Structural changes in alpha-crystallin and whole eye lens during heating, observed by low-angle X-ray diffraction.

Whole eye lens and alpha-crystallin gels and solutions were investigated using X-ray scattering techniques at temperatures ranging from 20 degrees C to 70 degrees C. In whole lens isolated in phosphate-buffered saline, the spacing of the dominant X-ray reflection seen with low-angle scattering was constant from 20 degrees C to 45 degrees C but increased at 50 degrees C from 15.2 nm to 16.5 nm. At room temperature, the small-angle X-ray diffraction pattern of the intact lens was very similar to the pattern of alpha-crystallin gels at near-physiological concentration (approximately 300 mg/ml), so it is reasonable to assume that the alpha-crystallin pattern dominates the pattern of the intact lens. Our results therefore indicate that in whole lens alpha-crystallin is capable of maintaining its structural properties over a wide range of temperature. This property would be useful in providing protection for other lens proteins super-aggregating. In the alpha-crystallin gels, a moderate increase in both the spacing and intensity of the reflection was observed from 20 degrees C to 45 degrees C, followed by an accelerated increase from 45 degrees C to 70 degrees C. Upon cooling, this effect was found to be irreversible over 11 hours. Qualitatively similar results were observed for alpha-crystallin solutions at a variety of lower concentrations.

Viscosity of alpha-crystallin solutions.

Accommodation in the mammalian lens requires flexure of lens fibres and some redistribution of their contents involving limited viscous flow. Shear-dependent viscosity of bovine alpha-crystallin solutions was determined with the Contraves Low-Shear Rheometer between 4.4 and 347 mg ml(-1), and at 15.5, 25, 30, and 37 degrees C. All solutions showed significant shear thinning, with markedly higher viscosity at physiological levels of approximately 300 mg ml(-1). Viscosity-concentration graphs were similar at low (1.0 s(-1)) and high (94.5 s(-1)) shear rates, indicating low molecular interaction in solution. Arrhenius plots which might have indicated the size of the energy barrier to displacement of molecules or aggregates were inconclusive.

Heat-induced quaternary transitions in hetero- and homo-polymers of alpha-crystallin.

PURPOSE: To compare the effects of heat incubation on the structure and function of native alpha-crystallin, urea denatured/renatured alpha-crystallin, and alphaA and alphaB-crystallin homo-polymers purified from bovine lenses. METHODS: Each of the alpha-crystallin samples were incubated for 1 h at temperatures ranging from 35 degrees C to 70 degrees C. After heat incubation structural perturbations in each of the samples were studied using non-denaturing gel electrophoresis, transmission electron microscopy (TEM) and far-UV circular dichroism. The chaperone-like activity of each of the heat-treated samples was measured using the DTT induced insulin aggregation assay. RESULTS: The native alpha-crystallin samples showed secondary structure perturbations, an increase in aggregate size and asymmetry, and an increase in chaperone-like activity after heat incubation above 50 degrees C. The other three sample types showed secondary structure perturbations beginning at lower incubation temperatures, and a progressive decrease in chaperone-like activity with exposure to increasing temperatures. TEM showed all samples formed large asymmetric high molecular weight aggregates after incubation at 65 degrees C. CONCLUSIONS: The urea denaturation/renaturation of alpha-crystallin has been shown to result in the loss of a small amount of alpha-helix, but to have no effect on chaperone-like activity under standard test conditions. The present results indicate this lost alpha-helix may be responsible for the differential effects of heat incubation on the different forms of alpha-crystallin.

Aging of the optics of the human eye: lens refraction models and principal plane locations.

Biometric data describing the geometry and spacings of emmetropic human eyes were combined with lens shape and placement within the globe to generate paraxial models of image formation as a function of age. Three different representations of the shape of the internal refractive index gradient of the lens were evaluated--a Gullstrand-type model consisting of cortical and nuclear regions with different refractive indices, a power series model, and a linear-gradient model. All three refractive models satisfy the requirements for focus for all the data sets, indicating that lenticular refractive index gradient shape is essentially underdetermined in the paraxial limit. Lens refractive power decreases by almost 2 D during a 50-year period, and the concomitant decrease in system refractive power is due almost entirely to this effect. The reduction in spacing between the lens principal planes is a function of this, as is their anterior movement with age, and suggests that the compensatory processes maintaining far focus at the expense of near are not exactly balanced. Despite these changes in the lens contribution and their effect on the location of the system principal planes, which also move anteriorly, the spacing between the system principal planes remains constant. However, the trend toward reduced overall system power with age indicates the primary role of the lens in mediating image formation onto the retina over time.

Aging of the human lens: changes in lens shape at zero-diopter accommodation.

Scheimpflug photographs of the zero-diopter-accommodated anterior segments of 100 human subjects, aged 18 to 70 yr and evenly spaced over this range, were digitized and analyzed to characterize lens and lens nucleus shape as a function of age by the Hough transform and other image analysis methods. Anterior and posterior lens surface curves exhibit a decrease in radius of curvature with increasing age, in qualitative but not quantitative agreement with the earlier observations of Brown [Exp. Eye Res. 19, 175 (1974)]. In contrast, the shape of the lens nuclear boundaries changes little with age. Overall lens volume at zero diopters increases with age, but the volume of the lens nucleus remains unchanged. The lens center of mass moves anteriorly with increasing age, as does the central clear region of the lens. Although these data sets were found to be more variable than those of Brown, the complementary variability of other factors, such as anterior chamber depth, for each subject leads to a very high statistical correlation between lens shape and lens location relative to the cornea. This supports the finding of previous work that image formation on the retina for a given individual results from the multifactorial balancing of related factors.

Correlation between the chaperone-like activity and aggregate size of alpha-crystallin with increasing temperature.

alpha-Crystallin, the major protein of the mammalian eye lens, is also found in the major tissues of the body, where one or the other of its two isoforms is characteristically expressed. Both isoform sequences are highly related to others of the small heat shock protein superfamily, leading to speculation about their functions in vivo outside of the lens. Tests of chaperone-like activity at 37 and 66 degrees C indicate that the protein can act to prevent the superaggregation of partially denatured proteins, but both alpha-crystallin aggregate size and shape are significantly altered with increasing temperature. Characterization of these changes indicates that secondary, tertiary, and quaternary structure are modified, with the latter effect especially striking above 50 degrees C. Furthermore, these changes appear to be irreversible when the temperature is returned to 25 or 37 degrees C. Functionally, the protein is effective in chaperone-like activity at all temperatures, but exhibits a somewhat increased capability after a cycle of heating and cooling. The results presented here indicate the heat-induced formation of high-molecular-weight aggregates of alpha-crystallin is a slow progressive process. The increased activity of these aggregates suggests that chaperone-like activity depends in part on the packing parameters of the aggregate and on conformation of the subunit within that aggregate.

The development and maintenance of emmetropia.

The human eye is programmed to achieve emmetropia in youth and to maintain emmetropia with advancing years. This is despite the changes in all eye dimensions during the period of growth and the continuing growth of the lens throughout life. The process of emmetropisation in the child's eye is indicated by a shift from the Gaussian distribution of refractive errors around a hypermetropic mean value at birth to the non-Gaussian leptokurtosis around an emmetropic mean value in the adult. Emmetropisation is the result of both passive and active processes. The passive process is that of proportional enlargement of the eye in the child. The proportional enlargement of the eye reduces the power of the dioptric system in proportion to the increasing axial length. The power of the cornea is reduced by lengthening of the radius of curvature. The power of the lens is reduced by lengthening radii of curvature and the effectivity of the lens is reduced by deepening of the anterior chamber. Ametropia results when these changes are not proportional. The active mechanism involves the feedback of image focus information from the retina and consequent adjustment of the axial length. Defective image formation interferes with this feedback and ametropia then results. Heredity determines the tendency to certain globe proportions and environment plays a part in influencing the action of active emmetropisation. The maintenance of emmetropia in the adult in spite of continuing lens growth with increasing lens thickness and increasing lens curvature, which is known as the lens paradox, is due to the refractive index changes balancing the effect of the increased curvature. These changes may be due to the differences between nucleus and cortex or to gradient changes within the cortex.

Environmental factors influencing the chaperone-like activity of alpha-crystallin.

The effects of mild environmental changes (e.g. the addition of divalent cations or EDTA, as well as variations of buffer pH) on the heat stability and chaperone-like activity of native alpha-crystallin, and denatured-renatured alpha-crystallin in the native molar isoform ratio, have been investigated using circular dichroism (CD) spectropolarimetry and functional assays. The presence or absence of divalent cations has little or no effect on the secondary structure of renatured samples, although chaperone-like activity levels can vary widely; the only relevant spectral difference observed is a loss of some alpha-helical content in all the renatured samples relative to the native protein, but this change has no impact on function. The range of concentration over which the inhibitory Mg2+ effect is observed is 10-fold higher for dialyzed fresh protein than for protein renatured into buffers containing Mg2+, but for both sets of samples, the full effect is established below physiological Mg2+ concentrations. Renaturing into various pH buffers, in contrast, affects both heat stability and chaperone-like activity below pH 7.0, with essentially no functionality observed at pH 6.0. CD spectra of these samples indicate that acidic conditions lead to some degree of unfolding, and that this unfolding correlates directly with functionality. Similar results are obtained for fresh protein dialyzed against these pH levels. Overall, these results suggest that heat stability is a function of the protein's secondary structure and folding state, while chaperone-like activity is primarily a function of factors at the tertiary and quaternary levels of organization.

Methods to obtain quantitative parametric descriptions of the optical surfaces of the human crystalline lens from Scheimpflug slit-lamp images. I. Image processing methods.

Of the methods developed (e.g., phakometry, magnetic resonance imaging, etc.) for noninvasive measurement of the geometry of the anterior segment of the human eye, Scheimpflug photography offers the best resolution and the highest precision. The primary obstacle encountered with this or any other image-based method has been in obtaining quantitative measurements directly from the images. Image enhancement (gray-scale gradient analysis) and pattern recognition methods (Hough transform and recursive least-squares algorithms) are developed so that parametric representations of lens surfaces and zone boundaries can be obtained directly from the images. Methods to correct for nonlinear Scheimpflug camera reproduction ratios and provide error estimates for geometrical parameters are also developed and will be presented separately. Combined, these techniques yield representations of lens geometry having sufficient precision, to which paraxial ray tracing can be applied to determine lens optical properties by using well-posed optical models with one unknown.

Investigation of the 'fines' hypothesis of primary open-angle glaucoma: the possible role of alpha-crystallin.

Primary open-angle glaucoma is a disease caused by an increase in intraocular pressure due to a decreased facility of outflow of aqueous humor through the trabecular meshwork. The etiology of primary open-angle glaucoma is currently unknown, but it has been suggested that one possible mechanism may be the obstruction of flow through the trabecular meshwork by small macromolecules, analogous to the effect of 'fines' in column chromatography. One such candidate is alpha-crystallin, a lens protein which may be released into the aqueous humor from lens fiber cells at concentrations below that necessary for the formation of the native aggregate. Results of in vitro binding experiments indicate that alpha-crystallin and serum albumin, which is secreted at the anterior root of the iris and is believed to act as a protein escort through the trabecular meshwork in mammalian eyes, will interact at concentrations of alpha-crystallin up to the critical micelle concentration for alpha-crystallin (3.5-5 mg/ml, or 0.18-0.25 mM). There is little interaction at or above this concentration. This binding could serve the necessary function of preventing interactions between alpha-crystallin monomers or small aggregates and hydrophobic surfaces within the trabecular meshwork. Since, however, the interaction between the two proteins is not extremely strong, the accumulation of unbound alpha-crystallin monomers and/or dimers could contribute to the development of primary open-angle glaucoma.

Studies of the denaturation patterns of bovine alpha-crystallin using an ionic denaturant, guanidine hydrochloride and a non-ionic denaturant, urea.

The effects of non-ionic and ionic denaturation and denaturation/renaturation on the native structure of alpha-crystallin at room temperature were examined. Native alpha-crystallin, at concentrations above and below the previously reported critical micelle concentration (CMC) range, was denatured by varying concentrations of urea and guanidine hydrochloride. The resulting denatured samples were examined by gel filtration fast performance liquid chromatography (FPLC), circular dichroism spectropolarimetry (CD), and transmission electron microscopy. Elution peak samples from gel filtration chromatography with sufficiently high concentrations were examined for subunit composition by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The studies presented herein demonstrate that the denaturation and renaturation of alpha-crystallin via non-ionic urea denaturation results in different renaturation species, depending upon the initial concentration of alpha-crystallin which is denatured and the concentration of urea, including certain species which, by gel filtration FPLC, have an apparent molecular weight greater than the native 800 kD aggregate. Transmission electron microscopy has also demonstrated the existence of a high molecular weight aggregate form for denatured samples. Ionic dissociation, in contrast, proceeds much in the same manner above and below the CMC range, the major difference occurring at 2 M guanidine hydrochloride. alpha B-crystallin is preferentially removed from the native alpha-crystallin aggregate upon treatment with 2 M guanidine hydrochloride indicating, once again, differences between the two subunits. Above and below the CMC range, dissociation with guanidine hydrochloride appears to plateau after 4 M guanidine hydrochloride as indicated by the presence of two apparent homotetrameric species and no further dissociation of these species with increasing guanidine hydrochloride concentrations. CD demonstrates that some secondary structure, which is lost with lower concentrations of alpha-crystallin, is still present when concentrations of alpha-crystallin, well above the critical micelle concentration range, are treated with high concentrations of urea at room temperature. In contrast, concentrations both above and below the CMC range demonstrate a significant loss of secondary structure upon treatment with 2 M guanidine hydrochloride. Finally, ionic denaturation and subsequent renaturation results in the formation of a species which is functionally incapable of protecting gamma-crystallin from heat-induced aggregation.

Preliminary studies on the aggregation process of alpha-crystallin.

The mechanism by which alpha-crystallin subunits form the native 800 kD aggregate is currently unknown. Experiments were performed to investigate the mechanism for this process. Gel-filtration Fast Performance Liquid Chromatography (FPLC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), with and without cross-linking with glutaraldehyde, indicate that alpha-crystallin undergoes a concentration-dependent aggregation process. The denaturation of alpha-crystallin, and its subsequent renaturation and reaggregation, lead to the formation of several different species. At very low concentrations (< 0.5 microM), only monomeric and/or dimeric species exist. With a ten-fold increase in alpha-crystallin concentration from 0.05 microM to 0.5 microM, the amount of the monomeric/dimeric species increases to a plateau coincident with the appearance of a tetrameric species at 0.5 microM. With an additional ten-fold increase in concentration from 0.5 microM to 5 microM, the amount of the tetrameric species increases and levels off to its own plateau coincident with the appearance of the native 800 kD alpha-crystallin aggregate at 5 microM. The amount of the native species is extremely small at this concentration, but increases sharply and linearly with increasing concentration, while the concentrations of monomeric/dimeric and tetrameric species remain constant. The concentration at which the relative amount of the native species begins to increase sharply is within the range of the critical micelle concentration previously characterized.

Accommodation and presbyopia in the human eye. Changes in the anterior segment and crystalline lens with focus.

PURPOSE: To characterize changes in the sagittal dimensions of the human crystalline lens and anterior segment as a function of accommodation, to determine the potential age dependence of these changes, and to evaluate these changes in relation to the development of presbyopia. METHODS: Scheimpflug slit-lamp photography, as well as a variety of standard ophthalmologic methods, was used to collect information about lens and anterior segment sagital dimensions in a population of 82 adults with refractive error < or = magnitude of 2.0 diopters and at least 0.25 diopter of accommodation for subjects 18 to 70 years of age. Data were analyzed statistically for dependence on accommodation, age, and age dependence of accommodative rate. RESULTS: The rate of change per diopter of accommodation for each measured variable within the lens is independent of age for the entire adult age range. With increasing accommodation, the lens becomes thicker and the anterior chamber shallower along the polar axis. This increase in sagittal lens thickness is entirely because of an increase in the thickness of the lens nucleus. Because the anterior and posterior halves of the nucleus increase in thickness at approximately the same rate with accommodation, the increase in lens thickness results from equal changes in the lengths of the anterior and posterior portions. CONCLUSIONS: Because changes along the sagittal axis of the anterior segment with accommodation are independent of age, any explanation of presbyopia that relies on simple changes in the rates of lens thickening and anterior chamber shallowing with age does not hold. In light of other age-related changes in the anterior segment and lens (e.g., increased sharpness of lens curvature, increased lens sagittal thickness, decreased anterior chamber depth), it appears that compensatory mechanisms to preserve far vision with age also preserve the rate of change per diopter of sagittal spacings.

Analysis of the factors involved in the loss and restoration of the chaperone-like function of alpha-crystallin.

alpha-crystallin, the major protein component of the crystallin lens of mammalian eyes, is found in vivo as two separate gene products. Both isoforms are expressed in different major tissues of the body, with the lens the only location where both are found together. Both sequences can be phosphorylated, though at different locations. Both exhibit a high sequence homology to the small heat shock proteins, and it has been shown that alpha-crystallin also resists heat-induced denaturation. Horwitz [J. Horowitz (1992) Proc. Natl. Acad. Sci. USA 89, 10449-10453] demonstrated that alpha-crystallin can exhibit chaperone-like protection against heat-induced turbidity increases, and it has been suggested that this may be an in vivo function as well. However, neither isoform, when purified, shows the same overall level of chaperone-like activity as the native species, except for one phosphorylated species [M. A. M. van Boekel, S. E. A. Hoogakker, J. J. Harding, and W. W. de Jong (1996) Ophthalmic Res. 28(Suppl. 1), 32-38]. Experiments designed to determine the factors leading to loss of chaperone-like activity indicate that strong ionic conditions, such as those used in isoform separation and/or the presence of divalent cations reduce the efficiency of this function and that the presence of EDTA fully restores it irrespective of prior treatment or buffer conditions. Heat stability is essentially preserved under all conditions. These results suggest that alpha-crystallin may serve primarily as a heat shock protein in vivo and that the chaperone-like function may be inhibited under physiological conditions.

Physiological strategies for emmetropia.

PURPOSE: To identify relationships among age-independent ocular biometric variables which contribute to ocular refraction in adult human eyes, and to identify differences in those relationships between emmetropes and ametropes. METHODS: Manifest refraction (DRS methodology), corneal refractive power (keratometry), and axial anterior segment (anterior cornea to posterior lens), vitreous cavity (posterior lens to anterior sclera) and total globe (anterior cornea to anterior sclera) length (A-scan ultrasonography) were determined in 185 unaccommodated right eyes of adult humans aged 18 to 70 years. There were 136 emmetropes with absolute refractive error < or = 2.0 diopter, and 49 ametropes (47 myopes, 2 hyperopes) with absolute refractive error of 2.25-11.0 diopters. RESULTS: Refraction decreased significantly with increasing globe and vitreous cavity length in emmetropes and ametropes. Anterior segment length was also significantly negatively correlated with refraction in emmetropes, but not in ametropes. Corneal refractive power was not correlated with refraction in either group, but decreased significantly with increasing globe length in both, more strongly in emmetropes. Globe and vitreous cavity length were significantly positively correlated in both groups, more strongly in ametropes. Anterior segment length increased significantly with increasing globe length in emmetropes but not in ametropes. Anterior segment length decreased significantly with increasing vitreous cavity length in ametropes but not in emmetropes. CONCLUSION: These findings indicate an "inflatable anterior segment" as well as the classic "inflatable globe" mechanism of emmetropization. This newly described anterior segment mechanism involving increased separation between the cornea and the lens with increasing globe size appears to be absent in adult human myopia.

Aging of the human crystalline lens and anterior segment.

Changes in the unaccommodated human crystalline lens were characterized as a function of subject age for 100 normal emmetropes over the age range 18-70 yr by Scheimpflug slit-lamp photography. With increasing age, the lens becomes thicker sagittally, but since the distance from the cornea to the posterior lens surface remains unchanged, this indicates that the center of lens mass moves anteriorly and the anterior chamber becomes shallower. Sagittal nuclear thickness is independent of age, but both anterior and posterior cortical thicknesses increase with age, shifting the location of the nucleus and the central sulcus in the anterior direction. The amount of light scattered by the lens at high angles, as represented by normalized and integrated lens densities from the digitized images, increases with increasing age in an exponential fashion. Similar relationships to age are observed for the major anterior zone of discontinuity (maximum density) and the central sulcus (minimum density). The relationships of these results to accommodation and presbyopia are discussed.

The zones of discontinuity in the human lens: development and distribution with age.

Statistical analysis of Scheimpflug images from the crystalline lenses of 100 emmetropic human subjects ranging in age from 18 to 70 yr confirms that specific zones of discontinuity are a function of lens development and growth. At and beyond the age of 40 yr, as many as four sharply demarcated and complementary zones are seen within the anterior and posterior lens cortex. The locations of the inner edges of the anterior cortical zones of discontinuity were characterized relative to the central sulcus of the lens. Consecutively from the central sulcus, the distances were 1.094, 1.415, 1.695, and 1.994 (+/- 0.11 mm). Since nuclear thickness in the adult lens is age-independent and the rate of cortical growth has been characterized, the location of the inner margins of the zones are indicative of the age at which they originated; these ages were 4 (+/- 1 yr), 9 (+/- 2 yr), 19 (+/- 4yr), and 46 (+/- 10 yr). All of the zones become broader along the outer margin and more dense upon aging, with specific zones appearing to merge in older presbyopic lenses. While lens fetal nuclear transparency decreases with age, it does not feature zones of discontinuity; instead, symmetrically amorphous regions appear centrally in the anterior and posterior nucleus. This demonstration of the onset of specific zones of discontinuity in emmetropic individuals, at defined periods of lens growth that are synchronous the production of successively more complex lens sutures, strongly suggests a causal relationship between lens sutures and the zones of discontinuity.

Filamentous aggregates of native titin and binding of C-protein and AMP-deaminase.

In solutions of high ionic strength, native titin-2, a large extractable fragment of the sarcomere matrix protein titin, appears as extremely long, flexible, and slender beaded strings. We report here that in solutions of lower ionic strength near neutral pH, titin-2 assembles into higher-order aggregates with surface projections. Solid phase binding assays show that two myosin-binding proteins, C-protein and AMP-deaminase, are also titin-binding proteins. Both proteins decorate titin aggregates, producing filaments of more uniform appearance. Numerical Fourier transforms of these decorated aggregates show approximately 12-nm periodicities. The interaction of titin with myosin-associated proteins such as C-protein may take part in the anchoring mechanism that prevents the stretching and extension of titin filaments in the A band.

Biophysical characterization of alpha-crystallin aggregates: validation of the micelle hypothesis.

The size of alpha-crystallin aggregates, as well as the structural organization of each particle's subunits, is currently unknown, although a number of different laboratories have suggested both structures and average molecular weights (Thomson, J.A. and Augusteyn, R.C. (1984) Proc. Int. Soc. Eye Res. 3, 152). One hypothesis, compatible with literature reports and consistent with what is known of subunit primary structure and physiological function, is that alpha-crystallin exists in vivo as a naturally occurring protein micelle (Sen, A.C. and Chakrabarti, B. (1991) Biophysical J. 59, 108a.) To test this hypothesis, experiments were performed on this protein to determine its behavior under increased hydrostatic pressure and the effect of its concentration on aqueous surface tension. With increasing hydrostatic pressure, the turbidity of an alpha-crystallin solution increases exponentially to a plateau at about 6000-8000 psi; upon release of pressure, the samples slowly return to their original turbidity level. Other naturally aggregating proteins, such as skeletal muscle myosin, demonstrate a decrease in turbidity under the same conditions. The surface tension of alpha-crystallin in aqueous solution decreases to a plateau with increasing protein subunit concentration, with an inflection point over the range 0.18-0.25 mM; cholate and other amphiphiles exhibit similar behavior. In contrast, plots of surface tension over the equivalent concentration range for other protein aggregates in the same buffer more closely approximate the types of curve obtained with short chain aliphatic acids. These results indicate that alpha-crystallin behaves like the protein version of a micelle.

Acquisition of the curves of the human crystalline lens from slit lamp images: an application of the Hough transform.

To accurately model lens-based functions such as accommodation and image formation on the retina, it is essential to know anterior chamber depth, anterior segment length, lens thickness, and, most importantly, lens curvature both on the surfaces and internally. With the exception of lens curvatures, all these data can be obtained with a high degree of precision by one or more techniques (i.e., A-scan ultrasonography and pachymetry). Lens curvatures can be collected by Scheimpflug slit lamp photography, but the curvature data must be extracted from these images, a problem complicated by both linear and nonlinear image distortion. Previous approaches have involved significant magnification of the image combined with major subjective input and judgment. We present here a computer-based application of the Hough technique for measurement of curvature of lens surfaces observed in Scheimpflug slit lamp photography and related evaluation of (and solutions for) the associated image distortion. Minimal user input is required for successful application of this method, but the time required to obtain a fit for each surface is >1 min. Results obtained by this technique on test images compare favorably with those obtained by independent methods.