Ein neues Konzept zur automatischen Erstellung von Mosaikbildern der Kornea für die Diagnose neuropathischer Erkrankungen

Die hochauflösende Abbildung der Nervenstrukturen im sub-basalen Nervenplexus der Kornea mittels der in-vivo-Konfokalmikroskopie birgt ein immenses Potential für die Diagnostik bei unterschiedlichsten Erkrankungen mit Beteiligung des peripheren Nervensystems. Bislang fehlt es jedoch an Verfahren zur zuverlässigen automatischen Erfassung eines ausreichend großen Bereichs des Nervenplexus als Grundlage einer belastbaren Diagnose. In der vorliegenden Dissertation wird hierfür ein neues Konzept zur automatischen Erstellung einer großflächigen Abbildung des sub-basalen Nervenplexus in der zentralen Kornea entwickelt. Wesentliche Anforderungen sind dabei ein möglichst hoher Automatisierungsgrad, die Abbildung einer kompakten zusammenhängenden Fläche und die Minimierung der dazu erforderlichen Aufnahmedauer. Das erarbeitete Konzept wird durch einen zweigeteilten Ablauf realisiert, mit einem Prozess zur Bilddatengewinnung einerseits und einem Prozess zur Erstellung einer großflächigen Darstellung andererseits. Beide Prozesse stellen in wesentlichen Teilen Neuentwicklungen dar, die ausführlich hergeleitet und beschrieben werden. Die Kernidee bei der Aufnahme der Bilddaten ist die kontinuierliche Führung der Blickrichtung des Patienten durch eine bewegte Fixationsmarke vor dem nicht untersuchten Auge während des Aufnahmeprozesses. Da die Augenbewegungen synchron erfolgen, überträgt sich die Bewegung auf das untersuchte Auge. Durch eine angepasste Bahnführung der Fixationsmarke wird eine kontinuierliche Erweiterung des aufgenommenen Areals der Kornea erreicht. Damit die Führung der Blickrichtung hinsichtlich der oben genannten Randbedingungen zielgerichtet geschieht, beinhaltet das Konzept ein Verfahren zur Analyse der akquirierten Bilddaten und zur Rückkopplung auf die Bahn der Fixationsmarke. Die zur Verarbeitung der Bilddaten zu großflächigen Mosaikbildern aufgebaute Prozesskette wird detailliert hergeleitet. Die Aufgabe der Mosaikbilderzeugung aus den akquirierten Aufnahmesequenzen kann in die Registrierung der Bilddaten und die anschließende Bildfusion unterteilt werden. Eine zentrale Herausforderung bei der vorliegenden Aufgabenstellung besteht in der Korrektur der Bewegungsartefakte, die durch die Bewegung des Auges während der Bildgebung verursacht werden. Die Bewegungsartefakte manifestieren sich in den Aufnahmen als für die Aufnahmetechnik charakteristische horizontale und vertikale Verzerrungen, die im Rahmen der Registrierung bestimmt und korrigiert werden. Die Beschreibung eines Labormusters zur experimentellen Erprobung der entwickelten Prozesse komplettiert die Darstellung des neuen Gesamtkonzepts zur Erzeugung großflächigen konfokalmikroskopischen in-vivo-Bildgebung des sub-basalen Nervenplexus der Kornea

Reproducibility of two calibration procedures for phase-measuring deflectometry

Phase-measuring deflectometry is an optical inspection technique for reflective surfaces. It enables absolute, quantitative surface measurements, given a calibrated measurement setup. Two general calibration approaches can be found in literature: First, the stepwise approach uses a calibration pattern and determines internal camera parameters and external geometrical parameters in separate, consecutive steps. Second, the holistic approach optimizes all parameters collectively, based on deflectometric measurements of a calibration mirror. Whereas both approaches have been compared regarding the accuracy of subsequent surface measurements, the present contribution focuses on experimental examination of their reproducibility. In experiment E1, we assess the parameter variability by repeating both calibration procedures ten times. In an additional experiment E2, we repeat all calibration measurements related to a mirror/pattern position ten times in a row before rearranging the mirror/pattern, in order to examine the purely noise-related parameter variability. Finally, we calculate the coordinate variability of a set of world points projected onto the image planes of the calibrated cameras. The measured variability is consistently higher in E1 than in E2 (average ratio: 3.2). Unexpectedly, in both experiments, the external parameter variability also turns out to be higher for the holistic approach compared to stepwise calibration (average ratio: 2.3). This is of importance, since the holistic approach is known from literature to be more accurate than the stepwise approach, regarding their respective application to surface measurements. The image coordinate variability is comparable for both calibration approaches with an average of 0.84 and 0.21 camera pixels for E1 and E2, respectively

The effects of pattern screen surface deformation on deflectometric measurements - A simulation study

Phase-measuring deflectometry (PMD) is an optical inspection technique for full-field topography measurements of reflective sample surfaces. The measurement principle relies on the analysis of specific patterns, reflected at the sample surface. Evaluation algorithms often model the respective pattern screen as a planar light source. However, the 32\u27\u27 pattern screen in our inspection setup exhibits a central bulge of its surface of about 2–3 mm. This paper presents a simulation framework for PMD to evaluate the effects of a deformed screen surface. The idea is to simulate image data acquired with screen surface deformations and to examine the effects on the PMD evaluation results. The simulated setup consists of a 32\u27\u27 pattern screen with an adjustable central bulge height of 0–3 mm and two cameras with a field of view (FOV) of approximately 225 mm by 172 mm on the sample surface. A first experiment examines the reconstruction errors for a planar sample surface if the reconstruction algorithm uses perfect calibration data (i.e. the same parameters used for the simulated image acquisition). The reconstructed surfaces exhibit a tilt with a maximum height difference of 174 μm across the FOV. A second experiment repeats the reconstruction process of the same sample surface, using camera parameters determined in a simulated calibration process. The resulting surfaces possess irregular, wave-like errors with amplitudes of up to 9 μm in the FOV. The presented simulation results reveal the accuracy limits if a deformation model of the pattern screen is not explicitly included in the reconstruction process

Robust phase unwrapping based on non-coprime fringe pattern periods for deflectometry measurements

Phase-measuring deflectometry is a technique for non-contact inspection of reflective surfaces. A camera setup captures the reflection of a sine-modulated fringe pattern shifted across a screen; the location-dependent measured phase effectively encodes the screen coordinates. As the used fringe patterns are much narrower than the screen dimension, the resulting phase maps are wrapped. The number-theoretical solution uses the Chinese remainder theorem to calculate an unwrapped phase map from repeated measurements with coprime fringe widths. The technique is highly susceptible to phase noise, i.e. small deviations of the measured phase values generally lead to unwrapped phase values with large errors. We propose a modification and show how non-coprime period widths make phase unwrapping robust against phase noise. Measurements with two non-coprime fringe period widths introduce the opportunity to discriminate between “legal” measured phase value pairs, that potentially originate from noise-free measurements, and “illegal” phase value pairs, that necessarily result from noise-affected measurements. Arranged as a matrix, the legal measurements lie on distinct diagonals. This insight not only allows to determine the legality of a measurement, but also to provide a correction by looking for the closest legal matrix entry. We present an experimental comparison of the resulting phase maps with reference phase maps. The presented results include descriptive statistics on the average rate of illegal phase measurements as well as on the deviation from the reference. The measured mean absolute deviation decreases from 1.99 pixels before correction to 0.21 pixels after correction, with a remaining maximum absolute deviation of 0.91 pixels

Corneal Subbasal Nerve Plexus Changes in Severe Diabetic Charcot Foot Deformity: A Pilot Study in Search for a DNOAP Biomarker

Introduction. Diabetic neuroosteoarthropathy (DNOAP) early symptoms are unspecific, mimicking general infectious symptoms and rendering a diagnosis challenging. Consequently, unfavourable outcomes occur frequently, with recurrent foot ulceration, infectious complications, and eventually amputation. Corneal confocal microscopy (CCM) of the subbasal nerve plexus (SNP) is used to detect early peripheral neuropathy in diabetic patients without diabetic retinopathy. This pilot study was designed to determine if specific SNP changes manifest in severe DNOAP in comparison to a healthy control group. Methods. This pilot study utilized a matched-pair analysis to investigate SNP changes by in vivo CCM for 26 patients (mean patient age 63.7 years, range 27 to 78) with severe DNOAP defined by condition after the need for reconstructive foot surgery (n=13) and a healthy control group (n=13). Corneal nerve fibre length (CNFL), nerve fibre density (CNFD), nerve branch density (CNBD), average weighted corneal nerve fibre thickness (CNFTh), nerve connecting points (CNCP), and average weighted corneal nerve fibre tortuosity (CNFTo) were assessed as well as the general clinical status, diabetic status, and ophthalmologic basic criteria. Results. In vivo CCM revealed significantly reduced SNP parameters in the DNOAP group for CNFL (p=0.010), CNFD (p=0.037), CNBD (p=0.049), and CNCP (p=0.012) when compared to the healthy control group. Six patients (46%) of the DNOAP group suffered from diabetic retinopathy and none of the control group. Conclusions. This pilot study revealed a rarefication of SNP in all measured parameters in patients with severe DNOAP. We see a potential value of CCM providing a SNP-based biomarker for early stages of DNOAP prior to the development of any foot deformities that needs to be evaluated in further studies. This trial is registered with German Clinical Trials Register (DKRS) DRKS00007537

Morphological characterization of the human corneal epithelium by in vivo confocal laser scanning microscopy

Background: Regarding the growing interest and importance of understanding the cellular changes of the cornea in diseases, a quantitative cellular characterization of the epithelium is becoming increasingly important. Towards this, the latest research offers considerable improvements in imaging of the cornea by confocal laser scanning microscopy (CLSM). This study presents a pipeline to generate normative morphological data of epithelial cell layers of healthy human corneas. Methods: 3D in vivo CLSM was performed on the eyes of volunteers (n=25) with a Heidelberg Retina Tomograph II equipped with an in-house modified version of the Rostock Cornea Module implementing two dedicated piezo actuators and a concave contact cap. Image data were acquired with nearly isotropic voxel resolution. After image registration, stacks of en-face sections were used to generate full-thickness volume data sets of the epithelium. Beyond that, an image analysis algorithm quantified en-face sections of epithelial cells regarding the depth-dependent mean of cell density, area, diameter, aggregation (Clark and Evans index of aggregation), neighbor count and polygonality. Results: Imaging and cell segmentation were successfully performed in all subjects. Thereby intermediated cells were efficiently recognized by the segmentation algorithm while efficiency for superficial and basal cells was reduced. Morphological parameters showed an increased mean cell density, decreased mean cell area and mean diameter from anterior to posterior (5,197.02 to 8,190.39 cells/mm²; 160.51 to 90.29 µm²; 15.9 to 12.3 µm respectively). Aggregation gradually increased from anterior to posterior ranging from 1.45 to 1.53. Average neighbor count increased from 5.50 to a maximum of 5.66 followed by a gradual decrease to 5.45 within the normalized depth from anterior to posterior. Polygonality gradually decreased ranging from 4.93 to 4.64 sides of cells. The neighbor count and polygonality parameters exhibited profound depth-dependent changes. Conclusions: This in vivo study demonstrates the successful implementation of a CLSM-based imaging pipeline for cellular characterization of the human corneal epithelium. The dedicated hardware in combination with an adapted image registration method to correct the remaining motion-induced image distortions followed by a dedicated algorithm to calculate characteristic quantities of different epithelial cell layers enabled the generation of normative data. Further significant effort is necessary to improve the algorithm for superficial and basal cell segmentation

Burst of corneal dendritic cells during Trastuzumab and Paclitaxel treatment

During breast cancer therapy, paclitaxel and trastuzumab are both associated with adverse effects such as chemotherapy-induced peripheral neuropathy and other systemic side effects including ocular complications. Corneal nerves are considered part of the peripheral nervous system and can be imaged non-invasively by confocal laser scanning microscopy (CLSM) on the cellular level. Thus, in vivo CLSM imaging of structures of the corneal subbasal nerve plexus (SNP) such as sensory nerves or dendritic cells (DCs) can be a powerful tool for the assessment of corneal complications during cancer treatment. During the present study, the SNP of a breast cancer patient was analyzed over time by using large-scale in vivo CLSM in the course of paclitaxel and trastuzumab therapy. The same corneal regions could be re-identified over time. While the subbasal nerve morphology did not alter significantly, a change in dendritic cell density and an additional local burst within the first 11 weeks of therapy was detected, indicating treatment-mediated corneal inflammatory processes. Ocular structures such as nerves and dendritic cells could represent useful biomarkers for the assessment of ocular adverse effects during cancer therapy and their management, leading to a better visual prognosis

3D confocal laser-scanning microscopy for large-area imaging of the corneal subbasal nerve plexus

The capability of corneal confocal microscopy (CCM) to acquire high-resolution in vivo images of the densely innervated human cornea has gained considerable interest in using this non-invasive technique as an objective diagnostic tool for staging peripheral neuropathies. Morphological alterations of the corneal subbasal nerve plexus (SNP) assessed by CCM have been shown to correlate well with the progression of neuropathic diseases and even predict future-incident neuropathy. Since the field of view of single CCM images is insufficient for reliable characterisation of nerve morphology, several image mosaicking techniques have been developed to facilitate the assessment of the SNP in large-area visualisations. Due to the limited depth of field of confocal microscopy, these approaches are highly sensitive to small deviations of the focus plane from the SNP layer. Our contribution proposes a new automated solution, combining guided eye movements for rapid expansion of the acquired SNP area and axial focus plane oscillations to guarantee complete imaging of the SNP. We present results of a feasibility study using the proposed setup to evaluate different oscillation settings. By comparing different image selection approaches, we show that automatic tissue classification algorithms are essential to create high-quality mosaic images from the acquired 3D dataset