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

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

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established