March 2012
Volume 53, Issue 3
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Cornea  |   March 2012
Corneal Surface Asphericity, Roughness, and Transverse Contraction after Uniform Scanning Excimer Laser Ablation
Author Affiliations & Notes
  • Sean J. McCafferty
    From Departments of Ophthalmology,
    Optical Science, and
  • Jim T. Schwiegerling
    From Departments of Ophthalmology,
    Optical Science, and
  • Eniko T. Enikov
    Mechanical/Aerospace Engineering, University of Arizona, Tucson, Arizona.
  • Corresponding author: Sean J. McCafferty, University of Arizona, 1630 East University Boulevard, Tucson, AZ 85721; sjmccafferty66@hotmail.com
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1296-1305. doi:10.1167/iovs.11-9267
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      Sean J. McCafferty, Jim T. Schwiegerling, Eniko T. Enikov; Corneal Surface Asphericity, Roughness, and Transverse Contraction after Uniform Scanning Excimer Laser Ablation. Invest. Ophthalmol. Vis. Sci. 2012;53(3):1296-1305. doi: 10.1167/iovs.11-9267.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To examine the interaction between the excimer laser and residual tissue.

Methods.: Ten cadaveric porcine eyes with exposed corneal stroma and plastic test spheres underwent uniform 6-mm ablation with a scanning excimer laser. Corneal profilometry of the central 3 mm was measured with submicrometer resolution optical interferometry, before and after uniform excimer ablation. Eleven surface-marked eyes were photomicrographed before and after excimer ablation. Images were superimposed, and mark positional changes were measured.

Results.: Uniform scanning excimer laser ablation of the corneal stroma produces a significant central steepening and peripheral flattening in the central 3-mm of the diameter. The central 1-mm corneal curvature radius (r) decreased from r = 10.07 ± 0.44 (95% CI) to 7.22 ± 0.30 mm, and the central 2-mm radius decreased from r = 10.16 ± 0.44 to 8.10 ± 0.43 mm. Q values, measuring asphericity in the 2-mm radius of the central cornea, were significantly lower before than after ablation (−5.03 ± 4.01 vs. −52.4 ± 18.7). Surface roughness increased significantly from 0.65 ± 0.06 to 1.75 ± 0.32 μm after ablation. The central 2 mm of the stromal surface contracted by 2.21% ± 0.80% at a sustained temperature of 5°C. Ablation of plastic spheres produced no significant change.

Conclusions.: The excimer laser interacts with the nonablated residual stromal surface in a characteristic fashion not seen with isotropic, inorganic material. Increases in asphericity, surface roughness, surface contraction, and stromal morphologic changes are supportive of this interaction. The surface changes demonstrated may be indicative of temperature-induced transverse collagen fibril contraction and stress redistribution, or the ablation threshold of the stromal surface may be altered. This phenomenon may be of increased importance using lasers with increased thermal load.

Modern refractive procedures using argon-fluoride (ArF) excimer wavelength (193 nm) laser radiation to change the surface shape of the human cornea, by ablative tissue removal, have been used successfully for more than 20 years. The procedures are elective, with high patient expectations. Often visual outcomes are acceptable to the patient. However, a significant minority of patients have poor or reduced visual outcomes, leading to a decrease in best corrected visual acuity (BCVA). It was originally assumed that a uniformly distributed laser profile would ablate a uniform thickness of the cornea, as it does with isotropic plastic materials, and the result of ablation would be the same as the original surface profile translated to a depth that was a function that total fluence applied. 1 However, it was noted early that the central cornea appeared to be significantly underablated, creating a severe asphericity, termed central islands (Parker PJ, et al. IOVS 1993;34:ARVO Abstract 509). These central irregularities correlated with visual disturbances, such as decreased best corrected visual acuity, glare, and halos. 2,3 Excimer-induced asphericity has been the accepted reason the original Munnerlyn formula works well on a plastic sphere, but not as well on a human cornea. Increased asphericity has been shown after commercial excimer myopic correction. Before the utilization of central overablation algorithms, the incidence of clinically evident central asphericity measured by placido ring topography in human eyes was between 50% and 88%. 4 Empiric adjustments to the ablation algorithm were made which mostly solved the existing problem, without a clear understanding of the cause. 5 8 Even with these interventions, research has noted that the largest contributor to postablation ocular aberrations, by a factor of 4, is spherical aberration. 9  
Over the past 15 years, three major theories have emerged as to the cause of the apparent underablation: (1) redeposition of material ejected from the ablation back onto the central cornea 10 12 ; (2) ejected plume material blocking the subsequent pulse 2 ; and (3) corneal edema preferential to the center of the cornea. 2,10,11 Additional theories have included that the ablation is a function of distance from the edge of the ablation 5,13 and that transverse energy along the ablated surface interacts with the tissue, decreasing the ablation threshold preferentially in the central cornea. 1 Several other theories have been considered, but have not gained support. 14 17 None of the theories adequately explains the apparent nonuniform ablation, and some authors have questioned their validity. 1,5  
The response of enucleated porcine corneal tissue is similar enough to that of live rabbit or live human eyes to justify their use and is supported in many studies. 12,13,18 22 There is evidence of increased amplitudes in the irregularities observed in enucleated eyes, possibly from increased hydration of the tissue. 12 Q values have been used clinically and mathematically to evaluate asphericity changes in living human corneas and mathematical models. 6,23 28 Studies have also looked at surface roughness after excimer ablation as a cause of subepithelial haze and suggested some interaction between the laser and residual corneal tissue. 29 The residual nonablated corneal tissue is noted to have numerous histologic changes suggestive of an interaction between the excimer laser and the residual corneal stromal surface. 12,22,30 34 Histologic changes include a thin “coagulation layer” on the residual surface and irregular stromal undulations with elevated temperature of the stroma. 
At least one researcher suggested that a thermal response is responsible for the central irregularities. 35 Some thermal response was noted with the excimer laser at its inception, but it was thought to be negligible. 14 Recent studies have also shown a thermal response to a brief temperature increase associated with the excimer laser, which was previously thought to be strictly ablative in nature. 20,21 This temperature increase is caused by ablation fluences used in both broad-beam and flying-spot lasers. 21 Other studies have shown this thermal response to be magnified with increasingly desirable pulse rates and laser fluences. 22 Some newer lasers running in excess of 1000 Hz and 500 mJ/cm2 are capable of reaching maximum temperature increases of almost 20°C. 36  
Thermal energy denatures the triple-helix structure of the corneal collagen, creating a contractile force along its lengthwise dimension. 37 Thermally denatured collagen can shrink between 8% and >30% of its original length, as seen in CO2 laser studies. 38 Collagen contraction and denaturation without thermal necrosis begins at 40°C and achieves its maximum at approximately 62°C. 38 40 The human cornea has been shown to be briefly heated to 60°C to 70°C during ablation with the ArF excimer laser. 20  
Despite the empiric changes to the excimer ablation algorithms, irregularities persist, albeit less frequently and with less severity. The major causes in symptomatic patients with poor visual outcomes are linked to persistent central corneal irregularities with associated aberrations, mostly spherical aberration. 9,41,42 Also, excimer laser ablations still lead to a decrease in the quality of vision, particularly at night with decreased contrast sensitivity. 41,43,44 Even recent studies highlight the postablative change in asphericity and the need to better understand its etiology. 26,45 48  
In this study, we examined and quantified the effects of the excimer laser on porcine corneal stromal tissue. It was designed specifically to look at changes in central surface shape, structure, and roughness, while also measuring potential causative factors such as temperature. The ultimate goal was a clearer understanding of this interaction, to facilitate predictive models that can assist in designing better excimer lasers using altered parameters, such as increased pulse rates and fluences. 
Methods
Ten whole, fresh, cadaveric porcine eyes were used less than 24 hours after death, stored in ice with preservative solution. All eyes were corneal disease and injury free, and the corneas were visually clear. The eyes were heated, via water bath, to 29°C just before use. A corneal pachymeter (Medtronic, Minneapolis, MN) was used to measure the central thickness, which was 1029 ± 58 μm. The porcine eyes were stabilized and maintained in a constructed chamber, leaving the cornea and surrounding sclera exposed (Fig. 1). The eyes were pressurized in the chamber with an intravenous gravity feed infusion setup of lactated Ringer's solution into the ocular vitreous. Intraocular pressure was maintained at 16 mm Hg and verified by pneumotonometry, correcting for corneal thickness (model 30; Medtronic). 
Figure 1.
 
Globe stabilization chamber with infusion.
Figure 1.
 
Globe stabilization chamber with infusion.
A microkeratome (Excellus Hansatome; Bausch & Lomb, Rochester, NY) with an 8.5-mm suction ring was used to create a corneal (LASIK type) flap of 180-μm thickness. This hinged flap was laid back to expose the bare stroma for ablation and measurement. At other times, the flap was replaced to prevent surface desiccation and stromal edema. In addition, hydration with basic salt solution in a sealed bag around the stabilizing chamber was used to prevent desiccation. 
An excimer laser 193-nm (argon-fluoride) commercial excimer laser (Visx S4; Abbott Laboratories, Milpitas, CA) was used to ablate the corneal stromal surface. The ablation pattern was strictly uniform without any adjustment algorithms for asphericity or central islands. This was achieved by using the laser's “block flat” test ablation without the block in place to ensure proper focus. The uniformity of the ablation was confirmed by both an optical PMMA test flat and PMMA test spheres which showed no change in surface profile from the nonablated surface. The laser performed 513 scanning pulses on the stromal (or plastic) surface at 160 mJ/cm2 at 10 Hz over a 6-mm diameter ablation. The residual tissue thickness was again measured to determine the depth of the ablation. 
The stromal surface topography of each eye was measured, both before and after excimer laser ablation, while the orientation of the eye was maintained, with a broad-band optical interferometry profilometer (model NT9800; Veeco, Tucson, AZ). This testing was completed to determine changes in profile and closest spherical fit as well as surface roughness. The profilometer software was used to provide raw profile data, as well as noise-reduced data with a Gaussian Fourier filter and tilt adjustment. 
The conic constant Q value was used as an indicator of asphericity of the central corneal stromal surface. The interferometer-measured profile was used to calculate the spherical fit of the central 1-mm diameter of the stromal surface, and the sag of the surface was determined at a 2-mm diameter. The Q value was calculated from a modification of Baker's equation.   where the shape factor P = 1 + Q, and R is the central 1-mm radius of curvature. 
The surface roughness was determined from the raw measured profile data, and a summed average of 10 randomly chosen places in the central 2 mm of the stroma. The following equation was used to calculate the roughness in micrometers, by determining the absolute value of the raw surface deviation x k from that of the smooth surface at n random locations.    
Transverse contraction (or expansion) of the corneal stromal surface was determined by using a diamond blade to make two parallel opposed positional marks in the stromal surface of 11 prepared eyes. The eyes were prepared as before with maintenance of intraocular pressure at 16 mm Hg and prevention of desiccation. The marks were made to a depth of 200 μm, approximately 2 mm apart, and centered in the 6-mm ablation crater. A CCD telecentric microscope with measurement software (Amscope, Irvine, CA) was used to produce standardized photomicrographs of the stromal surface maintaining a constant magnification and focal distance. Standardized photomicrographs of the stromal surface were taken before and after uniform excimer ablation, and the images were superimposed to examine transverse positional changes in the marks, possibly indicating a transverse mechanical alteration in the stromal surface. The apex medial and lateral edges of the V-shaped incisions were visible in photomicrographs (Fig. 2). Measurements were made from the medial edge of the incision to negate a false transverse contraction caused by apparent medial movement of the lateral edge, with ablation down around the V-shaped incision. Measurements of the distance between the two marks both before and after ablation were made at four to five separate locations by a blinded observer and averaged (Fig. 3). 
Figure 2.
 
Photomicrograph of postablation stromal corneal incision with diamond blade set at a 200-μm depth. Note that the apex as well as the medial and lateral edges of the V-shaped incision are visible.
Figure 2.
 
Photomicrograph of postablation stromal corneal incision with diamond blade set at a 200-μm depth. Note that the apex as well as the medial and lateral edges of the V-shaped incision are visible.
Figure 3.
 
Two samples of the superimposed images of pared stromal incisions before (blue) and after (red) treatment with uniform excimer laser ablation. The inner edge of incision is marked for clarity. Odd-numbered measurements are before treatment, and even ones are after treatment.
Figure 3.
 
Two samples of the superimposed images of pared stromal incisions before (blue) and after (red) treatment with uniform excimer laser ablation. The inner edge of incision is marked for clarity. Odd-numbered measurements are before treatment, and even ones are after treatment.
Transmission electron microscopy was performed on the postablation corneal stroma to examine morphologic changes in the residual stroma and possible histologic alterations consistent with temperature elevation or surface contraction. 
The temperature of the corneal surface during excimer ablation was determined by evaluating the irradiance recorded from an infrared camera (3–5 μm, indium antimonide; Radiance 1; Amber, Goleta, CA), running at a 60-Hz frame scan time with blackbody standardized software (MatLab; The MathWorks, Natick, MA), maintaining a constant image view angle and focal distance. 
To ensure the validity of the findings, 8-mm radius, optical quality, PMMA plastic test spheres were ablated in an identical fashion and measured for profile changes, surface roughness, and surface transverse positional changes. The same parameters were measured on the postablation corneal porcine stroma looking for changes occurring with desiccation over 30 minutes at 25°C and 30% relative humidity. Finally, the same parameters were measured on the postablation stroma with intraocular pressure elevated from 16 to 40 mm Hg, to determine whether the findings may be related to a mechanical weakening of the cornea. 
Results
Uniform scanning excimer laser ablation on the porcine corneal stroma produces a significant central (0.5 mm diameter) steepening and a comparable peripheral flattening in the optic zone (3-mm diameter) of the cornea. All ablated samples produced a central increase in curvature (decreased radius), which was significantly attenuated out to a 3-mm diameter. An examination of all 10 samples showed that the central 1-mm radius of curvature decreased from 10.07 ± 0.44 to 7.22 ± 0.30 mm (95% CI). Likewise, the central 2-mm radius of curvature decreased significantly from r = 10.16 ± 0.44 to 8.10 ± 0.43 mm. The larger diameter within the optic zone was affected to a lesser degree. Shown are examples of the optical interferometer-measured profile in the same orientation before and after uniform excimer ablation (Fig. 4). 
Figure 4.
 
Two corneal stromal profiles showing before and after uniform excimer laser ablation. The x-axis is in millimeters from the corneal apex. The y-axis is elevation in micrometers. Note that all profiles show a central steepening and peripheral flattening. The graphs were superimposed by setting the average of each profile to 0.
Figure 4.
 
Two corneal stromal profiles showing before and after uniform excimer laser ablation. The x-axis is in millimeters from the corneal apex. The y-axis is elevation in micrometers. Note that all profiles show a central steepening and peripheral flattening. The graphs were superimposed by setting the average of each profile to 0.
All samples showed an increase in asphericity when closest spherical fit was applied to the pre- and postablation profiles, as shown in Figure 5
Figure 5.
 
Corneal stromal profile showing deviation in micrometers (y-axis) from the closest spherical fit, both before and after uniform excimer ablation. (All samples had similar results.) The aspheric profile was greatly magnified after the uniform excimer ablation.
Figure 5.
 
Corneal stromal profile showing deviation in micrometers (y-axis) from the closest spherical fit, both before and after uniform excimer ablation. (All samples had similar results.) The aspheric profile was greatly magnified after the uniform excimer ablation.
Q values were calculated from measured profiles in the central 2 mm of the uniform ablation crater. All samples showed an increased negative Q value indicating a central steepening and comparative peripheral flattening of the postablation profile. Q values of all 10 samples measuring asphericity in the central 2 mm of the cornea, were significantly lower before than after ablation (−5.03 ± 4.01 vs. −52.4 ± 18.7, respectively) (Fig. 6). 
Figure 6.
 
Q values show a large, statistically significant flattening of the peripheral cornea (2-mm diameter) compared with the central cornea (1-mm diameter) after uniform excimer ablation. Peripheral flattening shows hyperboloid deformation (as opposed to paraboloid) with a negative Q value.
Figure 6.
 
Q values show a large, statistically significant flattening of the peripheral cornea (2-mm diameter) compared with the central cornea (1-mm diameter) after uniform excimer ablation. Peripheral flattening shows hyperboloid deformation (as opposed to paraboloid) with a negative Q value.
Stromal surface roughness increased visibly in all samples, as shown in the following raw topographies, before and after uniform excimer ablation, produced by optical interferometry (Figs. 7, 8). 
Figure 7.
 
Imaged surface of the corneal stroma (3 mm diameter) before and after uniform excimer ablation 531 pulses at 160 mJ/cm2. Increased roughness of the surface is evident in the postablation samples.
Figure 7.
 
Imaged surface of the corneal stroma (3 mm diameter) before and after uniform excimer ablation 531 pulses at 160 mJ/cm2. Increased roughness of the surface is evident in the postablation samples.
Figure 8.
 
Two postablation surfaces imaged with a Fourier Gaussian high-spatial-frequency filter and with the sphere term removed (closest spherical fit).
Figure 8.
 
Two postablation surfaces imaged with a Fourier Gaussian high-spatial-frequency filter and with the sphere term removed (closest spherical fit).
Surface roughness of the bare stroma increased significantly from 0.65 ± 0.06 to 1.75 ± 0.32 μm after ablation (Fig. 9). 
Figure 9.
 
Average surface roughness before and after excimer ablation of three samples.
Figure 9.
 
Average surface roughness before and after excimer ablation of three samples.
The temperature of the porcine stromal surface was measured during uniform ablation with the excimer laser. Again, the laser was used at 160 mJ/cm2, 10 Hz, 513 pulses, and a uniform 6-mm-diameter ablation. The maximum central corneal temperature elevated 5.0°C after 20 seconds of ablation. The temperature remained at these maximums during the remainder of the 37-second ablation time, with a pulsed variation (Figs. 10, 11). 
Figure 10.
 
Infrared camera, thermal maps (degrees) of the excimer ablated surface.
Figure 10.
 
Infrared camera, thermal maps (degrees) of the excimer ablated surface.
Figure 11.
 
Maximum temperature increase during excimer ablation. Note that it takes approximately 20 seconds to reach a steady state of an approximately 5°C increase from a baseline of 29°C.
Figure 11.
 
Maximum temperature increase during excimer ablation. Note that it takes approximately 20 seconds to reach a steady state of an approximately 5°C increase from a baseline of 29°C.
The 11 eyes all showed contraction of the stromal surface to various degrees after ablation. When the samples were evaluated together, the central 2 mm of the stromal surface contracted by 2.21% ± 0.80% with excimer ablation. Data show considerable scatter, as would be expected with biological tissue and complex procedures and measurement techniques. Eighteen eyes were also remeasured without ablation to examine variation in measurement technique. The nonablated, remeasured eyes indicated a change of −0.045% ± 0.235% (Fig. 12). 
Figure 12.
 
Percentage surface contraction with uniform excimer laser ablation of the stromal corneal surface and a constant maximum temperature increase of 5°C over 57 seconds.
Figure 12.
 
Percentage surface contraction with uniform excimer laser ablation of the stromal corneal surface and a constant maximum temperature increase of 5°C over 57 seconds.
Transmission electron microscopy was completed on the postablation corneal stroma to examine morphologic changes in the residual stroma. Photomicrographs of the ablated stromal surface indicate a 1- to 2-μm region of abnormal (undulating) collagen fibrils with underlying normal straight-layered fibril appearance. In addition, remnants of an electron-dense coagulation layer are seen on the surface (Fig. 13). 
Figure 13.
 
Transmission electron microscopy of the ablated stromal surface. Note a 2-μm depth region of abnormal “undulating” stromal fibrils and remnants of a “coagulation” layer on the surface (arrows).
Figure 13.
 
Transmission electron microscopy of the ablated stromal surface. Note a 2-μm depth region of abnormal “undulating” stromal fibrils and remnants of a “coagulation” layer on the surface (arrows).
Uniform excimer ablation of optical-quality, 8-mm radius, plastic PMMA spheres produced no significant changes in asphericity, surface roughness, or transverse contraction. The spheres were ablated as in the porcine corneas. Shown in Figures 14 and 15 are the raw profile of the PMMA sphere comparing nonablated, 265-pulse ablation, and 513-pulse ablation spheres. There is some suggestion of central flattening with increased uniform ablation. 
Figure 14.
 
PMMA sphere comparing profiles (micrometer elevation versus millimeters from the apex) of the nonablated sphere with a uniform excimer ablation of 265 or 513 pulses.
Figure 14.
 
PMMA sphere comparing profiles (micrometer elevation versus millimeters from the apex) of the nonablated sphere with a uniform excimer ablation of 265 or 513 pulses.
Figure 15.
 
PMMA sphere comparing closest spherical fit (micrometers deviation) of the nonablated sphere with a uniform excimer ablation of 265 or 513 pulses.
Figure 15.
 
PMMA sphere comparing closest spherical fit (micrometers deviation) of the nonablated sphere with a uniform excimer ablation of 265 or 513 pulses.
The uniformly ablated stromal surface was examined for changes in asphericity and transverse contraction, with moderate desiccation and elevation in intraocular pressure. The surface was not significantly affected by moderate desiccation (30 minutes, 29°C, and 30% relative humidity). Figures 16 and 17 are desiccation time graphs examining surface contraction and changes in profile. 
Figure 16.
 
Moderate desiccation over 30 minutes at 25°C and 30% relative humidity. Note variations in measurement without significance change in the surface.
Figure 16.
 
Moderate desiccation over 30 minutes at 25°C and 30% relative humidity. Note variations in measurement without significance change in the surface.
Figure 17.
 
Corneal stromal surface changes in profile (micrometers elevation versus millimeters from the apex) with desiccation up to 90 minutes at 25°C and 30% relative humidity. There is some suggestion of central flattening.
Figure 17.
 
Corneal stromal surface changes in profile (micrometers elevation versus millimeters from the apex) with desiccation up to 90 minutes at 25°C and 30% relative humidity. There is some suggestion of central flattening.
The uniformly ablated surface was examined for changes in profile and surface contraction (or expansion) with elevation in intraocular pressure (16–40 mm Hg). A corneal thickness of 1029 ± 58 μm was measured before ablation with a pachymeter. One corneal thickness was measured after ablation with the 180-μm-thick LASIK flap in place and found to be 891 μm. This thickness would indicate an ablation depth of 138 μm with at least 711 μm of residual tissue. Significant changes in profile or expansion would indicate a mechanical weakness of the central cornea as a partial cause for the corneal surface changes. No significant changes in profile or expansion were noted with elevation in intraocular pressure to 40 mm Hg (Figs. 18, 19). 
Figure 18.
 
Surface profile changes measuring the surface elevation (micrometers) versus distance from the apex at various intraocular pressures.
Figure 18.
 
Surface profile changes measuring the surface elevation (micrometers) versus distance from the apex at various intraocular pressures.
Figure 19.
 
Surface expansion changes measuring the distance between the postablation stromal incision marks with elevated intraocular pressure. They appear relatively constant.
Figure 19.
 
Surface expansion changes measuring the distance between the postablation stromal incision marks with elevated intraocular pressure. They appear relatively constant.
Discussion
Uniform excimer laser ablation interacts predictably and characteristically with the nonablated residual stromal surface. Many researchers have noted the changes in asphericity clinically, even with asphericity- and central island–correcting algorithms. 26,45 48 The use of Q values to quantify asphericity was helpful in this study, but cannot be directly compared to the clinical studies, as the ablation was completed on enucleated porcine eyes in a uniform manner without aspherical correction algorithms and measured the central 2-mm of the optic zone. Noack et al. 12 have also quantified the large changes in central apparent underablation with a uniform excimer ablation, and the results presented here agree with those findings. The changes in asphericity are not seen with isotropic, homogeneous, plastic materials. The increase seen only with corneal tissue supports the theories of preferential central corneal edema, of transverse energy on the corneal surface, and of asphericity as a function of distance from the edge of the ablation. 1,2,5,10,11,13 Also, increases in surface roughness and surface contraction are supportive of this interaction, but are likely to exclude corneal edema as an etiology. The uniformly ablated cornea increases its central curvature with a relative paracentral flattening out to a 3-mm diameter in the optic zone and has been demonstrated clinically with increased asphericity and central island formation. These clinical alterations have been predicted and treated empirically with all commercially available lasers by central overablation. 
Previous research has noted the increased roughness as a possible reason for subepithelial haze. 29 Increased apparent surface roughness after excimer ablation is observed clinically by LASIK surgeons, immediately after ablation and appears to be uniform across the stromal surface. Our raw profile data confirm these observations, which are suggestive of a uniform interaction between the excimer laser and the residual stroma. It is plausible that the increased corneal hydration seen in cadaveric porcine eyes could have an effect on the level of increased roughness on the stromal surface. Our study simply measured the change in surface roughness from before to after ablation. All measurements and ablation occur within a 1-hour period. Although increased hydration could occur during that period, we did not see changes in roughness in samples over an equal time period in which there was no ablation. Central corneal thickness in live pigs has been measured to be considerably less than in cadaveric samples. 49 The amplitudes of deformations after excimer ablation to the stromal surface appear to be magnified twofold in cadaveric eyes when compared to live human eyes. 13  
Histologically, undulations in the collagen sheets progressively increase from the normal straight horizontal pattern between 50° and 60°C. 50 Our transmission micrographs of the excimer-ablated surface also show these undulations to 2 μm below the ablated surface with a normal appearance to the underlying stroma. The postablation micrographs also show remnants of the thin, electron-dense coagulation layer reported previously. 51 The coagulation layer is consistent with gelatinization reported at temperatures above 70°C. 50 This finding is further evidence of the possibility of higher temperatures, albeit brief, that are able to denature and possibly contract the surface collagen fibrils. 
Contraction of the corneal stromal surface after excimer ablation has not been shown before this study. However, elevated temperatures of the corneal surface have been demonstrated with excimer ablation, and contraction of corneal stroma has been demonstrated at those temperatures. Researchers have seen temperature elevations with scanning lasers of 6°C to 8°C with a fluence of 118 mJ/cm2 at 30 Hz and between 6°C and 12°C with 81 mJ/cm2 at 30 Hz. 52,53 These researchers looked at temperatures averaged over long time constants. However, Ishihara et al., 21 examined the temperature elevation immediately after commonly used fluence excimer pulses in the microsecond domain and measured temperature elevations of 112°C. The stromal architecture is modified at several temperatures beginning at 45°C with high thermal damage noted at 62°. Linear corneal shrinkage was noted up to 2% at temperatures from 40°C to 60°C and from 2% to 25% between 60°C and 70°C. 50 In addition, this study showed that temperature induces histologic changes with associated contraction in collagen fibrils consistent with the changes seen in the top 2 μm of our corneal samples. 50 The linear corneal collagen shrinkage our study was approximately 2%, which correlates well with that seen at temperatures between 40°C and 60°C. However, our maximum steady state temperature was 35°C. With our infrared camera, it was not possible to measure the brief temperature increase after the pulse caused by the relatively long sensor integration time. This transient temperature increase probably exceeds 100°C. 21 Our histologic findings are consistent with higher temperatures. It is possible that the very rapid thermal-relaxing high temperatures may effectively add several degrees to the surface and is able to conduct this heat 1 to 2 μm into the stromal surface creating the surface shrinkage. 
No significant change in profile or asphericity was seen with increased intraocular pressure or surface desiccation. In other research, porcine corneas within the physiologic range of intraocular pressures (12–25 mm Hg) have shown negligible changes in corneal curvature or surface distention. 54 Even with a uniform excimer ablation and 70% of the corneal thickness remaining, we have seen no significant changes in curvature or surface expansion, which agrees with the prior study. These findings provide evidence that the asphericity induced with excimer ablation is not related to a mechanical weakening of the central cornea. If the central cornea were weakened and became ectopically displaced, the deformation should increase with elevated intraocular pressure as it does with radial keratotomy. 
Desiccation was examined as a possible factor that alters the surface profile or induces contraction. It has been shown that desiccation will change the amount of ablation per pulse. As we note here, it had no significant effect on asphericity or transverse contraction after excimer ablation. 
The porcine cornea is quite robust compared with the human cornea with a significantly increased corneal thickness of 1000 versus 550 μm, as well as increased tensile strength and modulus of elasticity. 55 Central corneal thickness in live pigs has been measured to be considerably less than in cadaveric samples. 49 The amplitudes of deformations after excimer ablation of the stromal surface appear to be magnified twofold in cadaveric eyes when compared with those in live human eyes. 13  
It could be postulated that the results reported herein would be attenuated in live human corneas. 
Optical interferometric profilometry is a direct measurement technique that does not rely on the rate of change in surface curvature and can also image discontinuities of the surface. It can provide high-resolution (submicrometer) surface elevation and lateral imaging of the exposed central corneal stroma within 0.5 mm of the corneal apex, which is not possible with most other topographic measurement techniques. The method is currently not useful clinically because of the time necessary for measurement. At least two other researchers have used this technique indirectly in the laboratory. 12,50  
One plausible theory is that corneal asphericity is a consequence of the stress redistribution in the stromal layer as a result of the thermal load produced by the excimer laser. The thermal load induces disruption of the integrity of the corneal collagen fibrils at the stromal surface and a subsequent deformation of the surface consistent with the new stress–strain equilibrium represented by a central peak and paracentral flattening. The cornea can be regarded as a thick membrane, with little resistance to shear and as a thick-walled pressure vessel where internal swelling (hydrostatic) stresses maintain equilibrium with the collagen fibril network. Modeled in this manner, it would conceivably require little contraction of the surface fibrils to create these deformations. 
Alternatively, these findings are not inconsistent with the previously presented theory that transverse energy induced by the excimer laser alters the ablation threshold and may certainly be a part of this mechanism. 1 It is possible that altered collagen fibrils and the surface contraction change the ablation threshold. A stress redistribution or altered ablation threshold theory is also consistent with prior theories relating the aspherical effect to the distance from the edge of the ablation, and it is likely that the same observations would have been seen had we varied the ablation diameter. 5,13  
A theory based on stress redistribution will be further evaluated by examining the isolated thermal response without ablation or a pulsed laser. A CO2 laser radiant energy source will be used to measure the cornea's response to an isolated thermal source. In addition, a mathematical model would be helpful. A corneal finite element model is being developed, which may confirm a contraction mechanism producing the aspherical changes. This phenomenon and its associated etiology may be of increased importance when using excimer lasers with increased thermal load, which has been demonstrated in newer, high-pulse-frequency, high-fluence lasers. 
Footnotes
 Disclosure: S.J. McCafferty, None; J.T. Schwiegerling, None; E.T. Enikov, None
The authors thank Charles Munnerlyn, PhD (Veeco Corporation), Eustace Dereniak, PhD, and Thomas Milster, PhD (OSC Remote Sensing Laboratory), and the Maskless Lithography Laboratory for lending their expertise and allowing the use of equipment; Tony Day, PhD, at the AHSC Imaging Facility for electron microscopy assistance; and Hodges Eye Care for use of the laser. 
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Figure 1.
 
Globe stabilization chamber with infusion.
Figure 1.
 
Globe stabilization chamber with infusion.
Figure 2.
 
Photomicrograph of postablation stromal corneal incision with diamond blade set at a 200-μm depth. Note that the apex as well as the medial and lateral edges of the V-shaped incision are visible.
Figure 2.
 
Photomicrograph of postablation stromal corneal incision with diamond blade set at a 200-μm depth. Note that the apex as well as the medial and lateral edges of the V-shaped incision are visible.
Figure 3.
 
Two samples of the superimposed images of pared stromal incisions before (blue) and after (red) treatment with uniform excimer laser ablation. The inner edge of incision is marked for clarity. Odd-numbered measurements are before treatment, and even ones are after treatment.
Figure 3.
 
Two samples of the superimposed images of pared stromal incisions before (blue) and after (red) treatment with uniform excimer laser ablation. The inner edge of incision is marked for clarity. Odd-numbered measurements are before treatment, and even ones are after treatment.
Figure 4.
 
Two corneal stromal profiles showing before and after uniform excimer laser ablation. The x-axis is in millimeters from the corneal apex. The y-axis is elevation in micrometers. Note that all profiles show a central steepening and peripheral flattening. The graphs were superimposed by setting the average of each profile to 0.
Figure 4.
 
Two corneal stromal profiles showing before and after uniform excimer laser ablation. The x-axis is in millimeters from the corneal apex. The y-axis is elevation in micrometers. Note that all profiles show a central steepening and peripheral flattening. The graphs were superimposed by setting the average of each profile to 0.
Figure 5.
 
Corneal stromal profile showing deviation in micrometers (y-axis) from the closest spherical fit, both before and after uniform excimer ablation. (All samples had similar results.) The aspheric profile was greatly magnified after the uniform excimer ablation.
Figure 5.
 
Corneal stromal profile showing deviation in micrometers (y-axis) from the closest spherical fit, both before and after uniform excimer ablation. (All samples had similar results.) The aspheric profile was greatly magnified after the uniform excimer ablation.
Figure 6.
 
Q values show a large, statistically significant flattening of the peripheral cornea (2-mm diameter) compared with the central cornea (1-mm diameter) after uniform excimer ablation. Peripheral flattening shows hyperboloid deformation (as opposed to paraboloid) with a negative Q value.
Figure 6.
 
Q values show a large, statistically significant flattening of the peripheral cornea (2-mm diameter) compared with the central cornea (1-mm diameter) after uniform excimer ablation. Peripheral flattening shows hyperboloid deformation (as opposed to paraboloid) with a negative Q value.
Figure 7.
 
Imaged surface of the corneal stroma (3 mm diameter) before and after uniform excimer ablation 531 pulses at 160 mJ/cm2. Increased roughness of the surface is evident in the postablation samples.
Figure 7.
 
Imaged surface of the corneal stroma (3 mm diameter) before and after uniform excimer ablation 531 pulses at 160 mJ/cm2. Increased roughness of the surface is evident in the postablation samples.
Figure 8.
 
Two postablation surfaces imaged with a Fourier Gaussian high-spatial-frequency filter and with the sphere term removed (closest spherical fit).
Figure 8.
 
Two postablation surfaces imaged with a Fourier Gaussian high-spatial-frequency filter and with the sphere term removed (closest spherical fit).
Figure 9.
 
Average surface roughness before and after excimer ablation of three samples.
Figure 9.
 
Average surface roughness before and after excimer ablation of three samples.
Figure 10.
 
Infrared camera, thermal maps (degrees) of the excimer ablated surface.
Figure 10.
 
Infrared camera, thermal maps (degrees) of the excimer ablated surface.
Figure 11.
 
Maximum temperature increase during excimer ablation. Note that it takes approximately 20 seconds to reach a steady state of an approximately 5°C increase from a baseline of 29°C.
Figure 11.
 
Maximum temperature increase during excimer ablation. Note that it takes approximately 20 seconds to reach a steady state of an approximately 5°C increase from a baseline of 29°C.
Figure 12.
 
Percentage surface contraction with uniform excimer laser ablation of the stromal corneal surface and a constant maximum temperature increase of 5°C over 57 seconds.
Figure 12.
 
Percentage surface contraction with uniform excimer laser ablation of the stromal corneal surface and a constant maximum temperature increase of 5°C over 57 seconds.
Figure 13.
 
Transmission electron microscopy of the ablated stromal surface. Note a 2-μm depth region of abnormal “undulating” stromal fibrils and remnants of a “coagulation” layer on the surface (arrows).
Figure 13.
 
Transmission electron microscopy of the ablated stromal surface. Note a 2-μm depth region of abnormal “undulating” stromal fibrils and remnants of a “coagulation” layer on the surface (arrows).
Figure 14.
 
PMMA sphere comparing profiles (micrometer elevation versus millimeters from the apex) of the nonablated sphere with a uniform excimer ablation of 265 or 513 pulses.
Figure 14.
 
PMMA sphere comparing profiles (micrometer elevation versus millimeters from the apex) of the nonablated sphere with a uniform excimer ablation of 265 or 513 pulses.
Figure 15.
 
PMMA sphere comparing closest spherical fit (micrometers deviation) of the nonablated sphere with a uniform excimer ablation of 265 or 513 pulses.
Figure 15.
 
PMMA sphere comparing closest spherical fit (micrometers deviation) of the nonablated sphere with a uniform excimer ablation of 265 or 513 pulses.
Figure 16.
 
Moderate desiccation over 30 minutes at 25°C and 30% relative humidity. Note variations in measurement without significance change in the surface.
Figure 16.
 
Moderate desiccation over 30 minutes at 25°C and 30% relative humidity. Note variations in measurement without significance change in the surface.
Figure 17.
 
Corneal stromal surface changes in profile (micrometers elevation versus millimeters from the apex) with desiccation up to 90 minutes at 25°C and 30% relative humidity. There is some suggestion of central flattening.
Figure 17.
 
Corneal stromal surface changes in profile (micrometers elevation versus millimeters from the apex) with desiccation up to 90 minutes at 25°C and 30% relative humidity. There is some suggestion of central flattening.
Figure 18.
 
Surface profile changes measuring the surface elevation (micrometers) versus distance from the apex at various intraocular pressures.
Figure 18.
 
Surface profile changes measuring the surface elevation (micrometers) versus distance from the apex at various intraocular pressures.
Figure 19.
 
Surface expansion changes measuring the distance between the postablation stromal incision marks with elevated intraocular pressure. They appear relatively constant.
Figure 19.
 
Surface expansion changes measuring the distance between the postablation stromal incision marks with elevated intraocular pressure. They appear relatively constant.
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