated vasodilation in the murine saphenous artery in vivo. This new approach revealed an altered flow-mediated dilation in Nox4(-/-) mice, indicating a role for Nox4 under physiological conditions in peripheral arteries in vivo. CONCLUSIONS: Nox4 plays an important role in maintaining endothelial function under physiological and pathological conditions. Loss of Nox4-derived H2O2 could be partially compensated for by nNOS upregulation, but severe endothelial dysfunction is not reversible. This leads to increased atherosclerosis under atherosclerotic prone conditions.

Modeling inducible neuropathologies of the retina with differential phenotypes in organoids.

Neurodegenerative diseases remain incompletely understood and therapies are needed. Stem cell-derived organoid models facilitate fundamental and translational medicine research. However, to which extent differential neuronal and glial pathologic processes can be reproduced in current systems is still unclear. Here, we tested 16 different chemical, physical, and cell functional manipulations in mouse retina organoids to further explore this. Some of the treatments induce differential phenotypes, indicating that organoids are competent to reproduce distinct pathologic processes. Notably, mouse retina organoids even reproduce a complex pathology phenotype with combined photoreceptor neurodegeneration and glial pathologies upon combined (not single) application of HBEGF and TNF, two factors previously associated with neurodegenerative diseases. Pharmacological inhibitors for MAPK signaling completely prevent photoreceptor and glial pathologies, while inhibitors for Rho/ROCK, NFkB, and CDK4 differentially affect them. In conclusion, mouse retina organoids facilitate reproduction of distinct and complex pathologies, mechanistic access, insights for further organoid optimization, and modeling of differential phenotypes for future applications in fundamental and translational medicine research.

An advanced algorithm for dispersion encoded full range frequency domain optical coherence tomography.

Dispersion encoded full range (DEFR) optical coherence tomography (OCT) has become highly attractive as it is a simple way to increase the measurement range of OCT systems. Full range OCT is especially favorable as it does not only increase the measurement range but also shifts the highest sensitivity into the center of the measurement range. While the early versions of DEFR were highly computational expensive, new versions reduce the number of necessary Fourier transforms. Recently it has been shown that a GPU based algorithm can perform DEFR with more than 20,000 A-lines per second. We present a new version of the DEFR algorithm that requires only one Fourier transform per A-scan and uses convolution in z-space instead of multiplication in k-space, therefore reducing the computational effort considerably. While dispersion encoding has so far only been used to suppress mirror artifacts, we show that, with dispersion encoding and only one more Fourier transform, autocorrelation terms can be removed likewise. Since very high values of dispersion reduce the effective measurement range in dispersion encoded OCT, we present an estimate for a sufficient amount of dispersion for a successful image recovery, which is depending on the thickness of the scattering layers. Furthermore, we demonstrate the usability of ZnSe as a new dispersive material with a very high dispersion and describe a simple method to extract the dispersive phase from the measurement of a single reflex of a glass surface. Using a standard consumer PC, an artifact-free recovery of 1000 - 2000 A-scans per second with 2048 depth values including autocorrelation removal was achieved. The dynamic range (sensitivity) is not reduced and the suppression ratio of mirror artifacts and autocorrelation signals is more than 50dB using ZnSe.

Optical coherence tomography in biomedical research.

Optical coherence tomography (OCT) is a noninvasive, high-resolution, interferometric imaging modality using near-infrared light to acquire cross-sections and three-dimensional images of the subsurface microstructure of biological specimens. Because of rapid improvement of the acquisition speed and axial resolution of OCT over recent years, OCT is becoming increasingly attractive for applications in biomedical research. Therefore, OCT is no longer used solely for structural investigations of biological samples but also for functional examination, making it potentially useful in bioanalytical science. The combination of in vivo structural and functional findings makes it possible to obtain thorough knowledge on basic physiological and pathological processes. Advanced applications, for example, optical biopsy in visceral cavities, have been enabled by combining OCT with established imaging modalities. This report gives an outline of the state of the art and novel trends of innovative OCT approaches in biomedical research in which the main focus is on applications in fundamental research and pre-clinical utilization.

Simultaneous dual-band optical coherence tomography in the spectral domain for high resolution in vivo imaging.

Optical coherence tomography (OCT) in the spectral domain is demonstrated simultaneously at two wavelength bands centered at 800 nm and 1250 nm. A novel commercial supercontinuum laser is applied as a single low coherence broadband light source. The emission spectrum of the source is shaped by optical and spatial filtering in order to achieve an adequate double peak spectrum containing the wavelength bands 700 - 900 nm and 1100 - 1400 nm for dual-band OCT imaging and thus reducing the radiation exposure of the sample. Each wavelength band is analyzed with an individual spectrometer at an A-scan rate of about 12 kHz which enables real-time imaging for the examination of moving samples. A common path optical setup optimized for both spectral regions with a separate single fiber-based scanning unit was realized which facilitates flexible handling and easy access to the measurement area. The free-space axial resolutions were measured to be less than 4.5 microm and 7 microm at 800 nm and 1250 nm, respectively. Three-dimensional imaging ten times faster than previously reported with a signal-to-noise-ratio of above 90 dB is achieved simultaneously in both wavelength bands. Spectral domain dual-band OCT combines real-time imaging with high resolution at 800 nm and enhanced penetration depth at 1250 nm and therefore provides a well suited tool for in vivo vasodynamic measurements. Further, spatially resolved spectral features of the sample are obtained by means of comparing the backscattering properties at two different wavelength bands. The ability of dual-band OCT to enhance tissue contrast and the sensitivity of this imaging modality to wavelength-dependent sample birefringence is demonstrated.

Stem Cell-Derived Photoreceptor Transplants Differentially Integrate Into Mouse Models of Cone-Rod Dystrophy.

PURPOSE: Preclinical studies on photoreceptor transplantation provided evidence for restoration of visual function with pluripotent stem cells considered as a potential source for sufficient amounts of donor material. Adequate preclinical models representing retinal disease conditions of potential future patients are needed for translation research. Here we compared transplant integration in mouse models with mild (prominin1-deficient; Prom1-/-) or severe (cone photoreceptor function loss 1/rhodopsin-deficient double-mutant; Cpfl1/Rho-/-) cone-rod degeneration. METHODS: For photoreceptor transplant production, we combined the mouse embryonic stem cell retinal organoid system with rhodopsin-driven GFP cell labeling by recombinant adeno-associated virus (AAV). Organoid-derived photoreceptors were enriched by CD73-based magnetic-activated cell sorting (MACS) and transplanted subretinally into wild-type, Prom1-/- and Cpfl1/Rho-/- hosts. The survival, maturation, and synapse formation of donor cells was analyzed by immunohistochemistry. RESULTS: Retinal organoids yielded high photoreceptor numbers that were further MACS-enriched to 85% purity. Grafted photoreceptors survived in the subretinal space of all mouse models. Some cells integrated into wild-type as well as Prom1-/- mouse retinas and acquired a mature morphology, expressing rod and synaptic markers in close proximity to second-order neurons. In contrast, in the novel Cpfl1/Rho-/- model with complete photoreceptor degeneration, transplants remained confined to the subretinal space, expressed rod-specific but only reduced synaptic markers, and did not acquire mature morphology. CONCLUSIONS: Comparison of photoreceptor grafts in preclinical models with incomplete or complete photoreceptor loss, showed differential transplant success with effective and impaired integration, respectively. Thus, Cpfl1/Rho-/- mice represent a potential benchmark model resembling patients with severe retinal degeneration to optimize photoreceptor replacement therapies.

Imaging of nanoparticle-labeled stem cells using magnetomotive optical coherence tomography, laser speckle reflectometry, and light microscopy.

Cell transplantation and stem cell therapy are promising approaches for regenerative medicine and are of interest to researchers and clinicians worldwide. However, currently, no imaging technique that allows three-dimensional in vivo inspection of therapeutically administered cells in host tissues is available. Therefore, we investigate magnetomotive optical coherence tomography (MM-OCT) of cells labeled with magnetic particles as a potential noninvasive cell tracking method. We develop magnetomotive imaging of mesenchymal stem cells for future cell therapy monitoring. Cells were labeled with fluorescent iron oxide nanoparticles, embedded in tissue-mimicking agar scaffolds, and imaged using a microscope setup with an integrated MM-OCT probe. Magnetic particle-induced motion in response to a pulsed magnetic field of 0.2 T was successfully detected by OCT speckle variance analysis, and cross-sectional and volumetric OCT scans with highlighted labeled cells were obtained. In parallel, fluorescence microscopy and laser speckle reflectometry were applied as two-dimensional reference modalities to image particle distribution and magnetically induced motion inside the sample, respectively. All three optical imaging modalities were in good agreement with each other. Thus, magnetomotive imaging using iron oxide nanoparticles as cellular contrast agents is a potential technique for enhanced visualization of selected cells in OCT.

Characterization of a mouse model with complete RPE loss and its use for RPE cell transplantation.

PURPOSE: Age-related macular degeneration (AMD) is a major leading cause of visual impairment and blindness with no cure currently established. Cell replacement of RPE is discussed as a potential therapy for AMD. Previous studies were performed in animal models with severe limitations in recapitulating the disease progression. In detail, we describe the effect of systemic injection of sodium iodate in the mouse retina. We further evaluate the usefulness of this animal model to analyze cell-specific effects following transplantation of human embryonic stem cell (hESC)-derived RPE cells. METHODS: Morphologic, functional, and behavioral changes following sodium iodate injection were monitored by histology, gene expression analysis, electroretinography, and optokinetic head tracking. Human embryonic stem cell-derived RPE cells were transplanted 1 week after sodium iodate injection and experimental retinae were analyzed 3 weeks later. RESULTS: Injection of sodium iodate caused complete RPE cell loss, photoreceptor degeneration, and altered gene and protein expression in outer and inner nuclear layers. Retinal function was severely affected by day 3 and abolished from day 14. Following transplantation, donor hESC-derived RPE cells formed extensive monolayers that displayed wild-type RPE cell morphology, organization, and function, including phagocytosis of host photoreceptor outer segments. CONCLUSIONS: Systemic injection of sodium iodate has considerable effects on RPE, photoreceptors, and inner nuclear layer neurons, and provides a model to assay reconstitution and maturation of RPE cell transplants. The availability of an RPE-free Bruch's membrane in this model likely allows the unprecedented formation of extensive polarized cell monolayers from donor hESC-derived RPE cell suspensions.

Quantitative fluorescence angiography for neurosurgical interventions.

Present methods for quantitative measurement of cerebral perfusion during neurosurgical operations require additional technology for measurement, data acquisition, and processing. This study used conventional fluorescence video angiography--as an established method to visualize blood flow in brain vessels--enhanced by a quantifying perfusion software tool. For these purposes, the fluorescence dye indocyanine green is given intravenously, and after activation by a near-infrared light source the fluorescence signal is recorded. Video data are analyzed by software algorithms to allow quantification of the blood flow. Additionally, perfusion is measured intraoperatively by a reference system. Furthermore, comparing reference measurements using a flow phantom were performed to verify the quantitative blood flow results of the software and to validate the software algorithm. Analysis of intraoperative video data provides characteristic biological parameters. These parameters were implemented in the special flow phantom for experimental validation of the developed software algorithms. Furthermore, various factors that influence the determination of perfusion parameters were analyzed by means of mathematical simulation. Comparing patient measurement, phantom experiment, and computer simulation under certain conditions (variable frame rate, vessel diameter, etc.), the results of the software algorithms are within the range of parameter accuracy of the reference methods. Therefore, the software algorithm for calculating cortical perfusion parameters from video data presents a helpful intraoperative tool without complex additional measurement technology.

Characterization of light lesion paradigms and optical coherence tomography as tools to study adult retina regeneration in zebrafish.

Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.

Endoscopic optical coherence tomography device for forward imaging with broad field of view.

One current challenge of studying human tympanic membranes (TM) with optical coherence tomography (OCT) is the implementation of optics that avoid direct contact with the inflamed tissue. At the moment, no commercial device is available. We report an optics design for contactless forward imaging endoscopic optical coherence tomography (EOCT) with a large working distance (WD) and a broad field of view (FOV) by restricting the overall diameter of the probe to be small (3.5 mm), ensuring a sufficient numerical aperture. Our system uses a gradient-index (GRIN) relay lens and a GRIN objective lens, and executes a fan-shaped optical scanning pattern. The WD and FOV can be adjusted by manually changing the distance between the triplet and the GRIN relay lens. The measured lateral resolution is ∼28 µm at a WD of 10 mm with a FOV of 10 mm. Additionally, a camera and an illumination beam path were implemented within the probe for image guidance during investigations of the TM. We demonstrated the performance of the EOCT design by 3-D imaging of a human TM ex vivo and in vivo with a k-linear spectral domain OCT system.

Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue.

Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 µm line pair feature and has an approximately 11 µm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 µm (laterally), and 5.9 µm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

Shear flow-induced optical inhomogeneity of blood assessed in vivo and in vitro by spectral domain optical coherence tomography in the 1.3 µm wavelength range.

The optical inhomogeneity of flowing blood, which appears as a waisted double fan-shaped intensity pattern inside vessels in cross-sectional optical coherence tomography (OCT) images, was investigated for the first time. High resolution spectral domain OCT in the 1.3 µm wavelength region is used to assess this inhomogeneous intravascular backscattering of light in an in vivo mouse model and flow phantom measurements. Based on a predicted alignment of the red blood cells toward laminar shear flow, an angular modulation of the corresponding backscattering cross-section inside the vessels is assumed. In combination with the signal attenuation in depth by absorption and scattering, a simple model of the intravascular intensity modulation is derived. The suitability of the model is successfully demonstrated in the in vivo experiments and confirmed by the in vitro measurements. The observed effect appears in flowing blood only and shows a strong dependency on the shear rate. In conclusion, the shear-induced red blood cell alignment in conjunction with the vessel geometry is responsible for the observed intensity distribution. This inherent effect of blood imaging has to be considered in attenuation measurements performed with OCT. Furthermore, the analysis of the intravascular intensity pattern might be useful to evaluate flow characteristics.