Hinnerk Schulz-Hildebrandt

Label-Free In Vivo Imaging of Corneal Lymphatic Vessels Using Microscopic Optical Coherence Tomography

Purpose Corneal neovascularization, in particular lymphangiogenesis, is a limiting factor in corneal transplant survival. Novel treatment approaches focus on (selective) inhibition and regression of lymphatic vessels. Imaging clinically invisible corneal lymphatic vessels is a prerequisite for these strategies. Using a murine model, this study investigates whether corneal lymphatic vessels can be imaged using microscopic optical coherence tomography (mOCT). Methods Corneal neovascularization was induced by intrastromal placement of 11.0 nylon sutures in one eye of BALB/c mice. After 2 weeks, cross-sectional images and volumes of the corneas with a 0.5 mm lateral and axial field of view were acquired using a custom-built mOCT system enabling a resolution of 1 μm at a B-scan rate of 165/s. Three of the six animals received an additional intrastromal injection of India ink 24 hours before the measurement to stain the corneal lymphatic system in vivo. Immunohistochemistry using CD31 and LYVE-1 was used to validate the mOCT findings. Results Using mOCT, lymphatic vessels were visible as dark vessel-like structures with the lumen lacking a hyperreflective wall and mostly lacking cells. However, individual, slowly moving particles, which most likely are immune cells, occasionally could be observed inside the lumen. In lymphatic vessels of ink-stained corneas, hyperreflection and shadowing underneath was observed. Ink-filled lymphatic vessels were colocalized in consecutive corneal flat mounts of the same specimen. Conclusions Corneal lymphatic vessels can be imaged using mOCT. This novel approach opens new options for noninvasive clinical imaging of corneal lymphatic vessels for diagnostic and therapeutic indications.

OCT verstehen – Teil 2: Praktische Aspekte und Anwendung

OCT ver be rr ec ht lic h ge sc hü tz t. Den Schwerpunkt dieses Artikels bildet die Vermittlung praktischen Wissens rund um die optische Kohärenztomografie (OCT). Während in Teil 1 [1] die physikalischen Grundlagen vorgestellt wurden, werden hier praktische Aspekte für den Anwender erläutert, unabhängig vom verwendeten Gerätetyp oder Hersteller. Grundsätzliche Parameter und Einstellmöglichkeiten werden vorgestellt. Es wird hinterfragt, was ein OCT‐Bild eigentlich zeigt und wann bei der Interpretation und Auswertung Vorsicht geboten ist. Typische Artefakte sowie deren Ursachen und Möglichkeiten der Vermeidung werden diskutiert. Zuletzt wird eine Übersicht über aktuelle Anwendungsfelder der OCT in der Augenheilkunde gegeben.

Optimized segmentation and characterization of capillary networks using OCT (Conference Presentation)

The ability to image the physiology of microvasculature with high spatial resolution in three dimensions while investigating structural changes of skin, is essential for understanding the complex processes of skin aging, wound healing and disease development. Further, the quantitative, automatic assessment of these changes enables to analyze large amounts of image data in an abstract but comprehensive manner. However, previous work using OCT with methods of angiography was imaging less scattering, hence more challenging tissue than skin, such as brain and retina tissue. The published methods for capillary segmentation were mostly non-automatic, poorly benchmarked against state-of-the-art methods of computer vision and not applied to investigate medical processes and studies in a comprehensive fashion. Here, segmentation of capillaries in skin is reported and its efficacy is demonstrated in both, a longitudinal mouse study and a preliminary study in humans. By combining state-of-the-art image processing methods in an optimized way, we were able to improve the segmentation results and analyze the impact of each post-processing step. Furthermore, this automatic segmentation enabled us to analyze big amounts of datasets automatically and derive meaningful conclusions for the planning of clinical studies. With this work, optical coherence tomography is combined with methods of computer vision to a diagnostic tool with unique capabilities to characterize vascular diversity and provide extraordinary opportunities for dermatological investigation in both, clinics and research.

Novel endoscope with increased depth of field for imaging human nasal tissue by microscopic optical coherence tomography

Intravital microscopy (IVM) offers the opportunity to visualize static and dynamic changes of tissue on a cellular level. It is a valuable tool in research and may considerably improve clinical diagnosis. In contrast to confocal and non-linear microscopy, optical coherence tomography (OCT) with microscopic resolution (mOCT) provides intrinsically cross-sectional imaging. Changing focus position is not needed, which simplifies especially endoscopic imaging. For in-vivo imaging, here we are presenting endo-microscopic OCT (emOCT). A graded-index-lens (GRIN) based 2.75 mm outer diameter rigid endoscope is providing 1.5 - 2 µm nearly isotropic resolution over an extended field of depth. Spherical and chromatic aberrations are used to elongate the focus length. Simulation of the OCT image formation, suggests a better overall image quality in this range compared to a focused Gaussian beam. Total imaging depth at a reduced sensitivity and lateral resolution is more than 200 µm. Using a frame rate of 80 Hz cross-sectional images of concha nasalis were demonstrated in humans, which could resolve cilial motion, cellular structures of the epithelium, vessels and blood cells. Mucus transport velocity was successfully determined. The endoscope may be used for diagnosis and treatment control of different lung diseases like cystic fibrosis or primary ciliary dyskinesia, which manifest already at the nasal mucosa.

An Approach for Needle Based Optical Coherence Elastography Measurements

While navigation and interventional guidance are typically based on image data, the images do not necessarily reflect mechanical tissue properties. Optical coherence elastography (OCE) presents a modality with high sensitivity and very high spatial and temporal resolution. However, OCE has a limited field of view of only 2–5 mm depth. We present a side-facing needle probe to image externally induced shear waves from within soft tissue. A first method of quantitative needle-based OCE is provided. Using a time of flight setup, we establish the shear wave velocity and estimate the tissue elasticity. For comparison, an external scan head is used for imaging. Results for four different phantoms indicate a good agreement between the shear wave velocities estimated from the needle probe at different depths and the scan head. The velocities ranging from 0.9–3.4 m/s agree with the expected values, illustrating that tissue elasticity estimates from within needle probes are feasible.

Imaging cold-induced vasodynamic behaviour in skin using OCT for microangiography (Conference Presentation)

In dermatology the reflexes of vasoconstriction and vasodilation are known as important mechanisms of thermoregulation of the inner body. Imaging the physiology of microvasculature of the skin with high spatial resolution in three dimensions while reacting to changes in temperature is crucial for understanding the complex processes of vasodynamics, which result in constriction and dilation of vessels. However, previous studies using Laser-Doppler flowmetry and -imaging could not provide reliable angiographic images which allow to quantify changes in blood vessel diameter. Here, we report a different approach for angiographic imaging of microvasculature of a anaesthetized rodent model using speckle variance optical coherence tomography (svOCT) during and after localized cooling. Therefore a commercial OCT with a center wavelength of 1.3 μm and a spatial resolution of 13µm was used in combination with a custom built cooling device to image such reflexes at the mouse ear pinna and dorsal skinfold. Cooling was applied in steps of 2−5◦ C starting at the baseline temperature of 27◦ C down to −10◦ C. To our surprise and in contrast to the general opinion in literature, we were able to observe that the majority of vessels with a diameter larger than 20 μm maintain perfused with a constant diameter when the tissue is cooled from baseline to subzero temperatures. However, vasoconstriction was observed very rarely and only in veins, which led to their occlusion. The results of this experiment lead us to reconsider essential aspects of previous understanding of temperature-induced vasodynamics in cutaneous microvasculature.

Improving imaging of the air-liquid interface in living mice by aberration-corrected optical coherence tomography (mOCT) (Conference Presentation)

Failure in mucociliary clearance is responsible for severe diseases like cystic fibroses, primary ciliary dyskinesia or asthma. Visualizing the mucous transport in-vivo will help to understanding transport mechanisms as well as developing and validating new therapeutic intervention. However, in-vivo imaging is complicated by the need of high spatial and temporal resolution. Recently, we developed microscopy optical coherence tomography (mOCT) for non-invasive imaging of the liquid-air interface in intact murine trachea from its outside. Whereas axial resolution of 1.5 µm is achieved by the spectral width of supercontinuum light source, lateral resolution is limited by aberrations caused by the cylindric shape of the trachea and optical inhomogenities of the tissue. Therefore, we extended our mOCT by a deformable mirror for compensation of the probe induced aberrations. Instead of using a wavefront sensor for measuring aberrations, we harnessed optimization of the image quality to determine the correction parameter. With the aberration corrected mOCT ciliary function and mucus transport was measured in wild type and βENaC overexpressing mice, which served as a model for cystic fibrosis.

Imaging of mucus clearance in the airways of living spontaneously breathing mice by optical coherence microscopy (Conference Presentation)

Mucus transport is essential to remove inhaled particles and pathogens from the lung. Impaired removal of mucus often results in worsening of lung diseases. To understand the mechanisms of mucus transport and to monitor the impact of therapeutic strategies, it is essential to visualize airways and mucus in living animals without disturbing transport processes by intubation or surgically opening the airways. We developed a custom-built optical coherence microscope (OCM) providing a lateral and axial resolution of approximately 1.5 µm with a field of view of 2 mm at up to 150 images/s. Images of the intact trachea and its mucus transport were recorded in anesthetized spontaneously breathing mice. NaCl solution (0.9% and 7%) or Lipopolysaccharide were applied intranasally. OCM resolved detailed structure of the trachea and enabled measuring the airway surface liquid (ASL) thickness through the tracheal wall. Without stimulation, the amount of ASL was only a few µm above the epithelium and remained constant. After intranasal application of 30 µl saline at different concentrations, an early fast cough-like fluid removal with velocities higher than 1 mm/s was observed that removed a high amount of liquid. The ASL thickness increased transiently and quickly returned to levels before stimulation. In contrast to saline, application of Lipopolysaccharide induced substantial mucus release and an additional slow mucus transport by ciliary beating (around 100 µm/s) towards the larynx was observed. In conclusion, OCM is appropriate unique tool to study mechanisms of mucus transport in the airways and effects of therapeutic interventions in living animals.

Imaging the upper airways in humans with endoscopic optical coherence microscopy at subcellular resolution

Having a noninvasive technique to assess the human airway wall with subcellular resolution directly in patients would be of great benefit for the assessment of airway diseases. We therefore developed an endoscopic optical coherence microscope (eOCM) and tested its performance in human subjects. Images were taken with a custom-built eOCM device consisting of a broadband supercontinuum light source, a customized 400 nm spectrometer and a rigid grin lens based objective (0.5 NA) with a length of 45 mm and a diameter of 3 mm mounted on a handheld probe that could take images at a rate of 120 frames/s. With eOCM we could image the respiratory epithelium with underlying structures in the nose of human subjects. The device allowed to examine the airway wall including overlying mucus, to discriminate different types of epithelium (squamous epithelium, transition epithelium, respiratory epithelium), to visualize subepithelial structures such as the underlying fiber network, subepithelilal glands and blood vessels and to visualize blood flow. The resolution and frame rate allowed to detect ciliary beating and to analyze ciliary beat frequency. Our results show that eOCM is a useful tool for examining human airways. This technique has a great potential for functional imaging of the intrapulmonary airways of patients if integrated in a flexible bronchoscope.