Remote-Controlled Scanning and Automated Confocal Microscopy Through Focusing Using a Modified HRT Rostock Corneal Module

Departments of Ophthalmology, UT Southwestern Medical Center, Dallas, TX 75390-9057, USA.
Eye & contact lens (Impact Factor: 1.47). 11/2009; 35(6):302-8. DOI: 10.1097/ICL.0b013e3181bdc435
Source: PubMed


To modify the HRT-II Confocal Microscope with Rostock Corneal Module (HRT-RCM) to allow computerized control of the focal plane position (depth) within the cornea.
A threaded housing on the HRT-RCM microscope is normally rotated by hand to change the focal plane position within the cornea. This piece was removed to allow the front housing of the microscope to move freely. A linear actuator (Oriel Encoder Mike) was then attached to the side of the microscope and coupled to a drive shaft that was connected to the front housing. The actuator was connected to an Oriel 18011 Encoder Mike controller, which was interfaced to a PC. Software was developed to allow control and display of the focal plane position using this PC, while image acquisition software was run on the HRT-RCM PC. The instrument was tested on one human volunteer.
The modified instrument successfully allowed computer-controlled focusing throughout the entire cornea. Through-focus sequences could be collected online and analyzed and reconstructed three dimensionally off-line using modified confocal microscopy through-focusing software.
Although this is only a prototype instrument, it significantly improves the examination procedure by allowing completely "hands-free" operation of the HRT-RCM microscope. The data also demonstrate the feasibility of performing quantitative z-axis scans through the full thickness of the cornea with the HRT-RCM. Given the higher contrast images and improved optical sectioning of the HRT-RCM as compared with other instruments, these capabilities could have widespread application.

Download full-text


Available from: Walter Matthew Petroll, Jul 11, 2014
  • Source
    [Show abstract] [Hide abstract]
    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.
    Cornea 10/2012; 32(4). DOI:10.1097/ICO.0b013e31825ec44e · 2.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Diabetic corneal neuropathy can result in chronic, sight-threatening corneal pathology. Although the exact etiology is unknown, it is believed that a reduction in corneal sensitivity and loss of neurotrophic support contributes to corneal disease. Information regarding the relationship between nerve loss and effects on the corneal epithelium is limited. We investigated changes in the corneal epithelium and nerve morphology using three-dimensional imaging in vivo and in situ in a streptozotocin-induced diabetic mouse model. Streptozotocin-treated mice showed increased levels of serum glucose and growth retardation consistent with a severe diabetic state. A reduction in the length of the subbasal nerve plexus was evident after 6 weeks of disease. Loss of the subbasal nerve plexus was associated with corneal epithelial thinning and a reduction in basal epithelial cell density. In contrast, loss of the terminal epithelial nerves was associated with animal age. Importantly, this is the first rodent model of type 1 diabetes that shows characteristics of corneal epithelial thinning and a reduction in basal epithelial cell density, both previously have been documented in humans with diabetic corneal neuropathy. These findings indicate that in type 1 diabetes, nerve fiber damage is evident in the subbasal nerve plexus before terminal epithelial nerve loss and that neurotrophic support from both the subbasal nerve plexus and terminal epithelial nerves is essential for the maintenance of corneal epithelial homeostasis.
    American Journal Of Pathology 08/2014; 184(10). DOI:10.1016/j.ajpath.2014.06.016 · 4.59 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Purpose: To establish a rabbit model of infectious Pseudomonas aeruginosa keratitis using ultrahigh oxygen transmissible rigid lenses and characterize the frequency and severity of infection when compared to a non-oxygen transmissible lens material. Methods: Rabbits were fit with rigid lenses composed of ultrahigh and non-oxygen transmissible materials. Prior to wear, lenses were inoculated with an invasive corneal isolate of P. aeruginosa stably conjugated to green fluorescent protein (GFP). Corneas were examined before and after lens wear using a modified Heidelberg Rostock Tomograph in vivo confocal microscope. Viable bacteria adherent to unworn and worn lenses were assessed by standard plate counts. The presence of P. aeruginosa-GFP and myeloperoxidase-labeled neutrophils in infected corneal tissue was evaluated using laser scanning confocal microscopy. Results: The frequency and severity of infectious keratitis was significantly greater with inoculated ultrahigh oxygen transmissible lenses. Infection severity was associated with increasing neutrophil infiltration and in severe cases, corneal melting. In vivo confocal microscopic analysis of control corneas following lens wear confirmed that hypoxic lens wear was associated with mechanical surface damage, whereas no ocular surface damage was evident in the high-oxygen lens group. Conclusions: These data indicate that in the absence of adequate tear clearance, the presence of P. aeruginosa trapped under the lens overrides the protective effects of oxygen on surface epithelial cells. These findings also suggest that alternative pathophysiological mechanisms exist whereby changes under the lens in the absence of frank hypoxic damage result in P. aeruginosa infection in the otherwise healthy corneal epithelium.
    Investigative Ophthalmology &amp Visual Science 08/2014; 55(9). DOI:10.1167/iovs.14-14235 · 3.40 Impact Factor
Show more