Summary
Linear microscopy is an established imaging technology for ocular surface imaging. Nonlinear microscopy is a new non‐invasive imaging technique, which allows a visualization of biological ...tissue with high signal contrast due to spectral separation combined with high resolution. This talk presents the physical principles of different contrast mechanisms. Exemplary experimental results based on various linear and nonlinear signals are shown, opportunities of this technology are discussed and the prospect of translating this imaging technique into a clinical application is addressed.
Summary
There are many modalities that acquire data about the cornea: shape, power, morphology, biomechanics, and so forth. This talk will review the technologies on corneal assessment techniques and ...devices available in experimental and clinical practice. Specifically, it will be discussed slit lamp biomicroscopy, ultrasound, magnet resonance tomography, confocal microscopy, ultrasound biomicroscopy, optical coherence tomography, keratometry, Scheimpflug imaging, and dynamic applanation procedures. In addition, I discuss the necessity of developing new technologies for assessing both the morphology and the physiology of the cornea.
To evaluate in vivo three-dimensional (3-D) confocal laser scanning microscopy (CLSM) as a technique for visualising the corneal surface and epithelium.
Ten human corneas (three from healthy ...volunteers, three with bullous keratopathy, three from patients following penetrating keratoplasty, and one with corneal erosion) were examined by 3-D CLSM. A novel polymethyl methacrylate (PMMA) contact cap was designed to minimise artefacts due to applanation pressure.
3-D reconstruction and different visualisation techniques (volume rendering, cross-section, en face view, oblique section and surface reconstruction) were performed to demonstrate alterations to corneal surface and epithelium. Image quality (cell identification, motion blur, absence of compression artefacts, imaging of superficial structures and of subepithelial nerve plexus) was considerably superior to that obtained using a conventional contact cap with a planar surface.
3-D CLSM permits in vivo visualisation and analysis of the corneal surface and of spatial arrangement at the cellular level in epithelium in normal and pathological corneas. The novel design of the contact cap minimises artefacts due to applanation pressure and improves the image quality of epithelial structures. The method provides a basis for further in vivo studies of alterations to corneal surface structure and its cellular arrangement.
Mosaicking the subbasal nerve plexus Allgeier, S.; Reichert, K.M.; Stachs, O. ...
Acta ophthalmologica (Oxford, England),
September 2017, 2017-09-00, 20170901, Letnik:
95, Številka:
S259
Journal Article
Recenzirano
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.
Purpose
In vivo corneal confocal laser scanning microscopy became a valuable tool for studying corneal morphology in health and disease. Enabling the optical dissection of the corneal architecture, ...this technique offers non‐invasive in vivo imaging at a cellular level being important for current research. Presently there is only one device available without undergoing technological changes in the last years. We are presenting new Rostock Cornea Module developments for anterior segment imaging using up to date confocal scanning laser technology.
Methods
We redesigned the RCM in a modular way. It is possible to use different microscope objectives for user defined requirements as well as to choose between contact and non‐contact application. Since the focal adjustment inside the cornea is often fiddly, we incorporated a fast piezo stage for closed‐loop focus control and adjustment. Further topics addressed: modular setup, extended field of view, reduction of eye movement and compression artifacts as well as software based noise reduction technologies.
Results
We tested the new RCM in combination with a Spectralis platform, offering a resolution of 1536 by 1536 pixel. Performing contact measurements, we get similar results compared to the well‐known RCM‐HRT combination, but with a higher resolution and wider field of view. In non‐contact measurements, the reflection on the cornea surface is very prominent and limits imaging of epithelial structures. We could achieve high resolution stromal imaging of e.g. keratocytes nuclei and stromal nerves.
Conclusions
The RCM 2.0 concept is a versatile extension for visualization corneal structures at a cellular level enabling instant focal plane shifts. Further improvements, wavelength adaption and dedicated customizations are subject of current research.
PurposeThis study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and ...immunohistochemical staining of ex vivo donor corneas.MethodsSeven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by β-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared.ResultsOur experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm
), CNFD (1358.8 vs 277.3 nerve fibers/mm
), CNBD (847.6 vs 163.5 branches/mm
), CNFTo (0.095 vs 0.081 μm
), and CNCP (49.4 vs 21.6 connections/mm
) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 μm). No significant difference was observed in CNFTh (2.22 vs 2.20 μm) between the two groups.ConclusionsThe results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.
Accommodation research increasingly uses engineering methods. This article presents the use of the finite element method in accommodation research.
Geometry, material data and boundary conditions are ...prerequisites for the application of the finite element method. Published data on geometry and materials are reviewed. It is shown how boundary conditions are important and how they influence the results.
Two dimensional and three dimensional models of the anterior chamber of the eye are presented. With simple two dimensional models, it is shown that realistic results for the accommodation amplitude can always be achieved. More complex three dimensional models of the accommodation mechanism - including the ciliary muscle - require further investigations of the material data and of the morphology of the ciliary muscle, if they are to achieve realistic results for accommodation.
The efficiency and the limitations of the finite element method are especially clear for accommodation. Application of the method requires extensive preparation, including acquisition of geometric and material data and experimental validation. However, a validated model can be used as a basis for parametric studies, by systematically varying material data and geometric dimensions. This allows systematic investigation of how essential input parameters influence the results.
High-resolution biometry of the anterior ocular segment is now becoming more and more important against a background of refractive surgery and the evaluation of potentially accommodative lens ...replacement materials. The aim of this study was a systematic investigation of the currently available non-contact methods for measuring the anterior chamber depth (ACD).
The ACDs of 50 phakic eyes of 27 patients aged between 19 and 59 years were measured with the IOL-Master (Zeiss), the AC-Master (Zeiss), the Pentacam (Oculus) and slit-lamp pachymetry by Jaeger (Haag-Streit).
The median anterior chamber depth in the investigated eyes was 3.63 mm for the IOL-Master (minimum 2.88 mm, maximum 4.22 mm), 3.802 mm for the AC-Master (2.816 mm-4.373 mm), 3.915 mm for the Pentacam (minimum 2.994 mm, maximum 4.614 mm) and 3.75 mm for Jaeger (2.887 mm-4.29 mm). With a probability of error of alpha=0.05 there were no significant differences concerning the ACD between the methods of Jaeger and AC-Master, Jaeger and IOL-Master, or Pentacam and AC-Master (Wilcoxon and Wilcox). The intra-individual variability was +/-5.4 microm for AC-Master, +/-12.7 microm for Pentacam, +/-24.5 microm for IOL-Master and +/-41.2 microm for Jaeger. The maximum method-dependent difference in ACD determination was 285 microm.
All the methods allow non-contact biometry, but the results might differ due to measuring principles inherent to the system, experience of the examiner and compliance of the patient. Partial coherence interferometry with the AC-Master offers the advantage of measurement exactly along the optical axis with the highest reproducibility and patient compliance.
Optical coherence tomography (OCT) and confocal laser scanning microscopy (CLSM) are light-based imaging techniques that allow for a visualization of microscopic tissue properties in vivo. Our study ...was to examine whether they allow for differentiation of inverted papilloma (IP) from nasal polyps (NP). Five cases of IP and NP, respectively, were investigated intraoperatively with OCT and CLSM. Biopsies were taken of the investigated area and were analyzed ex vivo with OCT and CLSM and then underwent HE-staining for standard light microscopy. On OCT images, IP showed the characteristic inverted character of the epithelium, that was thicker with a high degree of variability of thickness compared to the thin and homogenous epithelium of NP. In addition, the characteristic stromal edema of NP could be visualized. On CLSM images, the typical epithelial invaginations of IP appeared as crypts, while in NP the highly organized cylindric epithelium could be visualized. In vivo, OCT acquired images of sufficient quality to visualize these characteristics, while CLSM did not. Our study demonstrates that OCT and CLSM can distinguish IP from NP. Further technical development is required to apply the techniques clinically to guide intranasal biopsies or even to make them dispensable.
Summary
Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal ...innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon—a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70–90 μm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti‐neuronal β‐III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20–30 μm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface.
Lay description
Human cornea is one of the most highly innervated tissues in the body. Sectioning of the cornea for the quantitative analysis of its innervation by immunohistochemistry is necessary during health and disease. Attempts to obtain frozen or paraffin‐sections of whole corneas parallel to the plane of its innervation are technically challenging and also time consuming due to the curvature of the tissue. In an effort to overcome this difficulty, here we report a contact‐free and rapid femtosecond laser based cutting method to obtain high quality surface parallel sections of the human cornea in less than 15 min. Furthermore, femtosecond based laser microtomy can be adopted easily for sectioning of all kind of biological samples including hard tissues (teeth and bone) and offers a more standardized way of tissue sectioning by avoiding the laborious processes of sample preparation and embedding thereby preventing the loss of valuable tissue sample.
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