Measurement of the shape of the corneal surface after photorefractive keratectomy is critical to perfect the technique. So far ablation rates have been determined either by counting pulses until tissue perforation occurs or by micrometry performed on histologic sections.
Perforation trials do not take into account different ablation rates for distinct tissue layers (eg, cornea); the relevance of histologic micrometry is limited by tissue deformation and shrinkage induced by the preparation technique.
Figure 1: Experimental set-up.
In this article, we describe a nondestructive method to preserve and determine the shape of the corneal surface after photoablation. Using a twocomponent liquid silicon that polymerizes after mixing, this method allows repeated measurements on normally hydrated corneas at any time as well as of the tissue.
MATERIALS AND METHODS
A two-component silicon (Alpa Sil*, Alpina Dental Seibicke, D-8025 Munich-Unterhaching, Germany) was used; viscosity at room temperature of component 1 was 3000 mPoise (1 Poise = 1 gx Cm-1XS"1), for component two 3500 mP. Mixing the two components should take place bubble-free, therefore a vacuum stirrer (Alpina Dental Seibicke) was used. Immediately after laser irradiation, the silicon is filled into a metal ring (15 mm diameter, 10 mm height), which surrounds the ablated corneal area (Fig 1). After mixing, processing time is about 5 minutes. Polymerization is athermal and takes about 30 minutes at room temperature. Warming up the material in a moderate way (constant temperature was achieved using a water bath) shortens the polymerization time (see the Results section). The material is nontoxic. The resulting silicon block is flexible but stable in shape. No warping of the surface occurs when the position of the silicon block is changed. Long-term stability of the cast has been proved by the manufacturer (Alpina Dental Seibicke).
Resolution of the silicon cast was determined in the following manner: A flat polymethylmethacrylate (PMMA) piece was scratched with a polishing paste (Ivoclar, Ltd, D-7090 Ellwangen, Germany), containing particles of a diameter of 0.2 to 1.0 µm at most. A cast of the scratched surface was then molded and examined by light microscopy and scanning electron microscopy (SEM).
To prove this technique could reproduce cast profiles of PMMA after 193-nanometer excimer laser irradiation, a flat PMMA disc underwent a myopic ablation of 4.00 diopters (94 pulses, 180 mJ/qcm, repetition rate 10 Hz, Summit Excimed UV 200). The surfaces of the PMMA disc and the corresponding cast underwent three-dimensional topometry (Fig 2) using phase shift prism interferometry The resolution of the method is at least 0.2 µp?; the method is fully described elsewhere.1
To demonstrate that silicon casting may be used on corneal tissue, porcine and human donor eyes were ablated using an excimer laser (Lambda Physik 102 E: fluence 185 mJ/qcm, beam diameter 1 mm; repetition rate 5 Hz) with a nearly rectangular beam profile. With this configuration, ablation depth as a function of pulse number was measured. Pulse numbers ranged from 3 to 150. The casts of the ablated area were molded immediately after laser exposure. To prevent anterior chamber flattening, intraocular pressure was stabilized (25 cm H2O) by injecting saline. The resulting silicon blocks were then cut perpendicularly to the anterior surface and measured by calibrated light microscopy.
The two-component silicon gives an exact cast of the ablated pattern. The polymerized silicon has an extremely smooth and featureless surface (Fig 3A). Repeated casts of the scratched PMMA surfaces all showed scratches (minimum depth 0.2 µm, maximum depth 1 µ??) on the silicon cast; thus resolution is at least 0.25 µ??. By comparing scratched areas of the PMMA surface with the corresponding silicon cast by means of microscopic photography, we could demonstrate the reproduction quality of the gel (Fig 3B).
Polymerization time at room temperature is 25 to 30 minutes. Figure 3C shows the exponential function of polymerization time and temperature of the gel: At 37° C, the gel is fully polymerized after 5 minutes.
Figure 2 shows a topométrie map of a flat PMMA disc after a 4.00-diopter ablation and the corresponding silicon cast surface. Ablation pattern and cast give ultrastructural details that correspond exactly.
Area ablation (1-millimeter diameter) on the cornea was detectable on the corresponding silicon cast after a minimum number of four laser pulses, but not always measurable in histologic cuts. To prove whether results of the silicon cast method are comparable to known data, ablation depth as a function of pulse number was measured: the incubation effect is clearly confirmed by extrapolation (Fig 4), and no detectable photoablation occurs with the two first shots. The ablation rate with a radiant exposure of 185 mJ/cmp 2 was found by extrapolation to be 0.44 ± 0.12 µm/pulse for Bowman's layer and 0.67 ± 0.13 µm/pulse for stroma.
Figure 2: (A) Four-diopter myopic ablation in a flat (note concave geometry) PMMA piece. (B) Corresponding silicon cast; both measured by phase shift prism interferometry.
Thus far, ablation rates as well as ablation profiles of porcine or human corneas have been determined either by counting the number of pulses needed for perforation or by measuring perpendicularly cut histological specimens under light microscopy. The latter does not preserve the typical state of hydration of the cornea and perforation trials ignore differences in ablation rates between inhomogenous tissue (eg, Bowman's layer and stroma). The effect of small pulse numbers cannot be determined precisely. A nondestructive method that preserves the properties of the ablated area immediately after laser processing should provide more accurate measurement of profiles, ablation rate, and surface properties, and also allow three-dimensional topometry and volumetry.
Figure 3: (A) Featureless surface of polymerized silicon (dust particle of about 0.5 µm diameter). (B) One-micrometer scratches reproduced on silicon cast surface. (C) Polymerization time as a function of the temperature of the silicon gel.
The replica idea was originally described by Vrabec borrowing the idea from Wolf.2 He studied three-dimensional surface structures like the inner surface of the trabecular mesh work or the surface of the human iris. Silicon was used by Riera and Liotet years ago to reproduce epithelial surfaces of living tissues to demonstrate them by SEM.3 Resolution was high and the material proved to be nontoxic. Now, silicon has been used to quantify photoablation results of the cornea.
The silicon method has several advantages: the method's accuracy is better than 0.25 µm in PMMA and corneal tissue and is sufficient for practical purposes. Thermal effects or material expansion do not occur and the substance is easy to handle.
Measuring the silicon cast surface instead of the ablated tissue directly allows for repeated measurements of the same corneal ablation.
Questions related to effective tissue ablation with regard to repetition rate, shielding effect, and so on that arose in previous studies46 can be answered easily using silicon casting as a nonhistologic method.
Figure 4: Ablation rate as a function of pulse number. Measurement obtained on silicon casts: The incubation effect of the 193 irradiation is confirmed.
Documentation and measurement of complex photoablation patterns (eg, astigmatic corneal ablation, aspheric ablations) and measurement of surface roughness may also be facilitated by the silicon cast method.
Polymerization time at room temperature is about 30 minutes. Thus, corneal hydration may change, and the shape will likely change also. The time needed may be cut down by using different catalysts or by warming the gel slightly (eg, warming up the metal ring).
Provided that faster, nontoxic, and athermal polymerization could be combined with the same surface smoothness, silicon casts could also be tried to obtain objective measurements in wound-healing experiments (in anesthetized animals), thus allowing more accurate measurement of corneal shape in vivo.7
Automated topometry further facilitates objective measurement including volumetric data. So far it has been limited to measurement of flat silicon blocks. Advanced confocal surface topometry devices have qualified for easy and fast three-dimensional measurement (UBM Microfocus,8 D-7505 Ettlingen, Germany). In the same manner, measurement of surface roughness has been performed successfully. Further work needs to be done for evaluation of silicon cast volumetry.
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3. Riera M, Liotet S. An application of scanning electron microscopy for replicas of living tissue. Contactologia. 1979;1D:2125.
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