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A common severe neurotoxic side effect of breast cancer (BC) therapy is chemotherapy-induced peripheral neuropathy (CIPN) and intervention is highly needed for the detection, prevention, and treatment of CIPN at an early stage. As the eye is susceptible to neurotoxic stimuli, the present study aims to determine whether CIPN signs in paclitaxel-treated BC patients correlate with ocular changes by applying advanced non-invasive biophotonic in vivo imaging. Patients (n = 14, 10 controls) underwent monitoring sessions after diagnosis, during, and after therapy (T0-T3). Monitoring sessions included general anamnesis, assessment of their quality of life, neurological scores, ophthalmological status, macular optical coherence tomography (OCT), and imaging of their subbasal nerve plexus (SNP) by large-area confocal laser-scanning microscopy (CLSM). At T0, no significant differences were detected between patients and controls. During treatment, patients’ scores significantly changed while the greatest differences were found between T0 and T3. None of the patients developed severe CIPN but retinal thickenings could be detected. CLSM revealed large SNP mosaics with identical areas while corneal nerves remained stable. The study represents the first longitudinal study combining oncological examinations with advanced biophotonic imaging techniques, demonstrating a powerful tool for the objective assessment of the severity of neurotoxic events with ocular structures acting as potential biomarkers.

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.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.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 CXL, 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.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.Die konfokale In-vivo-Mikroskopie hat sich in der Augenheilkunde als Forschungsmethode etabliert. Wegen der Notwendigkeit einer Kontaktankopplung bei suboptimalen Justierungshilfen der Geräte wird die Methode bisher wenig im klinischen Routinebetrieb eingesetzt. Die konfokale Laserscanning-Mikroskopie (CLSM) des Auges konnte in den letzten Jahren durch die enge Zusammenarbeit von Physikern, Informatikern und Augenärzten weiterentwickelt werden und steht nun als ein am Patienten einsetzbarer Prototyp zur Verfügung. Im Rahmen dieser Studie wurden die Möglichkeiten der Methode im klinischen Einsatz evaluiert.Wesentliche Neuerungen der CLSM stellen (1) ein neugestaltetes Koppelelement mit einer der Hornhautkrümmung angepassten Oberfläche und (2) der Einsatz eines dualen computergestützen Piezoantriebs für eine schnelle präzise Fokusführung dar. Im Postprocessing und nach einer elastischen Bildregistrierung der oberflächenparallelen Einzelbilder gelingt es auch, sagittale, spaltlampenähnliche Schnitte mit einer Auflösung im Mikrometerbereich zu erzeugen. Die an enukleierten Schweinebulbi erprobte Konzeption wird bei Normalprobanden und ausgewählten Patienten mit Hornhauterkrankungen eingesetzt.Durch die simultane Darstellung von oberflächenparallelen und sagittalen Untersuchungsebenen können Zusatzinformationen zum Verständnis von Wundheilungsvorgängen aller Hornhautsubstrukturen und die Rolle von immunkompetenten Zellen zur Verfügung gestellt werden. Die klinischen Einsatzmöglichkeiten werden an einem Augengesunden und mehreren Beispielen (Keratokonus nach CXL, rezidivierende Keratitis, Zustand nach PRK) demonstriert. Diese zeigen, dass die morphologische Untersuchung der Hornhaut mit dem neuen Ansatz in jeder gewünschten und der Fragestellung angepassten Untersuchungsebene bei zellulärer Auflösung möglich ist.Es konnte gezeigt werden, dass die neue Konzeption der CLSM klinisch einsetzbar ist und sowohl den tierexperimentell arbeitenden Forschenden als auch dem an Hornhauterkrankungen interessierten Augenarzt bisher nicht verfügbare Informationen, wie z. B. die Langerhans-Zelldichte und die Epithelschichtung bei Oberflächenerkrankungen des Auges, zur Verfügung stellen kann.

The purpose of the present proof-of-concept study was to use large-areaThree healthy individuals were examined roughly weekly over a total period of six weeks by large-areaTotal investigation times of 10 minutes maximum per participant were used to generate mosaic images with an average size of 3.61 mmThe results of this proof-of-concept study have demonstrated the feasibility of using

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.

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.

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.

Printed electronics offer great potential for new applications such as Internet of Things devices and wearables. A methodology for integration of design and process assessment based on a stepwise transfer of the circuit to be printed from printed circuit board test vehicles to the final printed substrate is introduced. A multi-layer vector ink-jet printing process with three functional inks is presented using the example of the fabrication of a demonstrator circuit. This fabrication is realized using a newly set up printing system as enabling technology: Integration of a piezo print head into the path planning of the printing system and its control as virtual stepper axis enable highly precise vector printing yielding printed functional elements with low tolerances. Finally, process improvements for assembly of surface mounted devices on printed foil substrates are outlined.

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.

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'' 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'' 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.

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 multi-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 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.

Der subbasale Nervenplexus (SNP) der Kornea bietet die Moglichkeit, periphere Nervenstrukturen nichtinvasiv in vivo mithilfe der Konfokalmikroskopie (CCM) zu untersuchen. Morphologische Veranderungen des SNP konnen so unmittelbar detektiert und quantitativ charakterisiert werden. Aufgrund der inhomogenen Verteilung der Nervenfasern reicht eine einzelne CCM-Aufnahme fur eine valide Diagnose nicht aus. Gesucht werden daher Verfahren zur grosflachigen Erfassung des SNP. Dieser Beitrag gibt einen Uberblick uber publizierte Ansatze zur Losung dieses Problems. Aktuelle Entwicklungsarbeiten am Karlsruher Institut fur Technologie und der Universitatsaugenklinik in Rostock lassen zukunftig eine vereinfachte Handhabung der Technologie und eine weitere Verbesserung der Bildqualitat erwarten.

The use of deep neural networks ("deep learning") creates new possibilities in digital image processing. This approach has been widely applied and successfully used for the evaluation of image data in ophthalmology. In this article, the methodological approach of deep learning is examined and compared to the classical approach for digital image processing. The differences between the approaches are discussed and the increasingly important role of training data for model generation is explained. Furthermore, the approach of transfer learning for deep learning is presented with a representative data set from the field of corneal confocal microscopy. In this context, the advantages of the method and the specific problems when dealing with medical microscope data will be discussed.Der 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.

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 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.

Purpose/Aim of the study: A recently proposed technique enables the generation of continuously increasing mosaic images of the corneal sub-basal nerve plexus (SNP) using in vivo corneal confocal microscopy (CCM). The aim of the present study was to investigate the progression of the corneal nerve fiber length (CNFL) measured in the growing mosaic images with regard to their increasing area.Five large datasets from three healthy volunteers were examined using the proposed CCM technique. Intermediate mosaic images were created and assessed for CNFL.The measured CNFL progression shows both over- and underestimation of the CNFL for small observed areas. Increasing the mosaic image area stabilizes the CNFL values and reduces the moving variance in all five datasets. The relative deviation of means from values of first and second examination of two of the subjects shows high differences for an observed area of1.5 mmThe present examination provides two measures to quantify different area-dependent aspects of the CNFL measured in an expanding mosaic image. The moving variance measures how stable the CNFL can be considered at a certain mosaic size. The relative deviation of means from two repeated CCM examinations on the other hand gives some indication on the level of reliability that can be expected from the measured CNFL. The progression of CNFL in the examined datasets manifests a potentially very high variability for mosaic sizes of less than about 1.5 mm

Functional printing shows a huge potential for fabrication of flexible electronics on polymer films. However, not all electronic functionality can be realized by functional printing yet. The combination of functional printing and mounting of silicon electronic components is a pragmatic approach to realize new functionalities. This paper presents an integrated, automated workflow to derive fabrication data for a flexible hybrid printed circuit from a CAD-layout. These CAD data and prior knowledge from empirical process data are used to create motion programmes for subsequent fabrication and inspection steps. On the example of a demonstrator circuit, circuit layout and assignment of different functionalities to different CAD layers are illustrated. The exported CAD data are the input for a generator tool, which first verifies and then optimises the layout for the subsequent fabrication processes. Thereafter the tool automatically generates motion commands for a vector single nozzle ink-jet printing module, an optical inspection module and a SMT component placement module. The automated data and fabrication workflow is explained in detail, finally leading to the functional printed demonstrator circuit.

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 datasets.

Hintergrund: Die hohe Auflosung der konfokalen Laser-Scanning-Mikroskopie (CCM) ermoglicht die Abbildung des kornealen subbasalen Nervenplexus (SNP) in vivo. Aufgrund des kleinen Bildfelds (0,16 mm²) ist ein einzelnes CCM-Bild nicht ausreichend fur eine zuverlassige morphometrische SNP-Charakterisierung. Wir entwickeln deshalb derzeit ein automatisiertes Verfahren, das CCM-Bildsequenzen aufnimmt und daraus grosflachige SNP-Abbildungen erzeugt. Methoden: Zur Aufnahme einer Bildserie eines groseren SNP-Areals wird die Blickrichtung des Patienten durch eine computergesteuerte, bewegte Fixationsmarke auf einem Display vor dem nicht untersuchten Auge geeignet gefuhrt. Die CCM-Bildsequenz wird mit 30 Hz aufgezeichnet und bereits wahrend der Aufnahme wird ein Mosaikbild berechnet, an dem der Aufnahmeprozess beobachtet und die resultierende Bildgrose und -qualitat beurteilt werden kann. Verbleibende Bildfehler werden in einem anschliesenden Berechnungsschritt automatisch korrigiert. Ergebnisse: Bei geeignet gewahlter Fixationsmarkenbahn konnte mit einem ersten Prototypsystem ein mittlerer Bildgrosenzuwachs von 0,18 mm²/s erreicht werden. Schlussfolgerung: Mit dem vorgestellten Verfahren konnen grosflachige SNP-Abbildungen erzeugt werden. Das Verfahren ist gekennzeichnet durch einen hohen Automatisierungsgrad und durch kurze Untersuchungszeiten.

Summary Corneal confocal microscopy (CCM) has been established as a noninvasive, in vivo imaging technology that provides high-resolution images of the corneal tissue layers at a cellular level. In particular, several research groups focus on the nerve fiber bundles constituting the subbasal nerve plexus (SNP). The ability to visualize - and quantify - morphological alterations of the SNP provides a new and potentially sensitive diagnostic approach for peripheral neuropathies, e.g. associated with diabetes. However, because of the small field of view of current CCM systems (approx. 0.16 mm²) and the locally inhomogeneous distribution of the subbasal nerve structures, a single image does not reliably reflect the condition of the SNP. To address this issue, we present a highly automated and integrated system that facilitates the imaging of an extended contiguous SNP area in a short recording time by guiding the gaze direction of the patient with a computer-controlled moving fixation target. The system achieves growth rates of the scanned area of approximately 0.16 mm² per second. Following the image acquisition process, a high-quality mosaic image of the scanned area is computed using specialized image processing software.

The densely innervated human cornea is the only superficial tissue of the human body in which nerve fibres are accessible in vivo by corneal confocal microscopy (CCM). Morphological parameters of the corneal sub-basal nerve plexus (SNP) derived from CCM images can potentially serve as a sensitive biomarker for early diagnosis of various neurodegenerative diseases. The evaluation of a single image with a typical field of view of 0.16 mm2 is insufficient for robust morphometric assessment. Mosaicking approaches have therefore been proposed to examine the SNP on a larger scale. Here we present a highly automated technique that significantly facilitates the generation of mosaic images of the SNP and is suitable for clinical tests.

Grating-based x-ray differential phase-contrast imaging (DPCI) is capable of acquiring information based on phase-shift and dark-field signal, in addition to conventional x-ray absorption-contrast. Thus DPCI gives an advantage to investigate composite materials with component wise similar absorption properties like soft tissues. Due to technological challenges in fabricating high quality gratings over a large extent, the field of view (FoV) of the imaging systems is limited to a grating area of a couple of square centimeters. For many imaging applications (e.g. in medicine), however, a FoV that ranges over several ten centimeters is needed. In this manuscript we propose to create large area gratings of theoretically any extent by assembling a number of individual grating tiles. We discuss the precision needed for alignment of each microstructure tile in order to reduce image artifacts and to preserve minimum 90% of the sensitivity obtainable with a monolithic grating. To achieve a reliable high precision alignment a semiautomatic assembly system consisting of a laser autocollimator, a digital microscope and a force sensor together with positioning devices was built. The setup was used to tile a first four times four analyzer grating with a size of 200 mm × 200 mm together with a two times two phase grating. First imaging results prove the applicability and quality of the tiling concept.

PURPOSE A growing number of studies provide evidence that the morphology of the corneal subbasal nerve plexus (SNP), examined by corneal confocal microscopy (CCM), is a sensitive marker for diabetic peripheral neuropathy. However, it has been established that the field of view of a single CCM image (≈0.16 mm(2)) is insufficient for reliable assessment of corneal nerve fiber morphology. The present work proposes a highly automated technique for imaging an extended area of the SNP and creating large-scale montages. METHODS A moving fixation target is presented on a small display in front of the nonexamined eye. By guiding the viewing direction of the subject in an expanding spiral pattern, the scanned corneal area is continuously expanded. Specialized software algorithms subsequently assemble a mosaic image from the acquired CCM image data. The proposed technique was applied in 12 healthy subjects. RESULTS Montage images of the SNP were successfully created from all examinations performed. The mean imaged SNP area was 9.86 mm(2) (range, 1.62-18.31 mm(2)). The mean CCM duration was 65.33 seconds (range, 14.58-142.58 seconds). CONCLUSIONS The key advances embodied in the proposed technique are its high degree of integration and automation (both for image acquisition and image processing) and the resulting short duration of CCM. By providing an easy-to-use tool for obtaining large-scale mosaic images of the SNP, this technique has the potential to facilitate larger clinical trials where SNP morphology is used as a surrogate marker for peripheral neuropathy.

Confocal laser scanning microscopy (CLSM) provides a technique for in vivo imaging the sub-basal nerve plexus (SNP) of the cornea. For mapping an extended area of the SNP, currently the objective of the CLSM is manually moved laterally across the corneal surface during acquisition of overlapping images. This work presents a proof of concept for a new method that realizes the movements during the mapping process by guiding the eye movements with a computercontrolled fixation target on a display in front of the contralateral eye. The purpose is to generate a maximized and completely covered scan area with an overlapping area of adjacent images of more than 50%.