A CONFOCAL MICROSCOPY STUDY OF THE ANTERIOR CORNEAL MOSAIC IN THE SUB-BASAL NERVE PLEXUS S. Allgeier 1 , F. Eberle 2 , B. Köhler 2 , S. Maier 1 , J. van Wezel 3 , A. Zhivov 4 , G. Bretthauer 1,2 1 Institute for Applied Computer Science/Automation, Karlsruhe Institute of Technology (KIT), Germany 2 Intitute for Applied Computer Science, Karlsruhe Institute of Technology (KIT), Germany 3 Steinbuch Centre for Computing, Karlsruhe Institute of Technology (KIT), Germany 4 Department of Ophthalmology, University of Rostock, Germany stephan.allgeier@kit.edu Abstract: In vivo confocal microscopy of the corneal sub- basal nerve plexus and assessment of its morphology prom- ises to provide a novel, highly sensitive tool for early diagno- sis and therapy monitoring of diabetic peripheral neuropa- thy. Ridge-like tissue deformations in the region of the sub- basal nerve plexus, induced by pressure upon the corneal surface, can prevent the imaging of sub-basal nerves over the entire field of view and impede the assessment of nerve morphology. We present an investigation of the distribution of the deformation heights, yielding a mean value of 28.9 μm in our data. Given the depth of focus of the used confocal microscope of 6-7 μm, our results strongly suggest the need for three-dimensional imaging methods for the reliable as- sessment of sub-basal nerve morphology. Keywords: cornea, sub-basal nerve plexus, anterior cor- neal mosaic, confocal microscopy, image processing Introduction Since the introduction of in vivo confocal microscopy of the cornea, the corneal sub-basal nerve plexus (SNP) has re- ceived a great amount of attention. It consists of a network of small peripheral nerve fibres and nerve fibre bundles (diame- ters < 5 μm) and is located immediately at the anterior sur- face of the cornea’s basal epithelial membrane. Due to its general two-dimensional arrangement parallel to the ocular surface, the SNP lends itself ideally to the imaging by in vivo confocal microscopy. A multitude of different studies of corneal sub-basal nerve structures has been conducted and published, assessing morphological changes in ocular (e.g. keratoconus, dry eye, contact lens wear) and systemic condi- tions and diseases (e.g. diabetes, Fabry’s disease) and after corneal surgery. Recent studies point toward the potential of sub-basal nerve morphology as a sensitive surrogate marker for early diagnosis and monitoring of progression or therapy success in diabetic peripheral neuropathy [1]. The reliable assessment of morphological parameters of the SNP, however, depends on the quality and first and foremost on the general visibility of the imaged nerve structures. Es- pecially in the use of confocal laser scanning microscopy (CLSM) – the most commonly used technology for in vivo imaging of the SNP because of its high lateral resolution and very low depth of focus – ridge-like deformations occurring in the region of the SNP and the neighbouring tissues can prevent the imaging of sub-basal nerve fibres over the entire field of view (Fig. 1). Figure 1: CLSM images of ridge-like deformation in the region of the SNP; focus plane at SNP level (A), at 19 μm anterior to SNP level (B) and at 38 μm anterior to SNP level (C). These deformations are one expression of a phenomenon termed the anterior corneal mosaic (ACM) by Bron in 1968 [2], who was the first to systematically examine it. The first description of the ACM ridges in in vivo corneal confocal microscopy dates back to 2006 by Kobayashi et al. [3] who later proved that the observed ridges in fact align with the ACM visible in slit lamp images of the cornea and are there- fore closely connected to each other [4]. They also described fibrilar structures (which they termed K-structures after Kobayashi) beneath the ridges which they identify as colla- gen fibre bundles in the anterior stroma. Finally, our group published an image processing technique – using software algorithms specifically designed to the imaging technology – for reconstructing two-dimensional images of the SNP from CLSM volume scans [5]. These SNP reconstruction images show the sub-basal nerve structures over the entire image area despite the presence of ACM ridges in the imaged vol- ume. The mentioned image processing technique has since been used extensively with volume scans taken from 151 subjects. We here present an analysis of the measured heights of the ACM ridges. Methods The image data used herein was acquired from 151 subjects during a study investigating nerve alterations in early stages of diabetes. In vivo confocal microscopy was performed using an HRT II CLSM system in conjunction with the RCM objective mod- ule (both Heidelberg Engineering GmbH, Heidelberg, Ger- many). Using a modified volume scan operating mode which continually shifts the focus plane of the microscope back and forth, a number of image stacks (with an axial image dis- tance of ca. 0.5 μm) were acquired for each subject, each stack representing a partial volume of the subject’s cornea. A B C Biomed Tech 2013; 58 (Suppl. 1) © 2013 by Walter de Gruyter · Berlin · Boston. DOI 10.1515/bmt-2013-4270