The HRT Rostock Corneal Module provides images with better resolution and contrast than other confocal systems. However, in order to change the focal plane position over large distances, a cylindrical housing must be rotated by hand. This housing is within inches of the cornea, and can sometimes interfere with the examination process. Overall, changing the focal plane position is cumbersome, and represents an important limitation of the instrument. To address this problem, we modified the HRT in our laboratory so that the focal plane position could be controlled using a computer-controlled lens drive system.23
This modification significantly improved the ease of the examination procedure by allowing “hands-free” focusing of the HRT-II microscope; however, this prototype still had several important limitations. First, the connection between the motor drive and the microscope objective was not robust. Second, a time-consuming, multi-step procedure was required to load images and data into the CMTF program. Third, the software could only collect 100 images during a sequential acquire, resulting in a large step size between CMTF images (> 5 μm), which limits the resolution for quantitative analyses of sub-layer thickness. In this study, we report modifications to the HRT-RCM hardware and software that addresses all three of these limitations. We also tested the feasibility of performing in vivo
quantitative full-thickness corneal imaging using the new system.
Using the modified HRT-RCM system, clear peaks were identified on the CMTF curves that allowed us to make measurements of epithelial, stromal and corneal thickness. Corneal thickness measurements made with the HRT-RCM were in good agreement with those obtained using ultrasonic pachymetry (373 ± 25 μm vs. 374 ± 17 μm, respectively). However, the coefficients of variation obtained for repeated scans of the epithelium and cornea were higher than that previously reported using the TSCM system (8.2% and 2.1% versus 2.5% and 0.7%).26
This is most likely due to the different mechanisms used for changing the focal plane on these two systems. In the TSCM, the focal plane position is changed by moving lenses inside the objective casing, thus the tip of the objective remains stationary during scanning. In contrast, changing the focal plane position of the HRT requires movement of the Tomocap which is in contact with the cornea. Essentially, the cornea is moved through the stationary focal plane of the objective. This movement can potentially change the degree of applanation during a scan, as well as introduce backlash when changing directions. In the current study, all through focus scans collected were performed with the Tomocap moving forward, to ensure that the tip remained in contact with the corneal surface throughout the procedure. For this study, the Newport motor drive was controlled using PC-based software; thus a separate PC (in addition to the HRT PC) was required to perform confocal imaging. Alternatively, a joystick remote can be used with a more expensive controller (Newport ESP301-1N), which eliminates the need for a second PC (unpublished observation). We are also evaluating other approaches for focal plane control that can be more easily added onto existing HRT-RCM systems.
Other techniques such as high frequency ultrasound and spectral domain OCT can also provide accurate measurements of corneal sub-layer thicknesses.28-30
However, quantitative 3-D confocal microscopy additional provides a series of high resolution en face images which allow assessment of depth-dependent changes in cell morphology, density and reflectivity.12-14,31
For example, CMTF imaging with the TSCM has been used for studying the effects of refractive surgical procedures such as PRK and LASIK, in which measurements of sub-layer thickness and depth-dependent cell and ECM backscatter are important.32-37
CMTF has also been used to assess the corneal light scattering profile in transgenic mouse models with altered corneal clarity.38
Unlike most anterior segment imaging approaches, confocal microscopy can also be used for reconstruction of the subbasal nerve plexus, assessment of corneal endothelial density and morphology, and 3-D localization and monitoring of corneal infection and inflammation.1-3,22,39
The TSCM has also been used to monitor changes in keratocyte density during aging, in keratoconus patients and following surgery.36,40-43
Previous studies have identified significant, often unexpected changes in cell density that may have long-term clinical implications. 28,30-33
Cell density was estimated from single (2-D) images collected at different depths from within the cornea in these studies. In the current study, we used the entire CMTF dataset, so that the true 3-D position of each cell could be determined. A linear decrease in keratocyte density was measured from the anterior to posterior cornea, consistent with previous data generated from the rabbit cornea in vitro
In order to perform true 3-D cell counting, the images within the stack were registered to compensate for translational movement of the cornea that occurred during scanning. Thus scans without large amounts of movement had to be excluded. Although manual cell counting was performed in the current study, the HRT-RCM should be well suited for previously published automated cell counting approaches due to the high image contrast.40,44
We were also able to generate 3-D volume renderings of the rabbit cornea from the confocal scans using Imaris software. To our knowledge, these are the first such reconstructions that encompass the full-thickness of the in vivo cornea. It should be noted that such reconstructions would be more difficult in the human cornea, due to involuntary eye movements that occur during scanning. As recently demonstrated by Zhihov and coworkers, such movements can cause distortions within individual images, and therefore require more complex image registration techniques.22
It should also be noted that the HRT streaming software used in this study is a beta version not yet approved for human use. Without this software only 100 frames can be acquired during a sequential acquire, which is not sufficient for high resolution 3-D reconstructions of the tissue.
Overall, the hardware and software modifications to the HRT-RCM allow high resolution 3-D image stacks to be collected from the entire rabbit cornea in vivo. These datasets can be used for interactive visualization of corneal cell layers, quantitative assessment of sub-layer thickness and depth-dependent measurements of keratocyte density. Overall, the modifications should significantly expand the capabilities of the HRT-RCM for quantitative research applications.