[Chromatic Swept-Source Laser Scanning - Concept for a Cell-Resolving Confocal Laser Slit Lamp?] / Chromatic Swept-Source Laser Scanning ­ Konzept für eine zellauflösende konfokale Laserspaltlampe?

BACKGROUND: The in vivo characterisation of corneal epithelial tissue morphology is of considerable importance for diagnosis, disease prognosis, and the development of a treatment strategy for ocular surface diseases. In contrast to many alternative methods, in vivo corneal confocal microscopy (CCM) not only provides a macroscopic description of the corneal tissue but also allows its visualisation with cellular resolution. However, the translation of CCM from research to clinical practice is significantly limited by the complex and still largely manual operation of available CCM systems. In addition, for cross-sectional images, and analogously to conventional slit lamp microscopy, volume data must be acquired in time-consuming depth scans due to the frontal orientation of the image field in CCM, from which depth slices can subsequently be calculated. The pure acquisition time is already in the range of seconds, and additionally, motion artefacts have to be corrected in a sophisticated way. MATERIALS AND METHODS: This paper presents the concept and optics simulation of a new imaging technique based on a swept-source laser in combination with special chromatic optics. Here, the laser periodically changes its wavelength and is focused at different depths due to the wavelength-dependent aberration of the chromatic optics. RESULTS: The optics simulation results promise good optical resolution at a total imaging depth of 145 µm. CONCLUSION: The long-term goal is cell-resolving in vivo corneal confocal microscopy in real time with differently oriented sectioning directions.

Taxane-Induced Neuropathy and Its Ocular Effects-A Longitudinal Follow-up Study in Breast Cancer Patients.

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.

The Rostock Method for Qualitative and Quantitative Evaluation of Intraocular Lenses. / Die Rostocker Methode zur qualitativen und quantitativen Bewertung von Intraokularlinsen.

BACKGROUND: For quantitative and qualitative evaluation of the imaging properties of IOLs, axial cross-sectional images can be obtained from the 3-dimensional light distribution by means of an optical bench, as is known from light sheet recordings in fluorescein baths. This paper presents a new image-processing algorithm to enhance the quality of generated axial cross-sectional images, and the two methods are then compared. MATERIAL AND METHODS: The 3-dimensional point spread function of a diffractive trifocal IOL (AT LISA tri 839MP, Carl Zeiss Meditec AG, Jena, Germany) was recorded on an optical bench developed in Rostock for different pupil diameters. A specially adapted image processing algorithm was then applied to the measurements, allowing through-focus curves to be generated. In addition, cross-sectional images of the IOLs studied were acquired using the light sheet method in a fluorescein bath. RESULTS: The study clearly shows the superiority of the newly developed method over the light sheet method in terms of image quality. In addition to the individual focal points, fine focal structures as well as halos can be made visible in the cross-sectional images obtained using the new method. In the generated through-focus curves, 3 intensity peaks can be identified, which represent the near, intermediate and far focus of the tested MIOL and cannot be represented by light sheet methods. CONCLUSION: The interaction of the optical bench with the developed image processing algorithm allows a more detailed understanding of the image formation and false light phenomena of IOLs, which was restricted by the technical limitations of the existing light sheet method. In addition, other quantities such as the through-focus curve can be derived quantitatively.

Assessment of dynamic corneal nerve changes using static landmarks by in vivo large-area confocal microscopy-a longitudinal proof-of-concept study.

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.

In Vivo Monitoring of Corneal Dendritic Cells in the Subbasal Nerve Plexus during Trastuzumab and Paclitaxel Breast Cancer Therapy-A One-Year Follow-Up.

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.

Method for the generation and visualization of cross-sectional images of three-dimensional point spread functions for rotationally symmetric intraocular lenses.

Cross-sectional images of three-dimensional point spread functions of intraocular lenses are used to study their image formation. To obtain those, light sheet-based methods are established. Due to the non-negligible thicknesses of the light sheets, the image quality of the cross-sectional images is constrained. To overcome this hurdle, we present a dedicated evaluation algorithm to increase image quality in the post-processing step. Additionally, we compare the developed- with the light sheet method based on our own investigations of a multifocal diffractive intraocular lens conducted in an in-house designed optical bench. The comparative study showed the clear superiority of the newly developed method in terms of image quality, fine structure visibility, and signal-to-noise ratio compared to the light sheet based method. However, since the algorithm assumes a rotationally symmetrical point spread function, it is only suitable for all rotationally symmetrical lenses.

Real-time large-area imaging of the corneal subbasal nerve plexus.

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.

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.

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.

In vivo Histology of the Cornea - from the "Rostock Cornea Module" to the "Rostock Electronic Slit Lamp" - a Clinical "Proof of Concept" Study. / In-vivo-Histologie der Hornhaut ­ vom "Rostocker Cornea Modul" zur "Rostocker Elektronischen Spaltlampe" ­ eine klinische "Proof-of-Concept"-Studie.

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 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. 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.

Multiwavelength confocal laser scanning microscopy of the cornea.

Confocal reflectance microscopy has demonstrated the ability to produce in vivo images of corneal tissue with sufficient cellular resolution to diagnose a broad range of corneal conditions. To investigate the spectral behavior of corneal reflectance imaging, a modified laser ophthalmoscope was used. Imaging was performed in vivo on a human cornea as well as ex vivo on porcine and lamb corneae. Various corneal layers were imaged at the wavelengths 488 nm, 518 nm, and 815 nm and compared regarding image quality and differences in the depicted structures. Besides the wavelength- and depth-dependent scattering background, which impairs the image quality, a varying spectral reflectance of certain structures could be observed. Based on the obtained results, this paper emphasizes the importance of choosing the appropriate light source for corneal imaging. For the examination of the epithelial layers and the endothelium, shorter wavelengths should be preferred. In the remaining layers, longer wavelength light has the advantage of less scattering loss and a potentially higher subject compliance.

[Digital Image Processing and Deep Neural Networks in Ophthalmology - Current Trends]. / Digitale Bildverarbeitung und Tiefe Neuronale Netze in der Augenheilkunde ­ aktuelle Trends.

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.

In vivo corneal confocal microscopy aided by optical coherence tomography.

In vivo corneal confocal microscopy and its operability in scientific as well as in clinical applications is often impaired by the lack of information on imaging plane position and orientation inside the cornea during patient's examination. To overcome this hurdle, we have developed a novel corneal imaging system based on a commercial scanning device and a modified Rostock Cornea Module. The presented preliminary system produces en face images by confocal laser scanning microscopy and sagittal cross-section images by optical coherence tomography simultaneously. This enables imaging guidance during examinations, improved features for diagnostics along with thickness measurements of the cornea as well as corneal substructures from oblique sections.

[Pre-clinical Evaluation of Intraocular Lenses through Simulated Implantation]. / Präklinische Bewertung von Intraokularlinsen durch simulierte Implantation.

The development of new optical designs for intraocular lenses (IOL) is a major challenge for lens manufacturers, as postoperative patient satisfaction is difficult to assess a priori with new concepts. Until now, invasive clinical investigations were carried out for this purpose. In contrast, the simulated implantation of IOLs allows a risk-free determination of the achievable visual performance and a subjective evaluation of the visual impression of new optical concepts. For intraocular lens manufacturers, this offers, on the one hand, the possibility to take the subjective perception of test persons into account during the development and optimization of novel lens designs and, on the other hand, to carry out comparative performance tests with known products before the first implantation. By means of simulated implantation, subjectively optimized IOLs may lead to a better postoperative visual quality for the patients and minimize the risk of a cost-intensive development of products with clinically unacceptable visual perception achievements due to optical side effects. The application of the simulated implantation during the preoperative patient consultation may enable the determination of the patient's subjective visual preferences and result in a targeted IOL selection recommendation. In addition to an improved selection from an existing IOL portfolio, IOL manufacturers could also offer individualized multifocal IOLs that match the patient's previous preference pattern. This article explains the technical background and application scenarios of the simulated implantation and introduces current procedures and devices.

Cellular in vivo 3D imaging of the cornea by confocal laser scanning microscopy.

We present an in vivo confocal laser scanning microscopy based method for large 3D reconstruction of the cornea on a cellular level with cropped volume sizes up to 266 x 286 x 396 µm3. The microscope objective used is equipped with a piezo actuator for automated, fast and precise closed-loop focal plane control. Furthermore, we present a novel concave surface contact cap, which significantly reduces eye movements by up to 87%, hence increasing the overlapping image area of the whole stack. This increases the cuboid volume of the generated 3D reconstruction significantly. The possibility to generate oblique sections using isotropic volume stacks opens the window to slit lamp microscopy on a cellular level.

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 datasets.

[Effect of the Age-Related Corneal Elasticity on Applanation Tonometry]. / Auswirkungen des altersbezogenen kornealen Elastizitätsmoduls auf die Applanationstonometrie.

Elevated intraocular pressure (IOP) is accepted to be one important criterion for glaucoma and is usually measured by applanation or rebound tonometry. The individual uncertainty due to central cornea thickness (CCT) is thereby corrected, while the error induced by age-related elastic modulus (EM) change of the cornea is ignored. To investigate its influence on IOP measurement, we derive a model including also the elastic modulus. Our approach is based on known equations from experimental physics and several assumptions being justified in this paper. Our correction values are in good agreement with the Dresden correction table for low CCT values up to 650 µm using a mean EM of 0.29 MPa. An EM variation from 0.2 to 0.5 MPa, which relates to ages from infancy to 90 years, results in an IOP error of up to 10 mmHg. A variation of the cornea curvature from 7.4 mm to 8.0 mm results in a total IOP change of about 3 mmHg, which is usually neglected. The derived model shows that established correction formulas can be insufficient for a reliable IOP determination. In many cases, the conventionally measured IOP may be precise enough, but the uncertainty in IOP determination due to CCT and EM influence are almost in the same range. Measuring the IOP using applanation methods with established correction formulas should not be overestimated without to respect the EM of the cornea.