|Molecular Vision 2006;
Received 10 March 2006 | Accepted 29 June 2006 | Published 7 July 2006
Biometry of primate lenses during immersion in preservation media
Augusteyn,1,2 Alexandre M. Rosen,3,4 David Borja,3,4
Noel M. Ziebarth,3,4 Jean-Marie
1Vision Cooperative Research Centre, Sydney, Australia; 2Ophthalmology Department, University of Melbourne, Melbourne, Australia; 3Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL; 4Biomedical Optics and Laser Laboratory, Department of Biomedical Engineering, University of Miami, Coral Gables, FL
Correspondence to: Robert C. Augusteyn, PhD, Vision CRC, 30 Melcombe Road, Ivanhoe, Victoria 3079, Australia; Phone: 61 3 9499 1838; email: B.Augusteyn@visioncrc.org
Purpose: The purpose of this study was to assess the condition of human lenses (obtained from an eye bank) and of fresh monkey lenses, and to determine the effects of maintaining these lenses in various liquid preservation media.
Methods: Freshly excised human and monkey lenses were maintained for 5 h in one of four solutions (Balanced Saline Solution [BSS], Ringer's Solution, Dulbecco's Modified Eagle Medium with Ham's F-12 [DMEM/F-12/F-12], and Tissue Culture Medium 199 [TC-199]) using a custom-designed, temperature-regulated testing cell. A modified optical comparator and digital camera were used to photograph magnified lens profiles and measure lens diameter and thickness. Lens volume was then calculated assuming rotational symmetry about the optical axis.
Results: Seven of the 33 human lenses exhibited extensive swelling and separation of the capsule from the lens cell mass prior to the incubation. During incubation, for 12/22 of the remaining human and 27/27 of the monkey lenses, thickness increased by 1.0-1.8%, diameter decreased by 0.7-1.6% and the volume was essentially unchanged. Substantial swelling and capsular separation were observed in 10 of the 22 human lenses, 7/10 for those maintained in salt solutions, and 3/12 for those in tissue culture media. Lens volumes increased by an average of 6.8%, due to an 8.7% increase in the thickness, while the diameter decreased by 0.9%. These changes appeared to be independent of postmortem time and donor age.
Conclusions: Culture media are more effective than simple salt solutions in maintaining lens physical integrity during short-term incubations. Substantial uptake of water, accompanied by separation of the capsule from the lens cell mass, occurs at various stages during storage and experimental manipulations in >60% of human lenses obtained from the eye bank. Data obtained with such lenses will not be representative of the true ex vivo state. It is recommended that lenses be assessed to determine if swelling has taken place before acceptance of data.
In vitro studies on lens properties are generally conducted with the lens placed in liquid media or gels. It is assumed that these will maintain normal hydration and other lens properties, including shape, while measurements are being made. However, this assumption is rarely tested.
Because of the presence of high concentrations of large negatively charged protein molecules inside lens cells, there is a constant influx of positive ions and water. Unchecked, this would rapidly lead to swelling and rupture of the cells. However, the normal lens has comprehensive mechanisms for regulating cell volume, ion movements, and hydration levels. Components of these mechanisms include the semipermeable capsule and cell membranes as well as a number of epithelial ion pumps, which maintain the ionic difference (i.e., the Donnan Equilibrium) between the intra- and extracellular spaces [1,2]. Lens metabolism provides energy, mainly in the form of ATP, for the ion pumps.
Anything that compromises these mechanisms can lead to water retention and swelling of the tissue. In vivo, such changes typically lead to cortical cataract in which water spokes invade the lens and, eventually, the tissue becomes swollen. In vitro, these could alter lens shape and physical properties, apart from having an impact on metabolic processes. Avoiding such changes is becoming increasingly more important with the development of techniques for studying lens mechanics and other properties in enucleated eyes [3-5] or with isolated lenses [6-12].
While previous studies have examined the effects of culture conditions on lens metabolism and volume regulation [2,13-19] little is known about the effects of culture conditions on lens biometric or biophysical properties. Therefore, the present study was undertaken to determine if the nature of the incubation medium could affect ex-vivo lens shape during immersion. Our results indicated that culture media are better for short-term experiments than the salt solutions. Our data also revealed that water uptake and lamellar separation commonly occur in human lenses during storage or experimental manipulation, raising serious questions about the validity of many published studies.
Intact human cadaver eyes were donated by the Florida Lions Eye Bank. Rhesus and cynomolgus monkey eyes were provided by the Department of Veterinary Resources and the Diabetes Research Institute, both at the University of Miami. Only healthy monkey eyes were used. Age, sex, body weight, and postmortem time were recorded, when available. Using an operating microscope, we removed the cornea and iris before extracting the lens by carefully cutting the zonules and adherent vitreous using Vannas scissors. Wire lens spoons (Segal Instruments, Bombay, India), were used to place the lens, usually with the anterior surface up, on the ring holder in a testing cell which had been prefilled with the selected preserving solution. The time from lens extraction to immersion was approximately 6 min. Lens capsule integrity was visually inspected using the comparator. If capsular damage or cataract were observed, the lens was discarded.
Four preserving solutions were used: Balanced Salt Solution (BSS; Alcon Laboratories, Fort Worth, Tx), Standard Ringer's Solution (SRS; Baxter Healthcare Corporation, Deerfield, Il), Dulbecco's Modified Eagle Medium with Ham's F-12 (DMEM/F-12; Mediatech, Herndon, Va), and Tissue Culture Medium 199 (TC-199; Mediatech, Herndon, Va). All were tested to determine the effect of immersion on the shape of 33 human, 11 rhesus monkey, and 16 cynomolgus monkey lenses. According to the manufacturer's data sheets, all 4 solutions had osmolalities around 300 mOsmoles/l (reported range 293-317).
Measurement of lens shape
Lens dimensions were monitored using a modified optical comparator (Topcon BP-30S, Tokyo, Japan) which projects a 20x magnified shadow of objects onto a viewing screen. Detailed descriptions of the apparatus and its use in measuring human lens dimensions have been provided elsewhere .
The experiment began with the lens profiled in the coronal (top) view and a picture was taken with a digital camera (4.0 Mp Nikon Coolpix 4500, Tokyo, Japan). Since the resolution depends on how much lens profile fills the image, optical zoom was used until the camera's view was completely filled by the lens profile on the screen. This resulted in a resolution of approximately 13 μm/pixel. A ruler was also photographed in the image for scaling purposes. Next, the comparator was repositioned to provide a sagittal lens profile (along the equator), and another picture was taken. Sagittal profile pictures were then taken every five min for 1 h and every fifteen min, thereafter, for another 4 h. Six human lenses were examined at 36 °C using DMEM-F12. All other measurements were made at 25 °C. The optical comparator was then repositioned for a coronal view again, and a final picture was taken. After testing, the lens was weighed to the nearest milligram.
The sagittal profile of the lens was used for measurements of equatorial diameter and sagittal thickness. The digital images were transferred to a computer and analyzed with the graphics program, Adobe Photoshop 7. As described previously , lines were drawn along the sagittal thickness and equator of the scaled lens image and the line lengths were recorded. The distances from the anterior and posterior surfaces to the line, drawn along the equatorial diameter, were recorded as measures of anterior and posterior sagittal thickness, respectively. Dividing these various values by the comparator magnification (20x) and ruler magnification gave dimension in millimeters. Equatorial diameter was also measured from the coronal view. In all cases, this was within 0.1% of the value obtained from the sagittal view. The approximate lens volume was calculated using the volume equation for an ellipsoid, assuming perfect rotational symmetry about the minor (optical) axis. This may overestimate the volume by as much as 5% .
Lens curvatures were determined by fitting the digitized surface to the general conic equation . All of the anterior surface could be used for the fitting but only 3-4 mm of the posterior surface was accessible because of interference by the supporting ring.
At the completion of the experiment, lenses were placed in 10% buffered formalin, and 8 μm sections were prepared from the center of the lens following directions outlined by Ziebarth et al. . These were stained with Periodic Acid Schiff and hematoxylin and eosin stains. Pictures were taken using a digital camera (Optronics, Goleta, GA) connected to the light microscope (Nikon, Tokyo, Japan) at a magnification of 40x. Capsular thickness was measured from these .
Shadow photogrammetry was used to monitor the effects of short term culture on the shapes and dimensions of 60 primate lenses (33 humans, 11 rhesus monkeys, and 16 cynomolgus monkeys). This simple but accurate technique was developed for evaluating corneal shape  and has recently been applied to the measurement of human lens dimensions . Typical coronal and sagittal shadow photographs for the three species, taken immediately after removal from the eye, are shown in Figure 1. The clarity and sharpness of the images allowed measurements to a resolution of 0.013 mm.
The lenses were immersed in one of four supporting media, DMEM/F-12, TC199, BSS, and Ringers solution and incubated at 25 or 36 °C for 4-5 h during which shadow photographs were taken at regular intervals.
Inspection of the shadow photographs revealed that seven of the 33 human lenses examined were swollen before incubation and their capsules had separated from the cell mass. An example is shown in Figure 2. The 42-year-old lens exhibited substantial separation at the posterior surface (top) prior to the incubation. Twenty-two of the apparently unaffected lenses were incubated in the supporting media. Of these, a further 10 became swollen and developed capsular separation at various times during the incubation. The 67-year-old lens, shown in Figure 3C, is typical of these. It appears normal before the incubation but is obviously swollen afterwards (Figure 3D). The capsule can be seen to have also separated from the cellular mass at the posterior surface (bottom). The remaining 12 human lenses appeared to be unchanged, as were all of the monkey lenses. No separation is observed at either the anterior or posterior capsule in the unaffected 63-year-old lens (Figure 3A,B).
The effect of temperature was examined by incubating six of the lenses at 36 °C. Of these, three developed capsular separation and three were unaffected, similar proportions to the seven affected and nine unaffected at 25 °C. Although the sample number was small, it appeared that the swelling took place more rapidly and was greater at 36 °C than at 25 °C.
This separation was observed at the posterior surface in all affected lenses, and, in some cases, also at the anterior surface. Histological examination of lenses with capsular separation (Figure 4C,D) revealed substantial lamellar separation at both the anterior and posterior capsules but none in the unaffected lenses (Figure 4A,B). In the example shown, the anterior epithelial cell layer has separated from the capsule and become disordered (Figure 4C). In some of the other lenses, the epithelial cells remained attached to the capsule and a fluid-filled space appeared between the epithelial cells and overlying fiber cells. The anterior epithelial cells were similar in size (about 7x12 μm) to those of the unaffected lenses (Figure 4A,C), but the capsule appeared to be swollen. The anterior and posterior capsules in the affected lens were about 18 and 5.5 μm thick, respectively, compared with 11 and 3.5 μm for the unaffected lens. The thickness of the anterior capsule in the swollen lens is well outside of the normal range (6.5-15.2 μm) reported by Ziebarth et al. . Despite these changes, the lenses appeared clear and transparent to the unaided eye.
The incidence of capsular separation in the different media is summarized in Table 1. Ten of the 22 lenses tested were affected. The data reveal that, in tissue culture media (TC199 and DMEM/F-12), a smaller proportion of human lenses (3/12) developed capsular separation than in the salt solutions (7/10).
Since the human eyes had been stored for variable times in the eye bank, prior to lens extraction, the incidence of capsular separation as a function of postmortem time was examined. No dependence on postmortem time was apparent, either in the lenses, which exhibited separation at the beginning of the incubation, or in those which developed the separation during the subsequent incubation. From the combined data presented in Figure 5A, it may be seen that separation was observed in one lens removed as early as 12 h post mortem and three as late as 144 h. On the other hand, two of the four lenses removed at 120 h were unaffected.
Similarly, no relationship was found between donor age and the incidence of capsular separation (Figure 5B). Capsular separation was observed in 61% of over 60-year-old lenses and 62% of those under 60 years old.
Lens dimensions were monitored throughout the incubation period. Data for an affected and an unaffected human lens are presented in Figure 6, and typical data for the monkey lenses are shown in Figure 7. The changes for all lenses examined are summarized in Table 2. Also included in Table 2 are data for lenses, which had capsular separation at the beginning of the incubation but were still monitored.
In all four media and for all three species, sagittal thickness increased continuously during the incubation while equatorial diameter decreased (Figure 6, Figure 7, Table 2). In the unaffected human lenses, the increase in thickness (1.5% over 5 h; Figure 6A) was greater than the decrease in the diameter (0.7%; Figure 6B). In the nonhuman primates, the diameter changes (1.4-1.6%; Figure 7A) were closer to the thickness changes (1.0-1.8%). After 5 h incubation, diameter and thickness were still changing at 0.05-0.15%/h in all unaffected lenses.
By contrast, the thickness change in the human lenses, which developed capsular separation, was pronounced (Figure 6A,B), averaging 8.7% for the 10 lenses. Anterior and posterior thickness appeared to change at the same rate. At the same time, the equatorial diameter decreased by 0.9%, close to the change in the unaffected lenses. Consequently, the aspect ratio (D/t) decreased from 2.0 to 1.8. No differences were apparent between lenses incubated in balanced salt solutions and culture media.
Calculations from the measured dimensions, indicated that there were substantial increases, averaging 6.8% in the volumes of lenses with capsular separation, but little, if any, changes in the other lenses (Table 2). The apparent 1.4-1.7% decrease in monkey lens volume can probably be attributed to errors in the volume calculation, with changing lens shape, because of the departure of lens shape from the assumed ellipsoid with rotational symmetry (Figure 1).
Six of the lenses which were swollen and exhibited capsular separation at the beginning were also monitored for the 5 h incubation period. One burst after an hour and a second increased in volume by >20%, suggesting it may also soon burst. The other four lenses continued to increase in thickness and decrease in equatorial diameter, becoming more rounded, but their (high) volumes remained constant (Table 2).
The weights of the lenses were determined at the completion of the experiment. Comparison with weights reported by Smith  and Harding et al.  indicated that the unaffected human lenses were, on average, 4% heavier than comparably aged lenses. Reflecting the increased volume, the weights of lenses with capsular separation were around 16% (30-40 mg) heavier.
Curvatures and shape factors were measured for all lenses before and after incubation. The data for human lenses were highly variable and no significant trend could be discerned. The curvatures of the monkey lenses were much less variable and increased by 3% for both anterior and posterior surfaces.
The purpose of this study was to examine the effects of short-term culture on the shape and dimensions of primate lenses. However, it became apparent early in the study that a number of the human eye bank lenses were swollen and exhibited separation of capsule and lens cell mass.
All lenses changed shape continuously during the incubations, increasing in thickness and decreasing in equatorial diameter. They could be divided into two distinct groups, those in which there was no volume change, comprising all of the monkey lenses and 12/22 of the human lenses, and those in which the volume increased. It seems unlikely that the small shape change in the first group is due to residual lenticular elasticity since the dimensions were still changing slowly at the end of 5 h, long after the zonular tension would have been released. More likely, the change can be attributed to gravity-induced sag.
Many different media have been used for lens culture but these may be simply divided into two groups: balanced salt solutions and nutrient media. In the present study, two balanced salt solutions (SRS and BSS) and two nutrient media (DMEM/F-12 and TC199) were examined. As might have been expected, the culture media, which contain nutrients such as glucose, were more effective than the simple salt solutions at maintaining lens dimensions and volumes. Nine of the 12 human lenses tested in culture media were essentially unchanged at the conclusion of the 5 h incubation. By contrast, only 3/10 survived in the balanced salt solutions. Clearly, nutrient-containing media should be used for in vitro human lens studies.
None of the monkey lenses were affected. It is probable that this can be attributed to their freshness (<3 h post mortem) and young age (2-13 years) compared with the human (12-144 h post-mortem and 20-99 years). In conjunction with the report that capsular strength decreases with age , this would suggest that the older lenses may be less able to cope with external stresses. However, we were unable to detect any significant trends.
The reasons for the large increases in thickness and volume, as well as the lamellar separation, observed with 17 of the human lenses are not obvious. At first, it would appear likely that, even though great care was taken throughout, capsular damage during lens isolation could be responsible. However, several observations would argue against this. Many of the lenses were already swollen when removed from the eye. Lamellar separation and volume increases did not occur in any of the 27 monkey lenses examined. Capsular thickness in the monkey is similar to that in the human  but the risk of damage would have been greater with the smaller monkey eyes. In the human lenses, no obvious capsular damage could be detected in the regions where the lamellar separation took place nor did there appear to be any significant disruption of the fiber or epithelial cells. In addition, it might be expected that, if the capsule were compromised, water could flow in and out freely and would not accumulate under pressure.
More likely, the swelling can be attributed to failure of the volume regulating systems. Thus, it is envisaged that water accumulates in the fiber cells, causing swelling and leading to their eventual rupture and the formation of lakes. The fluid in the lakes would not readily escape from the lens because the capsule was still intact. Furthermore, it has been shown that the permeability of the capsule decreases with increasing pressure, making it more likely that the fluid will be retained as the lens swells . Which component(s) of the regulating system may be responsible and why only some of the lenses were affected remains to be determined.
It has previously been suggested that lowering the temperature of the lens could slow metabolism sufficiently to prevent replenishment of ATP supplies needed for the ion pumps . This would be an obvious outcome of prolonged storage at 4-6 °C in the eye bank. Incubation in the salt solutions would also result in a reduction in lenticular ATP as shown by Greiner et al. . Thus, it is likely that lack of ATP is responsible for failure of the ion pumps, leading to ion and water accumulation.
An intriguing question arising from our observations is "why does swelling take place only in the thickness and not in the diameter?" This would suggest that the equatorial region is better able to resist internal expansionary forces. It may be that the equatorial capsule is thicker and stronger than that at the anterior and posterior surfaces. The abundance of nearby nucleated epithelial cells at different stages of differentiation and elongation may impart additional rigidity to this area. Alternatively, the equatorial cells may be more robust or retain more ATP than the immature fiber cells and, therefore, do not swell or rupture.
Sheep lenses, left in the enucleated eye at 0 °C increase in weight by up to 16% in three days due to the accumulation of water . It may reasonable to expect this would also occur with human lenses. Indeed, the weights of the unaffected lenses were, on average, some 4% (8-12 mg) higher than the lowest published values for fresh tissues , indicating that they had probably already taken up water during storage in the eye bank. However, the postmortem water uptake did not predispose all of the lenses to swelling or lamellar separation during the subsequent incubation. Those lenses, which exhibited capsular separation, had the highest wet weights but there seemed to be no relationship with postmortem time or with donor age.
It would appear that some investigators believe storage of the eye under the carefully controlled conditions in the eye bank will protect the lens. It should be remembered that eye banks are designed to preserve the cornea and should have little impact on the lens. Our data indicate that >60% of lenses obtained from the local eye bank eyes had, or rapidly developed, capsular separation as a result of substantial water accumulation. Similar data would be expected for tissues obtained from other eye banks. Comparison of >500 reported lens weights from a large number of laboratories with the data of Smith  and Harding et al.  suggests that at least one third of the lenses were already swollen at the time of removal (unpublished).
The observation that human lenses when left in the eye, take up water is not new. It was first mentioned in the report by Smith  and reiterated by Scammon and Hesdorffer  and van Heyningen . Deussen and Pau  reported that the water gradient in the lens rapidly (within 24-48 h after death) dissipated following enucleation due to large increases in the nuclear and posterior cortex water contents. It appears to be generally known in the lens research community that this is a largely unavoidable difficulty in working with human tissues. Yet, the problem is frequently ignored, and there are many published data obtained with lenses that were obviously swollen. How this swelling may have affected the reported data cannot be easily ascertained.
The histology and dimension changes suggest that the water accumulation occurs in discreet lakes, located near the capsule and around the optic axis. Therefore, any measurements of lens optical properties (focal length, aberration, or transparency) along or near the optic axis are going to be significantly affected. Alterations in physical properties may be restricted to these areas, giving rise to misleading results when measuring properties such as refractive index, hardness and viscosity in different parts of the lens. If the water should spread, it could also lead to softening of the nucleus .
It is clear that great care needs to be taken when interpreting data from in vitro measurements on human lenses, unless it can be shown that there has been no swelling or capsular separation prior to removal. It is suggested that, where possible, lens thickness and diameter be measured before and after any experimental protocol and that data from lenses with an aspect (equatorial/sagittal) ratio of less than 2.00 be viewed with great caution.
The authors gratefully acknowledge the support of Dr. Jorge Pena of the Florida Lions Eye Bank, Dr. Norma Kenyon of the Diabetes Research Institute, as well as Dr. Patricia Gullett and Dr. Bobby Collins of the Department of Veterinary Resources for providing the tissue used in these experiments. Dr. Joseph Stoiber and Dr. Viviana Fernandez assisted with the removal of some lenses. Supported in part by the NIH consortium grant EY1425-01; Research to Prevent Blindness; the Vision CRC, Sydney, Australia; the Florida Lions Eye Bank; and the Henri and Flore Lesieur Foundation.
1. Duncan, G, Jacob TJC. The lens as a physicochemical system. In: Davson H, editor. The Eye. Vol. 1B. 3rd ed. Orlando: Academic Press; 1984. p.159-206.
2. Cotlier E, Kwan B, Beaty C. The lens as an osmometer and the effects of medium osmolarity on water transport, 86Rb efflux and 86Rb transport by the lens. Biochim Biophys Acta 1968; 150:705-22.
3. Parel JM, Manns F, Denham D, Acosta AC, Yamamoto H, Billotte C, Fernandez V, Abri A, Lamar P, Ziebarth N, Orozco M, Borja D, Hughes T, Ho A, Augusteyn R, Ehrman K, Yoo S, Holden B. Restoring accommodation: Lens capsule refilling (Phaco-Ersatz) vs accommodating IOLs. Am J Ophthalmol 2005; 139:S7.
4. Ehrman K, Ho A, Parel JM. Evaluation of porcine crystalline lenses in comparison with moulded polymer gel lenses with an improved ex-vivo accommodation simulator. In: Manns F, editor. Ophthalmic technologies XV. Bellingham (WA): International Society for Optical Engineering (SPIE); 2005. p. 240-51.
5. Glasser A, Campbell MC. Presbyopia and the optical changes in the human crystalline lens with age. Vision Res 1998; 38:209-29.
6. Glasser A, Campbell MC. Biometric, optical and physical changes in the isolated human crystalline lens with age in relation to presbyopia. Vision Res 1999; 39:1991-2015.
7. Moffat BA, Atchison DA, Pope JM. Age-related changes in refractive index distribution and power of the human lens as measured by magnetic resonance micro-imaging in vitro. Vision Res 2002; 42:1683-93.
8. Thaung J, Sjostrand J. Integrated light scattering as a function of wavelength in donor lenses. J Opt Soc Am A Opt Image Sci Vis 2002; 19:152-7.
9. Schachar RA. Central surface curvatures of postmortem-extracted intact human crystalline lenses: implications for understanding the mechanism of accommodation. Ophthalmology 2004; 111:1699-704.
10. Heys KR, Cram SL, Truscott RJ. Massive increase in the stiffness of the human lens nucleus with age: the basis for presbyopia? Mol Vis 2004; 10:956-63 <http://www.molvis.org/molvis/v10/a114/>.
11. Dovrat A, Sivak JG. Long-term lens organ culture system with a method for monitoring lens optical quality. Photochem Photobiol 2005; 81:502-5.
12. Rosen AM, Denham DB, Fernandez V, Borja D, Ho A, Manns F, Parel JM, Augusteyn RC. In vitro dimensions and curvatures of human lenses. Vision Res 2006; 46:1002-9.
13. Patterson JW, Fournier DJ. The effect of tonicity on lens volume. Invest Ophthalmol 1976; 15:866-9.
14. Patterson JW. Lens volume regulation in hypertonic medium. Exp Eye Res 1981; 32:151-62.
15. Patterson JW. Volume regulation in rat lenses in media with varying concentrations of potassium and sodium. Exp Eye Res 1983; 37:105-17.
16. Korte I, Brenner KP, Pollmann KP. A comparison of two buffering systems for lens organ culture. Ophthalmic Res 1982; 14:265-8.
17. Bagchi M, Caporale MJ. The effects of osmotic shock on the organ cultured mammalian ocular lens. Invest Ophthalmol Vis Sci 1984; 25:586-93.
18. Tumminia SJ, Qin C, Zigler JS Jr, Russell P. The integrity of mammalian lenses in organ culture. Exp Eye Res 1994; 58:367-74.
19. Azzam N, Dovrat A. Long-term lens organ culture system to determine age-related effects of UV irradiation on the eye lens. Exp Eye Res 2004; 79:903-11.
20. Ziebarth NM, Manns F, Uhlhorn SR, Venkatraman AS, Parel JM. Noncontact optical measurement of lens capsule thickness in human, monkey, and rabbit postmortem eyes. Invest Ophthalmol Vis Sci 2005; 46:1690-7.
21. Denham D, Mandelbaum S, Parel JM, Holland S, Pflugfelder S, Parel JM. Shadow photogrammetric apparatus for the quantitative evaluation of corneal buttons. Ophthalmic Surg 1989; 20:794-9.
22. Smith P. Diseases of crystalline lens and capsule. 1. On the growth of the crystalline lens. Trans Ophthalmol Soc U K 1883; 3:79-99.
23. Harding JJ, Rixon KC Marriott FHC. Men have heavier lenses than women of the same age. Exp Eye Res 1977; 25: 651.
24. Krag S, Andreassen TT. Biomechanical measurements of the porcine lens capsule. Exp Eye Res 1996; 62:253-60.
25. Fisher RF. The water permeability of basement membrane under increasing pressure: evidence for a new theory of permeability. Proc R Soc Lond B Biol Sci 1982; 216:475-96.
26. Augusteyn RC, Cake MA. Post-mortem water uptake by sheep lenses left in situ. Mol Vis 2005; 11:749-51 <http://www.molvis.org/molvis/v11/a89/>.
27. Greiner JV, Kopp SJ, Sanders DR, Glonek T. Organophosphates of the crystalline lens: a nuclear magnetic resonance spectroscopic study. Invest Ophthalmol Vis Sci 1981; 21:700-13.
28. Scammon RE, Hesdorffer MB. Growth in mass and volume of the human lens in postnatal life. Arch Ophthalmol 1937; 17:104-12.
29. Van Heyningen R. The human lens. 3. Some observations on the post-mortem lens. Exp Eye Res 1972; 13:155-60.
30. Deussen A, Pau H. Regional water content of clear and cataractous human lenses. Ophthalmic Res 1989; 21:374-80.
31. Tabandeh H, Karim A, Thompson GM. Effect of exposure to balanced salt solution upon the hardness of the crystalline lens. Graefes Arch Clin Exp Ophthalmol 1998; 236:890-3.