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.

Background: The purpose of the present proof-of-concept study was to use large-area in vivo confocal laser scanning microscopy (CLSM) mosaics to determine the migration rates of nerve branching points in the human corneal subbasal nerve plexus (SNP). Methods: Three healthy individuals were examined roughly weekly over a total period of six weeks by large-area in vivo confocal microscopy of the central cornea. An in-house developed prototype system for guided eye movement with an acquisition time of 40 s was used to image and generate large-area mosaics of the SNP. Kobayashi-structures and nerve entry points (EPs) were used as fixed structures to enable precise mosaic registration over time. The migration rate of 10 prominent nerve fiber branching points per participant was tracked and quantified over the longitudinal period. Results: Total investigation times of 10 minutes maximum per participant were used to generate mosaic images with an average size of 3.61 mm2 (range: 3.18–4.42 mm2). Overall mean branching point migration rates of (46.4±14.3), (48.8±15.5), and (50.9±13.9) µm/week were found for the three participants with no statistically significant difference. Longitudinal analyses of nerve branching point migration over time revealed significant time-dependent changes in migration rate only in participant 3 between the last two measurements [(63.7±12.3) and (43.0±12.5) µm/week, P<0.01]. Considering individual branching point dynamics, significant differences in nerve migration rate from the mean were only found in a few exceptions. Conclusions: The results of this proof-of-concept study have demonstrated the feasibility of using in vivo confocal microscopy to study the migration rates of corneal subbasal nerves within large areas of the central human cornea (>1 mm2). The ability to monitor dynamic changes in the SNP opens a window to future studies of corneal nerve health and regenerative capacity in a number of systemic and ocular diseases. Since corneal nerves are considered part of the peripheral nervous system, this technique could also offer an objective diagnostic tool and biomarker for disease- or treatment-induced neuropathic changes.

Paclitaxel and trastuzumab have been associated with adverse effects including chemotherapy-induced peripheral neuropathy (CIPN) or ocular complications. In vivo confocal laser scanning microscopy (CLSM) of the cornea could be suitable for assessing side effects since the cornea is susceptible to, i.e., neurotoxic stimuli. The study represents a one-year follow-up of a breast cancer patient including large-area in vivo CLSM of the subbasal nerve plexus (SNP), nerve function testing, and questionnaires during paclitaxel and trastuzumab therapy. Six monitoring sessions (one baseline, four during, and one after therapy) over 58 weeks were carried out. Large-area mosaics of the SNP were generated, and identical regions within all sessions were assigned. While corneal nerve morphology did not cause alterations, the number of dendritic cells (DCs) showed dynamic changes with a local burst at 11 weeks after baseline. Simultaneously, paclitaxel treatment was terminated due to side effects, which, together with DCs, returned to normal levels as the therapy progressed. Longitudinal in vivo CLSM of the SNP could complement routine examinations and be helpful to generate a comprehensive clinical picture. The applied techniques, with corneal structures acting as biomarkers could represent a diagnostic tool for the objective assessment of the severity of adverse events and the outcome.

The morphometric assessment of the corneal subbasal nerve plexus (SNP) by confocal microscopy holds great potential as a sensitive biomarker for various ocular and systemic conditions and diseases. Automated wide-field montages (or large-area mosaic images) of the SNP provide an opportunity to overcome the limited field of view of the available imaging systems without the need for manual, subjective image selection for morphometric characterization. However, current wide-field montaging solutions usually calculate the mosaic image after the examination session, without a reliable means for the clinician to predict or estimate the resulting mosaic image quality during the examination. This contribution describes a novel approach for a real-time creation and visualization of a mosaic image of the SNP that facilitates an informed evaluation of the quality of the acquired image data immediately at the time of recording. In cases of insufficient data quality, the examination can be aborted and repeated immediately, while the patient is still at the microscope. Online mosaicking also offers the chance to identify an overlap of the imaged tissue region with previous SNP mosaic images, which can be particularly advantageous for follow-up examinations.

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.

BackgroundRegarding 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.Methods3D 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.ResultsImaging 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.ConclusionsThis 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.

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 3200 pattern screen in our inspection setup exhibits a central bulge of its surface of about 2–3mm. 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 3200 pattern screen with an adjustable central bulge height of 0–3mm and two cameras with a field of view (FOV) of approximately 225mm by 172mm 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.

Small fiber neuropathy (SFN) has been suggested as a trigger of restless legs syndrome (RLS). An increased prevalence of peripheral neuropathy has been demonstrated in Parkinson’s disease (PD). We aimed to investigate, in a cross-sectional manner, whether SFN is overrepresented in PD patients with concurrent RLS relative to PD patients without RLS, using in vivo corneal confocal microscopy (IVCCM) and quantitative sensory testing (QST) as part of small fiber assessment. Study participants comprised of age- and sex-matched PD patients with (n = 21) and without RLS (n = 21), and controls (n = 13). Diagnosis of RLS was consolidated with the sensory suggested immobilization test. Assessments included nerve conduction studies (NCS), Utah Early Neuropathy Scale (UENS), QST, and IVCCM, with automated determination of corneal nerve fiber length (CNFL) and branch density (CNBD) from wide-area mosaics of the subbasal nerve plexus. Plasma neurofilament light (p-NfL) was determined as a measure of axonal degeneration. No significant differences were found between groups when comparing CNFL (p = 0.81), CNBD (p = 0.92), NCS (p = 0.82), and QST (minimum p = 0.54). UENS scores, however, differed significantly (p = 0.001), with post-hoc pairwise testing revealing higher scores in both PD groups relative to controls (p = 0.018 and p = 0.001). Analysis of all PD patients (n = 42) revealed a correlation between the duration of l-dopa therapy and CNBD (ρ = −0.36, p = 0.022), and p-NfL correlated with UENS (ρ = 0.35, p = 0.026) and NCS (ρ = −0.51, p = 0.001). Small and large fiber neuropathy do not appear to be associated with RLS in PD. Whether peripheral small and/or large fiber pathology associates with central neurodegeneration in PD merits further longitudinal studies.

In vivo confocal microscopy (IVCM) is a non-invasive imaging technique facilitating real-time acquisition of images from the live cornea and its layers with high resolution (1–2 µm) and high magnification (600 to 800-fold). IVCM is extensively used to examine the cornea at a cellular level, including the subbasal nerve plexus (SBNP). IVCM of the cornea has thus gained intense interest for probing ophthalmic and systemic diseases affecting peripheral nerves. One of the main drawbacks, however, is the small field of view of IVCM, preventing an overview of SBNP architecture and necessitating subjective image sampling of small areas of the SBNP for analysis. Here, we provide a high-quality dataset of the corneal SBNP reconstructed by automated mosaicking, with an average mosaic image size corresponding to 48 individual IVCM fields of view. The mosaic dataset represents a group of 42 individuals with Parkinson’s disease (PD) with and without concurrent restless leg syndrome. Additionally, mosaics from a control group (n = 13) without PD are also provided, along with clinical data for all included participants.

Introduction Confocal in vivo microscopy is an established method in ophthalmology research. As it requires contact coupling and calibration of the instruments is suboptimal, this method has been only rarely used in clinical routine work. As a result of close collaboration between physicists, information scientists and ophthalmologists, confocal laser scanning microscopy (CLSM) of the eye has been developed in recent years and a prototype can now be used in patients. The present study evaluates possible clinical uses of this method. Material and Methods The essential innovations in CLSM are (1) a newly designed coupling element with superficial adaptation to corneal curvature and (2) the use of a dual computerised piezo drive for rapid and precise focusing. In post-processing and after elastic imaging registration of the individual images parallel to the surface, it is also possible to produce sagittal sections resembling a split lamp and with resolution in the micrometer range. The concept was tested on enucleated pig bulbi and tested on normal volunteers and selected patients with diseases of the cornea. Results Simultaneous imaging in planes parallel to the surface and in sagittal planes provided additional information that can help us to understand the processes of wound healing in all substructures of the cornea and the role of immune competent cells. Possible clinical uses were demonstrated in a volunteer with healthy eyes and several groups of patients (keratoconus after al, recurrent keratitis, status after PRK). These show that this new approach can be used in morphological studies at cellular level in any desired and appropriate test plane. Conclusions It could be shown that this new concept of CLSM can be used clinically. It can provide valuable and novel information to both preclinical researchers and to ophthalmologists interested in corneal disease, e.g. density of Langerhans cells and epithelial stratification in ocular surface diseases.

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.

Printed electronics offer great potential for new applications such as Internet of Things devices and wearables. However, to date, only a limited number of electronic functions and integration densities can be realised by printing processes. Hence, hybrid printed electronic circuits are actually created by mounting silicon electronic components. Since both printed materials and processes are continuously evolving, an accompanying structured development methodology is required. This paper highlights a digital workflow from design to automated fabrication using the example of a demonstrator circuit. A mull-layer vector ink-jet printing process to print electronic devices onto foil substrates with three functional inks is presented. This printing process is improved using a newly set-up printing system: Integrating a piezo print head into the path planning of the printing system and its control as a virtual stepper axis enable highly precise vector printing. This leads to printed resistors with low tolerances. Adaptations of surface mount technology for assembling silicon electronic components onto printed foil substrates are discussed. Finally, image processing methods to cope with deformations of the flexible foil substrates in the fabrication process are introduced.

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.

ZusammenfassungDer Einsatz von Tiefen Neuronalen Netzen (Deep Learning) eröffnet neue Möglichkeiten in der digitalen Bildverarbeitung. Auch für die Auswertung von Bilddaten in der Ophthalmologie wird diese Methode erfolgreich eingesetzt und findet weite Verbreitung. In diesem Artikel wird die methodische Vorgehensweise beim Deep Learning betrachtet und der klassischen Vorgehensweise für die Entwicklung von Methoden für die digitale Bildverarbeitung gegenübergestellt. Dabei wird auf Unterschiede eingegangen und die wichtiger werdende Rolle von Trainingsdaten für die Modellbildung erklärt. Weiterhin wird die Vorgehensweise des Transfer-Lernens (Transfer Learning) für Deep Learning am Beispiel eines Datensatzes aus der kornealen Konfokalmikroskopie vorgestellt. Dabei wird auf die Vorteile der Methode und auf Besonderheiten beim Umgang mit medizinischen Mikroskopdaten eingegangen.

Corneal confocal laser scanning microscopy is a promising method for in vivo investigation of cellular structures, e.g., of nerve fibers in the sub-basal nerve plexus. During recording, even slight displacements of the focal plane lead to images of adjacent tissue layers. In this work, we propose a closed-loop control of the focal plane. To detect and evaluate the visible tissues, we utilize the Bag of Visual Words approach to implement a customizable image processing pipeline for real-time applications. Furthermore, we show that the proposed model can be trained with small classification datasets and can be applied as a segmentation method. The proposed control loop, including tissue detection, is implemented in a proof-of-concept setup and shows promising results in a first evaluation with a human subject.

For corneal confocal microscopy (CCM) we use the Heidelberg Retina Tomograph (HRT) and the Rostock Cornea Module (RCM; both Heidelberg Engineering GmbH, Heidelberg, Germany). A modification of the HRT software enables the recording of focal image stacks by continually oscillating the focus depth within up to 80 μm. To align the CCM images for volume reconstruction we developed special purpose image registration algorithms, which also correct the motion induced distortions in the CCM images.