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. 2013 Apr;32(4):e36-43.
doi: 10.1097/ICO.0b013e31825ec44e.

Quantitative 3-dimensional corneal imaging in vivo using a modified HRT-RCM confocal microscope

W Matthew Petroll  1 , Matthew Weaver, Saurabh Vaidya, James P McCulley, H Dwight Cavanagh
Affiliations

Quantitative 3-dimensional corneal imaging in vivo using a modified HRT-RCM confocal microscope

W Matthew Petroll et al. Cornea. 2013 Apr.

Abstract

Purpose: The purpose of this study was to develop and test hardware and software modifications to allow quantitative full-thickness corneal imaging using the Heidelberg Retina Tomograph (HRT) Rostock Corneal Module.

Methods: A personal computer-controlled motor drive with positional feedback was integrated into the system to allow automated focusing through the entire cornea. The left eyes of 10 New Zealand white rabbits were scanned from endothelium to epithelium. Image sequences were read into a custom-developed program for depth calculation and measurement of sublayer thicknesses. Three-dimensional visualizations were also generated using Imaris. In 6 rabbits, stack images were registered, and depth-dependent counts of keratocyte nuclei were made using Metamorph.

Results: The mean epithelial and corneal thickness measured in the rabbit were 47 ± 5 μm and 373 ± 25 μm, respectively (n = 10 corneas); coefficients of variation for repeated scans were 8.2% and 2.1%. Corneal thickness measured using ultrasonic pachymetry was 374 + 17 μm. The mean overall keratocyte density measured in the rabbit was 43,246 ± 5603 cells per cubic millimeter in vivo (n = 6 corneas). There was a gradual decrease in keratocyte density from the anterior to posterior cornea (R = 0.99), consistent with previous data generated in vitro.

Conclusion: This modified system allows high-resolution 3-dimensional image stacks to be collected from the full-thickness rabbit cornea in vivo. These data sets can be used for interactive visualization of corneal cell layers, measurement of sublayer thickness, and depth-dependent keratocyte density measurements. Overall, the modifications significantly expand the potential quantitative research applications of the HRT Rostock Cornea Module microscope.

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Figures

Figure 1
Figure 1
(A) Heidelberg Engineering HRT-RCM confocal microscope (From Heidelberg Engineering Website). (B) Modified HRT-RCM with motor drive to allow automated through focusing, and modified support structure (slit lamp stand) to facilitate positioning.
Figure 2
Figure 2
Comparison of focal plane position determined from the inductive displacement transducer on the HRT-RCM (HRT Depth Display) and the depth reading from the CMTF Program. The Newport linear actuator was used to change the focal plane position.
Figure 3
Figure 3
Screen shot of CMTF program. Right side shows corneal intensity curve with intensity peaks at the superficial epithelium (Epi), basal lamina (BL), and endothelium (Endo). Images on the left are reconstructions of the image stack collected by CMTF imaging shown at different projection angles. Scan shown was collected at a speed of 30 μm/second.
Figure 4
Figure 4
A sampling of images from a CMTF scan taken from a rabbit cornea in vivo. The position displayed in the upper right cornea of each image is the depth relative to the front surface of the Tomocap. A speed of 60 μm/second was used for the CMTF scan.
Figure 5
Figure 5
A) Maximum Intensity Projection along the x-axis of an in vivo image stack collecting using the CMTF program. B) Graph showing mean cell densities through the stromal thickness of six corneas. Both the image and the graph show progressively decreasing cell density through the stroma from the basal lamina to the endothelium. Graph shows mean and standard deviation of measurements from six corneas taken in vivo (N=6).
Figure 6
Figure 6
Volume renderings of CMTF data. Images were cropped in 3-D to focus on a region of interest, and rendered using an orthogonal maximum intensity projection within the Surpass module of Imaris.
See this image and copyright information in PMC

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