Abstract
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.
Highlights
A well-established method for acquiring corneal images at cellular level is the combination of the Heidelberg Retina Tomograph (HRT) and the Rostock Cornea Module (RCM; both Heidelberg Engineering GmbH, Heidelberg, Germany), which is a confocal laser scanning microscope
Since its first presentation in 2002 [1], the HRT + RCM serves as a reliable instrument [2] and plays an important role in ex vivo and in vivo studies of human or animal corneas for a qualitative and quantitative analysis based on 2D imaging and/or 3D image reconstruction, e.g. the anatomical comparison of laboratory animal corneas [3], the assessment of stromal modifications of patients with progressive keratoconus after treatment by riboflavin-UVA-induced cross-linking of corneal collagen [4], quantitative full-thickness corneal 3D imaging [5], automated quantification of morphologic features of different epithelial cell layers [6], 2D reconstruction of the subbasal nerve plexus (SNP) from volume scans in the presence of ridge-like tissue deformations [7] and large-scale image reconstruction of the SNP [8,9,10,11,12,13]
Besides confocal laser scanning microscopy based methods, the full-field optical coherence tomography (FF-OCT) [14] is a promising attempt to obtain en face corneal images
Summary
A well-established method for acquiring corneal images at cellular level is the combination of the Heidelberg Retina Tomograph (HRT) and the Rostock Cornea Module (RCM; both Heidelberg Engineering GmbH, Heidelberg, Germany), which is a confocal laser scanning microscope. Image stacks through the entire cornea are presented in [16] and were acquired with a tandem scanning confocal light microscope and without image alignment, leading to lower image quality and lower resolution compared to confocal laser scanning based methods. When the TomoCap moves towards the cornea, the increasing applanation pressure on the cornea induces compression artifacts that manifest as ridge-like deformations in the SNP and the adjacent tissue [19,20,21,22] These deformations are detrimental for imaging thin layers such as the SNP, which cannot be kept in focus over the entire field of view once the deformation height exceeds the depth of field of the RCM
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