Lens capsule structure assessed with atomic force microscopy

• Introduction
• Methods
• Results
• Discussion

Abstract

Purpose: To image the ultrastructure of the anterior lens capsule at the nanoscale level using atomic force microscopy (AFM).

Methods: Experiments were performed on anterior lens capsules maintained in their in situ location surrounding the lens from six human cadavers (donor age range: 44–88 years), four cynomolgus monkeys (Macaca fascicularis age range: 4.83–8.92 years), and seven pigs (<6 months). Hydration of all samples was maintained using Dulbecco’s Modified Eagle Medium (DMEM). Whole lenses were removed from the eye and placed anterior side up in agarose gel before gel hardening where only the posterior half of the lens was contained within the gel. After the gel hardened, the Petri dish was filled with DMEM until the point where the intact lens was fully submerged. AFM was used to image the anterior lens surface in contact mode. An integrated analysis program was used to calculate the interfibrillar spacing, fiber diameter, and surface roughness of the samples.

Results: The AFM images depict a highly ordered fibrous structure at the surface of the lens capsule in all three species. The interfibrillar spacing for the porcine, cynomolgus monkey, and human lens capsules was 0.68±0.25, 1.80±0.39, and 1.08±0.25 μm, respectively. In the primate, interfibrillar spacing significantly decreased linearly as a function of age. The fiber diameters ranged from 50 to 950 nm. Comparison of the root mean square (RMS) and average deviation demonstrate that the surface of the porcine lens capsule is the smoothest, and that the human and cynomolgus monkey capsules are significantly rougher.

Conclusions: AFM was successful in providing high-resolution images of the nanostructure of the lens capsule samples. Species-dependent differences were observed in the overall structure and surface roughness.

The lens capsule is an acellular basement membrane that completely encapsulates the lens of the eye. The capsule maintains the structural integrity of the lens material and serves as an anchorage point for the zonules [1]. The capsule, together with the ciliary muscles and zonules, alters the shape of the lens material during accommodation [1]. It separates the lens from other ocular tissues while protecting it from viruses and bacteria [2]. The capsule also functions as a medium of biochemical interchange [3]. It allows for the passive exchange of metabolic substrates and waste between the ocular environment and the lens cells, while selectively filtering molecules based on their size and charge [2,4]. Finally, interactions between the lens capsule and the lens epithelial and fiber cells are required for normal lens development and growth [5].

The molecular composition of the lens capsule is similar to other basement membranes throughout the body, although the lens capsule is the thickest basement membrane in the body due to the slow turnover rate and continuous deposition of matrix material [4,6,7]. The lens capsule is composed of networks of laminin [8-12], collagen IV [8,13,14], entactin/nidogen [8,9,15], and some heparin sulfate proteoglycans, including perlecan [8,16-18], collagen XVIII [19], collagen XV [20], and agrin [21]. Collagen type IV is the most abundant component [14], making up 30–40% of the lens capsule’s dry weight [6]. Additional studies on the bovine lens capsule have shown a similar composition to that of the human [22]. The three-dimensional, interwoven networks of the various components of the lens capsule endow it with strength, flexibility, and elasticity, thus enabling the capsule to function successfully. Imaging studies have shown that this general network is the same in multiple mammalian species, including porcine, human, and ox [23].

Images of the ultrastructure of the anterior lens capsule surface have been previously acquired using cryoelectron microscopy [5,23], scanning helium ion microscopy [4], and atomic force microscopy (AFM) [24-27]. For cryoelectron microscopy and scanning helium ion microscopy techniques, irreversible modifications are made to the tissue when preparing it for imaging, such as freezing, fixation, sectioning, or critical point drying. With AFM, the tissue remains fully hydrated and intact up to and throughout the imaging process. However, these methods have not captured the three-dimensional ultrastructure of the fully hydrated lens capsule in its in situ location surrounding the lens material. The purpose of the current study was to use AFM to image the structure of the fully hydrated lens capsule at the nanoscale level without excising the capsule from the lens.

The height and three-dimensional images obtained from the Z-sensor signal after post-processing are shown in Figure 1. These images depict the vertical displacement of the cantilever tip as it is scanned across the topography of the sample. The images show a highly ordered fibrous structure at the surface of the lens capsule in all three species. This organization was expected, since the lens capsule is comprised predominately of fibrous proteins (collagen IV and laminin) networked with entactin/nidogen and perlecan, forming the meshwork seen in Figure 1. The white lines (areas of higher topography) correspond to fibers whereas the darker gray and black areas correspond to openings in the mesh.

The interfibrillar spacing for the porcine, cynomolgus monkey, and human lens capsules was 0.68±0.25, 1.80±0.39, and 1.08±0.25 μm, respectively (Table 2). The spacing was significantly different among the porcine, monkey, and human lens capsules (p=0.002 and p=0.008, respectively). The spacing of the human and monkey lens capsules was also significantly different (p=0.01). However, the human and non-human primate data were grouped together using a scaling factor of 1 monkey year to 3 human years [28-30] to determine if age was the reason for the statistical difference. This analysis showed that interfibrillar spacing significantly decreased linearly as a function of age in the primate (p=0.028).

The fiber diameters for the porcine, cynomolgus monkey, and human lens capsules were 306 nm±239 nm (range: 50 nm–950 nm), 397 nm±159 nm (range: 176 nm–687 nm), and 339 nm±135 nm (range: 67 nm–691 nm), respectively (Table 2). There were no statistical differences between the fiber diameters in any of the three species (p=0.235 for porcine–cynomolgus, p=0.381 for porcine–human, p=0.286 for cynomolgus–human). There was also no relationship between fiber diameter and age for the primate (p=0.529).

The roughness parameters for the three species are summarized in Table 2. Comparison of the RMS and average deviation demonstrate that the surface of the porcine lens capsule is the smoothest, and that the human and cynomolgus monkey capsules are significantly rougher (p=0.057 and 0.040, respectively, for RMS deviation; 0.058 and 0.046, respectively, for average deviation). The roughness of the human and monkey lens capsules are comparable (p=0.171 and 0.188 for RMS deviation and average deviation, respectively). No significant relationship was found with age for the human and monkey lens capsules, although there was a trend toward decreasing surface roughness with age. Kurtosis values indicate that the human lens capsules have the most gradual transitions from high to low area topographies.

Prior investigations into the structure of the lens capsule used cryoelectron microscopy and helium ion microscopy, and therefore required extensive tissue preparation before imaging. A few additional studies used atomic force microscopy to image the surface of the lens capsule, but the experiments were performed on excised samples. In the current study, images of the lens capsule, in its native, unprocessed state, were captured. Furthermore, AFM allowed the lens capsule to be maintained in a hydrated state throughout the imaging process, mitigating time-sensitive morphological changes that occur ex vivo. Finally, the current study expanded upon previous AFM studies by imaging the lens capsule in its in situ location surrounding the lens material.

The fibrous network at the surface of the lens capsule was observed in all three of the species imaged in this study: humans, cynomolgus monkeys, and pigs. The images clearly show that the porcine lens capsule had the most densely packed structure, whereas the cynomolgus monkey had clearly defined openings or pores. The structure of the human lens capsule was intermediate, with some porous areas surrounded by a densely interwoven fibrous structure. Previous studies investigating anterior lens capsule mechanical properties [30] and thickness [31] found no statistical differences between cynomolgus monkeys and humans for either of these two parameters. Since the mechanical properties of tissues are directly correlated to the organization of the individual components [32-34], it is reasonable that the lens capsule structure would also be comparable for humans and cynomolgus monkeys. For the porcine lens capsule, a study by Danielsen [35] found that the capsule is approximately four times thicker but 50% less stiff than the human lens capsule. Therefore, these differences in thickness and mechanical properties should be manifested in differences in structure as well, as shown in this study (see Figure 1). Interestingly, the fiber diameters were not significantly different among the species. This suggests that the actual network comprises the same components, but the organization of these components with varying interfibrillar spacing and additional layers to increase thickness endows the lens capsule with different mechanical properties seen in previous studies. Furthermore, the pig lens is unable to accommodate [36]. Since the lens capsule distributes the forces of accommodation on the lens material during accommodation [37], the organizational layout of the collagen network may be different in species that accommodate versus those that do not.

The structural differences seen in this study could be explained by differences in age. The age range of the human capsules was 44–88 years, whereas the cynomolgus monkeys ranged from 14 to 17 equivalent human years, using a conversion factor of 1 monkey year equal to 3 human years [28-30]. When the human and cynomolgus interfiber spacing was analyzed together, a significant decrease was found with age. Although the data support the theory that interfibrillary spacing has a direct correlation with the primate’s age, additional interspecies differences, independent of age, might have influenced the results. For example, compositional variations in the lens capsule between species could have affected the imaged structure; however, the absence of previous studies of additional species in the literature makes it difficult to draw any further conclusions.

Previous studies investigating the lens capsule ultrastructure showed a similar fibrous structure as seen in the current study [23-27,38]. Atomic force microscopy images of the excised lens capsule showed fiber-like surface structures [24-27] with diameters ranging from 1 to 100 nm wide [26,27], which is at the low end of the range found in the current study. In a classic study, Barnard et al. used cryoelectron microscopy [23] and observed images of a highly ordered collagen IV matrix, with interfibrillar spaces measuring 30 nm. In another study, Danysh et al. used helium ion microscopy and observed pore-like structures measuring 6–25 nm at the surface of the anterior lens capsule [38]. The current study found interfibrillar spaces on the order of 1 μm. Although differences in the technique used could result in differences in the dimensions, it is most likely that the AFM images in the current study captured a larger-scale view of the lens capsule surface than those in previous studies. It is possible that imaging at higher magnification would provide more details about a structure within the structures observed in this study. Nevertheless, the current study provides insight into the lens capsule structure in three different species.

During AFM scans, the piezoelectric mechanism makes vertical adjustments to keep the force applied to the sample constant, which provides information on the sample height. The surface roughness can be calculated from these vertical displacements of the cantilever with subnanometer precision. Analysis of the roughness data (RMS roughness and average deviation) showed that the surface of the porcine lens capsules was significantly smoother than that of cynomolgus monkeys or humans. This could be because the fibrous structure is so dense in porcine lenses, with very small pore-like openings (pure black area in the image). The height differences measured go from a higher fiber to a lower fiber, as opposed to an opening in the structure, as was seen in the cynomolgus monkey and human lens capsules (see Figure 1). The RMS roughness values found in the current study for humans (12–58 nm) are comparable to those found in a previous AFM investigation by Creasey et al. on excised anterior lens capsules (5–50 nm) [26]. An additional AFM study by Choi et al. [25] found that anterior lens capsules from patients with cataract were less rough than those from non-cataract patients. Interestingly, our results for the human lenses showed a decreasing trend in roughness with age, although this was not statistically significant due to the small range in age (44–88 years). The degree of cataract progression in the lenses included in the current study was not noted, so a correlation with cataract, as in the Choi et al. study, could not be made. A larger value for roughness could indicate that the samples were deteriorating as a function of sample postmortem time. However, the postmortem time of the monkey samples was comparable to that of the porcine samples, but the monkey samples were significantly rougher.

With AFM, we image and analyze the outer topography of the lens capsule. Because of the continuous deposition of the capsule by the fiber cells and the lens epithelium lining the anterior capsule’s posterior surface [2], the outer layer of the lens capsule would have been formed during the early stages of the organism’s development. For the specific species investigated in this study, the fibrous networks of the anterior lens capsule imaged in the porcine lenses were formed months before the AFM imaging whereas the outer layer of the primate lens capsules were formed years to decades before the AFM imaging. Such ample time leaves the possibility for protein oxidation to occur [39,40], possibly affecting the roughness and structure of the lens capsule’s outer layer. In addition, pigs are unable to accommodate [36]. Therefore, the porcine lens capsule is not subjected to the continual mechanical stresses throughout its lifetime to which the lens capsule of a primate is accustomed [37]. It is possible that mechanical stress correlates to the surface roughness at the nanoscale. In fact, this study found an increase in roughness with increasing amplitude of accommodation (porcine, human, monkey).

All images of the lens capsule were obtained in contact mode operation of the AFM. Contact mode has been criticized because it can create lateral drag forces between the cantilever tip and the sample’s surface that may actually damage the sample. However, selecting a slower scan rate can minimize these lateral forces and the occurrence of sample damage and drag artifacts. To further mitigate possible damage to the sample from the cantilever, the set point, corresponding to the cantilever’s vertical force on the sample, was kept below 1.875 V (approximately 5 nN). Adjustments to the set point also helped compensate for changes to the tissue postmortem, including water diffusion into the lens that causes lens capsule delamination. A previous study looked at lens biometry while immersed in various preservation media, including DMEM [41]. This study found that monkey and human lenses swelled less than 10% after 5 h in the solution. Therefore, although the network would expand correspondingly, we anticipate the changes would be small.

In summary, atomic force microscopy was used to successfully image the fully hydrated lens capsule. Species-dependent differences were observed in the overall structure and surface roughness. Further studies should investigate structure as a function of age to determine how the lens capsule changes with increased age in different species.