|Year : 1983 | Volume
| Issue : 5 | Page : 476-485
Application of the scanning electron microscopy in ophthalmic research
Department of Ophthalmology, University of Toronto, Canada
P K Basu
Department of Ophthalmology, University of Toronto, 1 Spadina Crescent, Toronto, Ontario M5S 2J5
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Basu P K. Application of the scanning electron microscopy in ophthalmic research. Indian J Ophthalmol 1983;31:476-85
|How to cite this URL:|
Basu P K. Application of the scanning electron microscopy in ophthalmic research. Indian J Ophthalmol [serial online] 1983 [cited 2019 Aug 23];31:476-85. Available from: http://www.ijo.in/text.asp?1983/31/5/476/29526
| Introduction|| |
The eye consists of many layers of tissues. Therefore, its constituents present many surfaces, the integrity of which are essential for the maintenance of the health of the organ. The surfaces of the ocular tissues alter in diseases and thus, a study of these surfaces become important in the understanding of the pathological process. For this reason, scanning of the surfaces of the cornea, conjunctiva, iris, lens, retina and optic nerve head is routinely done by the ophthalmologist. The surfaces of the eye tissues are generally studied under the slit lamp, specular microscope and ophthalmoscope. These instruments do not give a great magnification. Pathologists can study the ocular tissue surfaces under a dissecting microscope under a greater magnification. For a pathological diagnosis, he, however, depends mainly on the light microscopy. Occassionaliy he uses the transmission electron microscope. However, these techniques are not convenient to study the topography of a tissue unless a very large number of serial sections are examined. Some tissues can be however, examined under the light microscope using a flat preparation  but for various technical reasons this technique has limited use and cannot be applied to all kinds of ocular tissue.
The scanning electron microscopy (SEM) in recent years is slowly but steadily becoming an important research tool in ophthalmology. This is because with this method, the ocular tissue surfaces can be observed not only under a great magnification but also in three dimensions.
In this presentation, I shall give a brief account of some of my investigations to illustrate how SEM has been very useful to me in the understanding of certain ocular pathological processes.
I am interested in devising methods to determine the adverse effects of the air and water pollutants, drugs and contact lenses on the surfaces of the conjunctiva and cornea.
The conjunctival and corneal surfaces are normally covered with microplicae and micro ridges [Figure - 1]. These surface furnishings, the intercellular junctions, orifices of tear ducts and mucous glands in the conjunctiva, and certain hole-like structures on the epithelial cells can be clearly seen by the SEM in three dimensions.
In order to see the effect of cigarette smoke on the cells of the external ocular surfaces, in one experiment, we placed hamsters in a smoke chamber. Cigarette smoke produced a matting of the surface ultrastructures  The result of this study gave an explanation of what we had observed in human volunteers who were exposed to cigarette smoke. In this human study, we found that the smoke caused a lowering of the tear break-up-time.  This abnormality is probably related to the ocular irritation that we feel when we enter a smoke-filled room. From our SEM study, we hypothesized that an alteration of the microplicae systems of the external ocular tissues could be a reason for the impairment of the tear holding capacity by the conjunctival and corneal cells, resulting in the uncovering of the sensitive ocular nerves.
I have been studying the effects of various methods of storage of the cornea in the Eye Bank. Some of the works in which the SEM was specially useful are briefly described below.
The corneal endothelium is a monolayer of cells. Its viability and integrity are vital for the clarity of the cornea and thus for the success of the corneal grafting operation. We have been testing various preservation methods trying to determine the best one that will keep this important cellular layer from autolysis and other kinds of damage.
The endothelial cells are beset with microvilli. Their intercellular borders are clearly defined [Figure - 2]. When the cells break down due to adverse treatment, the sequence of the damaging effect can be very clearly seen with the SEM.
We used the SEM extensively in studying the effects of various agents on the prevention of corneal endothelial damage during storage-
We found that the steroids (probably acting as a lysosomal membrane stabilizer) prevented structural alteration of the endothelial cells of stored donor eyes [Figure - 3][Figure - 4].
Donor eyes are kept and transported routinely in eye bank bottles in a moist atmosphere. Due to human error, sometimes eyes come in dry bottles. We studied the effects of drying of the corneal surface. For this experiment, one eye of a donor was kept in a moist chamber and the other in a dry chamber for a given time and temperature, and then the cornea surfaces were examined by the SEM. We found that drying of the cornea not only affected epithelium adversely, but also damaged its endothelial surface [Figure - 5][Figure - 6][Figure - 7].
We also used the SEM for studying the effects on the corneal endothelium of the movement of the donor eye during long distance transportation. We found the corneal endothelium is not affected by the movement of the donor eye.
We tested by the SEM the edge of corneal grafts cut by different methods front the donor eye. We tested a punch technique developed in our laboratory.  We confirmed that when the cornea after being isolated from the donor eye was placed on a firm silicone surface, and then was punched from the endothelial surfaces, the graft had a very uniform and regular edge with minimum damage to the adjoining cells [Figure - 8].
Transplantation reaction is one of the main causes of corneal graft failure. Cytotoxic cells e.g. lymphocytes, macrophages and in the early stage of the reaction, the neutrophiles, come into the anterior chamber of the eye from the blood and then attack the graft endothelium.
In an organ culture model,  we studied the interaction between the endothelial cells and specifically sensitized lymphocytes in organ culture [Figure - 9][Figure - 10]. The nature of damage caused by the cytotoxic lymphocytes on the target corneal cells could be clearly observed by the SEM. Similar experiments using the corneal endothelial cells in tissue culture - [Figure - 11] gave identical results.
For the cytological diagnosis of certain kinds of external eye diseases e.g. conjunctivitis and corneal ulcer, cellular samples are taken by pressing a cover slip on the target area.  We used the SEM to study the nature of adhesion of cells to different kinds of cover slips to determine the best kind of material that would collect the greatest number of ocular and blood cells for examination [Figure - 12].
Iris and trabecular meshwork
We have been studying the effectiveness of laser treatment for glaucoma.  The operations consisted of making holes through the iris, and the trabecular meshwork at the angle of the anterior chamber. The SEM gave a clear demonstration about the efficiency of the machine in making holes through the target tissues.. Minute haemorrhagic spots at the angle of the anterior chamber was revealed by SEM [Figure - 13].
In connection with the laser treatment of the iris' it was important to see whether or not the treatment damaged the lens and zonules. The SEM study showed that they were not injured.
With SEM the nature of the damage to the corneal endothelium in a case of chronic iritis was readily studied [Figure - 14]. In a case of a premature infant that died of congenital heart disease, we found that trabecular meshwork were covered with a filmy material, the origin and significance of which is not known [Figure - 15].
Neural Retina and Retinal Pigment Epithelium
As our eye bank receives eyes of many kinds of donors, we had the opportunity of examining the surfaces of the retina and retinal pigment epithelium of some eyes of donors who had unusual medical histories. One eye came from a patient having toxoplasmosis. Another eye came from a victim of retinitis pigmentosa. In the first eye definite retinal holes were seen in the macular region. In the other eye, evidence of degeneration of rods and cones of the retina and damage to the surface ultrastructures (e.g. the microvilli) of the retinal pigment epithelium (RPE) were seen. The second eyes, because of the spontaneous damage of the RPE, we had an opportunity to visualize the inner surface of the Bruch's membrane. As this aspect of my study has been presented in the Symposium on Macula, I refrain to elaborate this topic here.
We have been studying the mechanism of phagocytosis of foreign particles, including the outer photoreceptor segments [Figure - 16] by the retinal epithelial cells using different models. We used the SEM to study the adhesion of latex particles (0.8-11 µm) during their phagocytosis on to the surface of the RPE cells as well as to see the effect of washing in the removal of the extracellular particles from the cell surface. By this technique we were also able to identify phagocytosed intracellular particles both in organ culture  and tissue culture  and differentiate the round latex particles from the fusiform melanin pigments.
Suture reaction and suture degeneration
We are attempting to determine which type of corneal and conjunctival sutures produce the least tissue reaction. In tissue reaction, leucocytes are attracted to the suture. With the use of the SEM we have found that the sutures which produce a greater tissue reaction in vivo tended to be associated with a greater number of polymorphonuclear leucocytes if both are brought together in vitro. The cellular attachments to transparent sutures e.g. nylon suture, can be determined to some extent by the light microscopy but the cells on an opaque suture such as the silk and catgut sutures cannot be visualized by it. In this connection, the SEM has been of tremendous help to us visualizing cells as well as platelets on both the transparent and opaque sutures [Figure - 17].
For reasons yet unknown, some sutures fall off from the conjunctival and corneal wound sites following cataract surgery and corneal grafting. Using the SEM we have been studying the nature of damage in these fallen off sutures that we have been collecting from human patients.
Different kinds of deposits accumulated on the contact lens surface and these may give rise to ocular irritation if these contact lenses are worn. We have studied a patient who had worn contact lenses and had a unilateral ocular irritation. Using the SEM, when we compared the contact lenses of two eyes we found a greater amount of deposits of different sizes and patterns at the peripheral regions of the anterior surface of the contact lens of the irritated eye [Figure - 18]. The nature of these deposits could be further analysed by the elemental analysis which involved the SEM. This we plan to do in our future studies on the rejected contact and intraocular lenses.
In conclusion, I have tried to give you some examples to show how I have benefited in my research on ocular tissues by using the SEM. I have been able to visualize at a very high magnification the surfaces of many of the eye tissues. One disadvantage that I have felt was that the SEM is an expensive proposition. Another disadvantage was that the identification of cells was not always easy and that artifacts may be sometimes very confusing. However, when the SEM is used in conjunction with other techniques, it offers new information and thus opens up new lines of investigation.
| Summary|| |
The purpose of this report is to show how the author has profitably applied the scanning electron microscopy (SEM) in his research on the surface ultrastructure of different healthy and diseased eye tissues of humans and animals. The SEM has been useful in studying not only the surface of a tissue in situ but also the surfaces of the ocular cells in the tissue and organ cultures. The SEM has been valuable in the studies on the surface of ophthalmic sutures with reference to their role in tissue reaction and their postoperative degradation. The SEM was also useful in the examination of the contact lens deposits with relation to eye irritation.
When used in conjunction with other methods of investigation, the SEM technique offers new information and thus opens up new fields of investigation.
| Acknowledgements|| |
I am thankful to Prof. F.W. Doane, Miss N. Anderson and Mr. K. Schultes, Department of Microbiology and Parasitology, Mr. Eric Lin, Department of Zoology, and Mr. Peter Lea, Electron Microscopy of the Faculty of Medicine, University of Toronto, and Dr. P.K. Basrur, Department of Biomedical Sciences, University of Guelph, for various assistance in my investigations. Dr. M. Avaria, Mr. S.M. Hasany, Mr. F. Carre and Mr. A Cutz gave valuable assistance.
| References|| |
Basu, P.K., Hasany, S.M., Doane, F.W. and Schultes, K. : Can steroid reduce endothelial damage in stored corneas? Effect on cell viability and ultrastructure. Can. J. Ophthalmol., 13: 31-38, 1978.
Basrur, P.K. and Basu, P.K. : The effect of cigarette smoke on the surface structure of the conjunctival epithelium. Can. J. Ophthalmol., 15: 20-23, 1980.
McCulloch, C.: A silicone plastic cup' for cutting corneal transplants. Can. J. Ophthalmol., 7: 366-367, 1972.
Basu, P.K., Hasany, S.M. and Ohashi, K.. : A corneal model for studying the interaction between the endothelium and sensitized lymphocytes. Can. J. Ophthalmol., 14: 29-42, 1979.
Hershenfeld, S., Kazdan, J.J., Macer, K., FeugasP., Basu, P.K., Basu. P.K., Avaria, M.: Impres, sion cytology in conjunctivitis. Can. J. Ophthalmol., 16: 76-78, 1981.
Landecker, L., Mortimer, C. and Basu, P.K. Laser iridectomy and trabeculectomy in monkeys. A morphological study. Can. J. Ophthahnjl., 13: 45-49, 1978.
Rosenstock, T., Basu, P.K. and Ranadive, N.S. Quantiative assay of phagocytosis by retinal pigment epithelium. An organ culture model. Experimental Eye Research, 30: 719-729, 1980.
Basu, P.K., Sarkar, P., Menon, I., Carre, F. and Persad, S. : Bovine retinal pigment epithelial cells cultured in vitro: growth characteristics, morphology, chromosomes, phagocytosis ability. tyrosinase activity and effect of freezing. Submitted for publication, 1981.
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7], [Figure - 8], [Figure - 9], [Figure - 10], [Figure - 11], [Figure - 12], [Figure - 13], [Figure - 14], [Figure - 15], [Figure - 16], [Figure - 17], [Figure - 18]