Experimental Eye Research 121 (2014) 58e65

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Elemental analysis of sunflower cataract in Wilson’s disease: A study using scanning transmission electron microscopy and energy dispersive spectroscopy Hyo Ju Jang, Joon Mo Kim, Chul Young Choi* Department of Ophthalmology, Kangbuk Samsung Hospital, Sungkyunkwan University College of Medicine, Seoul, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 September 2013 Accepted in revised form 6 February 2014 Available online 15 February 2014

Signature ophthalmic characteristics of Wilson’s disease (WD) are regarded as diagnostically important manifestations of the disease. Previous studies have proved the common occurrence of copper accumulation in the liver of patients with WD. However, in the case of sunflower cataracts, one of the rare diagnostic signs of WD, no study has demonstrated copper accumulation in the lens capsules of sunflower cataracts in WD patients. To investigate the nanostructure and elemental composition of sunflower cataracts in WD, transmission electron microscopy (TEM) was done on the capsulorhexised anterior lens capsule of sunflower cataracts in WD in order to evaluate anatomical variation and elemental changes. We utilized energy dispersive X-ray spectroscopy (EDS) to investigate the elemental composition of the lens capsule using both point and mapping spectroscopy. Quantitative analysis was performed for relative comparison of the elements. TEM showed the presence of granular deposits of varying size (20e350 nm), appearing mainly in the posterior one third of the anterior capsule. The deposits appeared in linear patterns with scattered dots. There were no electron-dense particles in the epithelial cell layer of the lens. Copper and sulfur peaks were consistently revealed in electron-dense granular deposits. In contrast, copper and sulfur peaks were absent in other tissues, including granule-free lens capsules and epithelial tissue. Most copper was exclusively located in clusters of electron-dense particles, and the copper distribution overlapped with sulfur on mapping spectroscopy. Quantitative analysis presented inconsistent ratios of copper to sulfur in each electron-dense granule. The mean ratio of copper to sulfur was about 3.25 (with a range of 2.39e3.78). This is the first elemental analysis of single electron particles in sunflower cataracts using EDS in the ophthalmic area. Sunflower cataracts with WD are assumed to be the result of accumulation of heterogeneous compounds composed of several materials, including copper, sulfur, and/or copper-binding proteins. Linear patterns of copper and sulfur deposition were detected in various sizes and composition ratios with these elements in cases of WD. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: sunflower cataract energy dispersive X-ray spectroscopy elemental analysis scanning transmission electron microscopy Wilson’s disease

1. Introduction Wilson’s disease (WD) or hepatolenticular degeneration is a rare autosomal recessive genetic disorder that causes defective copper excretion, resulting in toxic accumulation of copper in multiple

Abbreviations: EDS, energy dispersive X-ray spectroscopy; KeF, Kaysere Fleischer; MT, metallothionein; STEM, scanning transmission electron microscopy; TEM, transmission electron microscopy; WD, Wilson’s disease. * Corresponding author. Department of Ophthalmology, Kangbuk Samsung Hospital, Sungkyunkwan University College of Medicine, Pyeong-dong, Jongno-gu, Seoul 110-746, Republic of Korea. Tel.: þ82 2 2001 2250; fax: þ82 2 2001 2262. E-mail address: [email protected] (C.Y. Choi). http://dx.doi.org/10.1016/j.exer.2014.02.003 0014-4835/Ó 2014 Elsevier Ltd. All rights reserved.

organs, particularly in the liver, brain (primarily the basal ganglia and cortex), kidneys, and eyes. Signature ophthalmic characteristics of WD are known to be diagnostically important manifestations. KaysereFleischer (KeF) rings are caused by copper accumulation in the Descemet’s membrane of the eye’s sclerocorneal junction. A combination of KeF rings and low serum ceruloplasmin are pathognomonic signs of WD. Sunflower cataracts, one of the few diagnostic signs of WD, were first described as “cataracts like rays of the sun” by Siemerling and Oloff (1922). Previous studies (Harry and Tripathi, 1970; Johnson, 1973; Tousimis and Adler, 1963; Uzman and Jakus, 1957), including studies that utilize transmission electron microscopy

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(TEM), electron probe X-ray microanalysis, and histochemistry, identified copper deposits in the characteristic KeF rings of WD. No previous study, however, has analyzed the elemental composition of the lens capsule of a sunflower cataract in a patient with WD. TEM with energy dispersive X-ray spectroscopy (EDS) is a method of elemental microanalysis that is widely applied in various fields, and is capable of identifying and quantifying all elements in the periodic table (with the exception of H, He, and Li) with 0.1 nm resolution. To our knowledge, this is the first study using TEM with EDS for sunflower cataracts in WD. The purpose of this study is to investigate the nanostructure and elemental composition of granular deposits in ophthalmic lenses in cases of sunflower cataracts in WD, as previously reported. 2. Material and methods A 37-year-old male was referred to the department of ophthalmology due to visual disturbance. He reported a history of ascites and liver cirrhosis seven years ago, with no apparent etiology. On neurologic and psychiatric examination, a mild hand tremor and mild cognitive impairment were noted. He also had a family history of liver cirrhosis from his mother and sister. On his ophthalmic examination, a yellow-brown colored ring was seen in the peripheral cornea and in the pupillary area, central disk-shaped, radiating spoke-like golden-brown colorations were noticed on the anterior lens capsules in both eyes (Fig. 1A and B). Golden-brown pigment deposits were located at the Descemet’s membrane level, and began to appear peripherally at Schwalbe’s line upon gonioscopic examination (Fig. 2). Based on laboratory tests, ophthalmic findings, and brain MRI, a diagnosis of WD was established. The patient’s preoperative best-corrected visual acuity was 0.15 in the right eye and 0.4 in the left eye. Intraocular pressure was 7 mmHg in both eyes.

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2.2. Transmission electron microscopy and energy-dispersive X-ray spectroscopy For our TEM study, the ultrathin sections were analyzed by the TitanÔ 80e300 microscope (FEI Company, Netherlands, with electron probe size < 0.09 nm) which achieves lateral resolution better than 0.1 nm. To investigate the elemental composition of the lens capsule, elemental analysis was performed using the TitanÔ 80e300 equipped with EDS. To avoid radiation damage caused by electron beams, the TitanÔ 80e300 was operated at the lowest possible voltage of 80 kV. The EDS analysis was carried out in scanning transmission electron microscopy (STEM)-mode, using both point and mapping spectroscopy, where data were obtained by automatically stepping the electron probe from one pixel to the next. To avoid the effects of lead and uranyl, which are materials commonly used for staining, EDS analysis was conducted under unstained conditions. The unstained EDS analysis in particular provided uncontaminated, accurate quantitative results for the relative elemental composition of atomic percentage of atomic ratio. EDS data were obtained using a Cs corrector on a TitanÔ 80e 300. Data analysis was carried out using automatic peak identification software, ES Vision (Emispec, Beaverton, OR, USA). 3. Results The anterior capsule was studied by TEM in stained sections. The majority of granular deposits were present at the posterior one third of the anterior capsule, and the deposits appeared in linear patterns with scattered dots (Fig. 3). Although the majority of granular deposits presented in the posterior one third, a few of them were detected in the anterior two thirds of the capsule. As shown in Fig. 4, multiple electron-dense particles (7e8 nm) formed clusters, and the cluster groups displayed linear-shaped granular masses. The size of electron-dense granules was in the range of 20e 350 nm. There were no electron-dense particles in the epithelial cell layer of the lens.

2.1. Surgical procedures and preparation for TEM 3.1. STEMeEDS analysis The patient’s right eye underwent conventional phacoemulsification. This extracted anterior lens capsule was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for TEM. After being washed in the buffer, the tissue blocks were post fixed with 1% osmium tetroxide (OsO4) for one hour. After dehydration in a graded series of alcohol, the samples were embedded in Epon812. Ultrathin sections (of 70 nm thickness) were prepared with an ultramicrotome (ULTRACUT UCT, LEICA, Installed at Korea Basic Science Institute), and the sections were mounted onto formvarcarbon support film-coated mesh nickel grids for TEM and EDS. Subsequently, the sections were stained with uranyl acetate and lead citrate before examination on TEM.

Elemental analysis by EDS was performed on the lens capsule and several intracellular regions, including the nucleus and cytoplasm in both the stained (Figs. 4 and 5) and unstained sections (Fig. 6). An EDS point analysis of electron-dense granular deposits from the anterior capsule revealed consistent peaks for copper and sulfur in all electron-dense granules in all magnification views (Figs. 4e6). However, the peak intensities of electron-dense granules were different from each other. Additional STEM images showed higher peak intensities with brighter electron-dense granules, whereas less bright electron-dense granules showed lower peak intensities. Moreover, peaks of copper and sulfur were

Fig. 1. A KeF ring was seen in the peripheral cornea (white arrow). A sunflower cataract was observed on the anterior lens capsule in both eyes (black arrow) (A e right eye, B e left eye).

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Fig. 2. Gonioscopy on the right eye (A e 6 o’clock, B e 3 o’clock, C e 12 o’clock, D e 9 o’clock). The golden-brown pigment deposits appeared at the Descemet’s membrane level and began peripherally at Schwalbe’s line.

absent in certain other parts, including the granule-free lens capsules and epithelial cells, and only lead and uranyl peaks in the stained tissues were weakly observed. Lead, uranyl, carbon, oxygen, and nickel peaks were visible as background peaks detected in the tissue herein. Lead and uranyl peaks originate from uranyl acetate and lead citrate, which is used for purposes of staining, so these peaks were not observed in the unstained sections of the sample. Carbon and oxygen peaks occurred in the region of the formvar-carbon supporting film (see the area marked on panel 7a). The nickel peaks are due to the

composition of the grid on which the tissue was mounted for analysis. In order to provide more detailed information, elemental chemical mapping of the lens capsule for copper and sulfur was carried out on different regions of the electron-dense granular deposits. As a first important piece of information, most of the copper was exclusively located in clusters of electron-dense particles (Fig. 7). The images presented in the figures in this study reveal that copper distribution overlapped with sulfur (Fig. 8). In contrast, the other background peaks of associated elements

Fig. 3. Transmission electron microscopy (TEM) taken from stained sections of the anterior capsule. Granular deposits were located mostly in the posterior third of the anterior lens capsule in a discontinuous linear fashion (A). Heterogeneous dense granules were observed in various sizes (B). The sizes of granules varied from 20 to 350 nm (C). The granule deposits consisted of clusters of electron-dense particles. Sizes of the particles were 7e8 nm (D).

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Fig. 4. An EDS point analysis performed at the marked point on the STEM image (aef) is matched in Panel aef. Copper and sulfur peaks were observed in all electron-dense granules. Peak intensities of electron dense granules were different from each other.

(i.e., carbon, oxygen, uranyl, and lead) showed diffuse distribution, regardless of copper distribution. 3.2. Quantitative analysis The relative elemental composition of some electron-dense granules at different points was analyzed by EDS (Table 1). To

investigate the relative ratio of copper and sulfur, pointing spectroscopy under highly magnified unstained section images was done at 13 points (Table 1). Carbon was regarded as the reference value, and the peak intensities of copper and sulfur were compared to the peak intensities of carbon. Quantitative analysis presents an inconsistent ratio of copper to sulfur from each electron-dense granule. The

Fig. 5. An EDS point analysis of electron-dense granular deposits under higher magnification (aec) is shown in Panel aec. Peak intensities of copper and sulfur were much higher in more electron-dense granules (a) than in relatively less dense granules (b). Peaks of copper and sulfur were absent in the granule-free lens capsule, however, background carbon peaks were visible, as well as lead, uranyl, and nickel peaks (c).

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Fig. 6. An EDS point analysis under unstained condition (a, b) is shown in Panel a,b. Copper and sulfur peaks were observed in electron-dense granules, as well as background peaks of carbon, nickel and oxygen (a, b). Uranyl and lead peaks were absent in the granules (a, b).

mean ratio of copper to sulfur in the electron-dense granules was about 3.25 (with a range of 2.39e3.78). 4. Discussion KeF rings are the most common ophthalmologic findings in WD and are an important clinical tool in diagnosis. There have been many previous studies about KeF rings that report copper deposits in the Descemet’s membrane of the eye (Harry and Tripathi, 1970; Johnson, 1973; Johnson and Campbell, 1982; Tousimis and Adler, 1963; Tso et al., 1975; Uzman and Jakus, 1957). The granular copper deposits accounting for KeF rings have been demonstrated histochemically by rubeanic acid (Harry and Tripathi, 1970; Johnson, 1973; Uzman and Jakus, 1957) and rhodanine (Tso et al., 1975), as well as electron-probe X-ray microanalysis (Johnson and Campbell, 1982; Tousimis and Adler, 1963). Some previous studies with TEM show similar findings that electron-dense granules appear mainly in the region of the Descemet’s membrane, endothelial cytoplasm, and occasionally the stroma (Johnson, 1973; Johnson and Campbell, 1982; Tso et al., 1975). As mentioned, of the existing studies on KeF rings, none have performed specific elemental analysis of each granule. Though relatively rare, the sunflower cataract is a characteristic of WD (Wiebers et al., 1977). Incidences of sunflower cataracts are fewer than the incidences of KeF rings because sunflower cataracts are observed in more advanced stages of the disease. Furthermore, because molecular genetic studies are becoming available for early diagnosis of WD, incidences of advanced WD are currently decreasing (Roberts and Schilsky, 2008). Accordingly, there have been few reports focusing on analysis of sunflower cataracts. A study by Tso et al. (1975) describes the initial appearance of sunflower cataracts in a patient with Wilson’s disease. The authors of the study obtained, via autopsy, eyes with KeF rings and sunflower cataracts caused by WD, and reported that granular deposits that seemed identical to granular deposits in the Descemet’s membrane were also present in the lens capsule. These deposits also stained positively with rubeanic acid and with rhodanine stain. Similar to our findings, the granules were located mostly in a plane within the posterior third of the anterior lens capsule, leaving the equatorial capsule relatively free of accumulation. No deposits were noted within the lens epithelium, the lens cortex, or in the adjacent

iris, ciliary body, retina, or elsewhere in the eye. Although electrondense granules similar to KeF rings were found via TEM, chemical composition such as copper deposits and electron-dense granules could not be demonstrated by the research group. Neither rubeanic acid nor rhodanine stains are specific for copper alone, and the stains also detect Co, Ni, Pt, and Ru in addition to Cu. In order to detect copper in particular, granular deposits should be investigated by EDS. To our knowledge, there have been histochemical and transmission electron microscopic studies of sunflower cataracts, but no elemental analytic study has been described for sunflower cataracts. Our EDS study demonstrates the presence of copper and sulfur within electron-dense granular deposits in both stained and unstained lens capsules. It also shows carbon, oxygen, lead, uranyl, and nickel peaks, but these peaks originate from the stain (Pb, U), the composition of the grid (Ni), and the supporting film (C, O). We suggest that the electron-dense granules are chemically composed of copper and sulfur, with the exception of background peaks due to the stain, the grid, and the supporting film. The findings in elemental mapping show copper distribution overlapping with sulfur. Peak intensities from multiple electron-dense granules are inconsistent with each other, and further, even within one electrondense granule, peak intensities are different between each discrete electron-dense particle. Sulfur can bind with copper, reducing its toxic effects. We think that this electron-dense granule originates from copper-binding proteins such as metallothioneins. Metallothioneins (MTs), one type of copper-binding protein, are sulfur-rich and low-molecularmass proteins that are localized mainly in the cytoplasm (Nordberg and Nordberg, 2009). The excessive amount of copper that occurs with WD is stored in tissues partly bound to MT. Toxicity from this accumulation of copper in ophthalmic tissue is explained by participation of MTs and active oxygen species that are produced upon reactions involving copper (Nordberg and Nordberg, 2009). According to a study by Johnson and Campbell, the association of copper with sulfur suggests that a sulfur-containing moiety functions in binding copper (Johnson and Campbell, 1982). The authors demonstrated copper and sulfur peaks in electron-dense granules of KeF rings by TEM with X-ray energy spectroscopy, similar to our study of sunflower cataracts.

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Fig. 7. An EDS area analysis performed at the marked area on the STEM image (aef) is shown in Panel aef. Carbon, oxygen, and nickel peaks were observed in the region of the formvar-carbon supporting film (a). In the granule-free lens capsule (b) and epithelial tissues (d, e, f, g), only lead and uranyl peaks weakly appeared.

The TEM used in this study (TitanÔ 80e300) is a more accurate method for the investigation of elemental composition of single particles than the TEM method in the study by Johnson and Campbell. The probe corrector in the current study allows for a focused electron probe with a diameter of around 0.07 nm (i.e., less than the size of an atom) for atomic resolution imaging and chemical analysis. These nanosized probe diameters allow the identification of elemental compositions at sub-cellular levels with extremely high quantitative accuracy and spatial resolution. Furthermore, the TitanÔ 80e300 is also able to analyze in both spot and mapping modes, in which the electron probe is localized on the area of a single particle within the field of view. The development mechanism of sunflower cataracts in WD is not yet known. Tso et al. (1975) did not explain why the equatorial capsule is relatively free from accumulation. They suggested that cell activity plays a significant role in accumulation of copper granules, in contrast to the concept of simple diffusion from the aqueous humor. Due to the absence of blood supply in the lens of

the eye, diffusion can be considered as a cause of the penetration of free ionic copper into the lens epithelium through the lens capsule from the aqueous humor. The lens epithelial cells secrete the components of the basal lamina, which serves to thicken the lens capsule (Parmigiani and McAvoy, 1991; Silver and Wakely, 1974). Clinical observations have also supported the view that the human lens capsule is synthesized from the inside out. An experimental animal study about the synthesis of the lens capsule shows that newly synthesized capsular materials originally accumulate close to the basal ends of epithelial and fiber cells (Young and Ocumpaugh, 1966). Accordingly, we suggest how granules composed of copper and sulfur accumulate in lens capsules close to the lens epithelium as follows. (1) In the equatorial region, ionic copper uptake into the lens epithelium occurs by diffusion. (2) The sulfhydryl groups in MTs or other copper-binding proteins form a chelate complex with the ionic copper in the cytoplasm. (3) With long-term exposure to copper, there is a slow release of copper-thionein from lens

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Fig. 8. EDS elemental chemical mapping of the lens capsule (A) on a single large granular deposit as marked, and the surrounding area of linear granular deposits (B). The distribution ratio of copper and sulfur overlapped and was similar. Other elements visible as background peaks, including carbon, oxygen, lead, and uranyl, showed diffuse distribution, regardless of electron-dense granules.

epithelial cells to the lens capsule during secretion of the basal lamina at the germinative area of the lens. Most histochemical or imaging studies of WD that have examined KeF rings and sunflower cataracts were performed by autopsy of the liver and the brain (Johnson and Campbell, 1982; Tousimis and Adler, 1963; Tso et al., 1975; Uzman and Jakus, 1957). These autopsy studies tend to be limited because postmortem autolytic changes preclude more definite interpretation. In this study, we exclude postmortem changes because the anterior capsule was obtained from the biopsy of a living patient during cataract surgery. There are definite advantages in collecting samples for examination from the lens equator region during this type of procedure, but it is against medical ethics. There are some limitations to the present study. Theoretically, EDS analysis is able to detect all elements from atomic number 4(Be) to 92(U). Elements with atomic numbers less than 11(Na), Table 1 Relative element composition by atomic percentage (copper, sulfur) of electrondense granules from unstained sections. Point

Carbon (atomic %)

Copper (atomic %)

Sulfur (atomic %)

Copper/sulfur atomic ratio

1 2 3 4 5 6 7 8 9 10 11 Fig. 6a Fig. 6b

90.07 83.64 91.21 91.49 92.61 92.47 90.83 85.32 87.46 89.81 92.09 89.43 88.51

7.44 12.11 6.50 6.73 5.82 5.71 7.02 11.46 8.84 7.82 6.25 8.20 8.76

2.49 4.25 2.29 1.78 1.57 1.82 2.15 3.22 3.70 2.37 1.66 2.37 2.73

2.99 2.85 2.83 3.78 3.71 3.14 3.26 3.56 2.39 3.30 3.78 3.46 3.21

however, cannot be analyzed by EDS. The reason for this is because the excitation volume of light elements is generally larger than the excitation volume of heavier elements (Berlin, 2011). Because copper can bind to metallothionein, a protein containing nitrogen, EDS analysis should reveal the presence of nitrogen. For the above reason, however, nitrogen was not detected. Sunflower cataracts are very rare compared to KeF rings in WD, and because patients without visual disturbance are not considered for surgical procedures, more specimens of sunflower cataracts were unfortunately not obtained. This is the first study to introduce elemental analysis of single electron-dense particles in sunflower cataracts using EDS capable of analysis of 0.1 nm units in the ophthalmic area. As previous studies have shown, KeF rings are composed of copper and sulfur. This research reports that copper and sulfur are first demonstrated in sunflower cataracts, but also presents the chemical characteristics of electron-dense particles. In addition, the component proportional ratio of each electron-dense granule of both copper and sulfur is analyzed herein. In conclusion, sunflower cataracts associated with WD are assumed to be the result of the accumulation of heterogeneous compounds composed of several materials, including copper, sulfur, and/or copper-binding proteins. We suggest that the accumulation of electron-dense granules is related to both diffusion into, and synthesis of, the lens capsule by lens epithelial cells. If the condition of copper accumulation of sunflower cataracts and KeF rings were to be analyzed in multiple organs, it would be easier to understand the pathophysiology of WD from an ophthalmic perspective. Conflict of interest Nothing to report.

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Acknowledgments The authors thank Korea Basic Science Institute (KBSI) for microtoming and preparing the grid for TEM and the Korea Institute of Science and Technology (KIST) for analysis of elemental composition with the TitanÔ 80e300 by EDS. Additional thanks to Jeong Kee Seo, M.D. (Division of Pediatric Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, College of Medicine, Seoul National University, Seoul, Korea) and Byung Ik Kim, M.D. (Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University College of Medicine, Seoul, Korea) for their valued comments on the manuscript.

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Johnson, R.E., Campbell, R.J., 1982. Wilson’s disease. Electron microscopic, X-ray energy spectroscopic, and atomic absorption spectroscopic studies of corneal copper deposition and distribution. Lab. Investig. 46, 564e569. Nordberg, M., Nordberg, G.F., 2009. Metallothioneins: historical development and overview. Met. Ions Life Sci. 5, 1e29. Parmigiani, C.M., McAvoy, J.W., 1991. The roles of laminin and fibronectin in the development of the lens capsule. Curr. Eye Res. 10, 501e511. Roberts, E.A., Schilsky, M.L., 2008. Diagnosis and treatment of Wilson disease: an update. Hepatology 47, 2089e2111. Siemerling, E., Oloff, H., 1922. Pseudosklerose (Westphal-Strümpell) mit Cornealring (Kayser-Fleischer) und doppelseitiger Scheinkatarakt, die nur bei seitlicher Beleuchtung sichtbar ist und die dem nach Verletzung durch Kupfersplitter entstehenden Katarakt ähnlichist. Klin. Wochenschr. 1, 1087e1089. Silver, P.H., Wakely, J., 1974. The initial stage in the development of the lens capsule in chick and mouse embryos. Exp. Eye Res. 19, 73e77. Tousimis, A., Adler, I., 1963. Electron probe X-ray microanalyzer study of copper within Descemet’s membrane of Wilson’s disease. J. Histochem. Cytochem. 11, 40. Tso, M.O., Fine, B.S., Thorpe, H.E., 1975. Kayser-Fleischer ring and associated cataract in Wilson’s disease. Am. J. Ophthalmol. 79, 479e488. Uzman, L.L., Jakus, M.A., 1957. The Kayser-Fleischer ring; a histochemical and electron microscope study. Neurology 7, 341e355. Wiebers, D.O., Hollenhorst, R.W., Goldstein, N.P., 1977. The ophthalmologic manifestations of Wilson’s disease. Mayo Clin. Proc. 52, 409e416. Young, R.W., Ocumpaugh, D.E., 1966. Autoradiographic studies on the growth and development of the lens capsule in the rat. Investig. Ophthalmol. 5, 583e589.

Elemental analysis of sunflower cataract in Wilson's disease: a study using scanning transmission electron microscopy and energy dispersive spectroscopy.

Signature ophthalmic characteristics of Wilson's disease (WD) are regarded as diagnostically important manifestations of the disease. Previous studies...
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