THE ANATOMICAL RECORD 205~363-37311983) Scanning Electron Microscopy of Monkey Foveal Photoreceptors BESSIE BORWEIN Department ofdnatomy, University of Western Ontario, London, Ontario, Canada N6A 5Cl ABSTRACT Rhesus monkey retina and especially the foveal photoreceptors (PR) were investigated by scanning electron microscopy (SEMI. There are a few scattered SEM photomicrographs of the primate retina in the literature but this is the first detailed and comprehensive view by SEM of a primate retina. Some new aspects of surface morphology are displayed and the study also highlights and emphasizes some aspects of photoreceptor structure that have either been overlooked or not clearly displayed in studies using transmission electron microscopy only. For examination by SEM retinas were fixed in glutaraldehyde-formaldehyde, osmicated, immersed in thiocarbohydrazide, dehydrated in alcohols, and goldcoated. The fovea appears as a sharply defined pit with steep slopes, and its vitreal surface looks different from that of the rest of the retina. It appears to have a matted surface. The rest of the vitreal surface is relatively smooth and displays distinct lines which diverge in a radiating pattern from the foveal slopes. The choroid has a spongelike appearance; the sclera appears fibrous with the fibers running parallel to the vitreal surface. Photoreceptor nuclei are sometimes lost during tissue processing. They leave a discrete “nuclear nest” formed from Muller cell processes. Henle fibers turn at a sharp angle from the cones to run parallel to the vitreal surface. The external limiting membrane is seen as a clear line. Immediatelv vitreal to it, the Miiller cell microvilli surround the proximal inner segments. The cone inner segment (CIS) narrows toward the cilium where the cell is markedly constricted. The ciliary connectives are aligned and appear as a shadowy, slightly wavy zone when the retina is viewed in vertical section. The freestanding, tapering calycal processes (CP) arise from and are continuous with longitudinal CIS ridges. CP surround the proximal parts of the outer segments (OS), but there are no CP around the ciliary backbone. Some CP bear small protrusions. 0s break off and remain embedded among the pigment epithelium microvilli (PEM) more often than PEM remain attached to 0s distal ends. The foveal 0s tapers slightly from its proximal to its distal end. The 0s may bear knoblike swellings and convolutions in their more distal regions but not a t their tips. There are very few SEM studies of normal photoreceptors of monkey (Borwein et al., 1980; Dickson et al., 1973; Kuwabara, 1970; Smith and Finke, 1972; Wickham and Adams, 1979) and man (Breipohl et al., 1974b; Follman and Radnot, 1979; Kuwabara, 1970; Radnot, 1978; Wickham and Adams, 1979). Very few good photographs are available. Several of these studies provide only one or two pictures (Breipohl et al., 1974b; Follman and Radnot, 1979; Kuwabara, 1970; Radnot, 1978; Smith and Finke, 1972) and one is an abstract without illustrations (Wickham and Adams, 1979). 0 1983ALAN R. LISS, INC. This paper presents details about monkey foveal retinal ultrastructure as seen by scanning electron microscopy (SEMI that have not been presented before, and it also corroborates and elaborates some aspects of primate photoreceptor morphology that have been established by studies using transmission electron microscopy (TEM) (Cohen, 1963, 1972; Dowling, 1965; Dunn, 1973; Fine and Yanoff, 1979; Hogan et al., 1971; Missotten, 1965; Young, 1969). It is the first extensive Received April 20,1982; accepted November 11, 1982. 364 B. BORWEIN view by SEM of a primate retina and enhances our visual conceptions of these structures. It extends our knowledge of primate photoreceptors. It is specifically complementary to a previous TEM study of monkey foveal photoreceptors by the author and coworkers (Borwein et al., 1980). SEM provides photographs of photoreceptors that are not only striking but which also emphasize details of surface morphology that have been overlooked or underemphasized when the three-dimensional picture depended wholly or mainly on mental reconstructions from small, thin sections. The special advantage of the SEM is its depth of focus over a relatively large area a t fairly high magnifications. Hollenberg, 1973; Dickson and Hollenberg, 1971; Leuenberger, 1971; Radnot, 19781, the most commonly used procedure is aldehyde fixative, usually followed by osmication, and critical point drying. We have found that thiocarbohydrazide (Malick et al., 1975) improved the cleanliness and clarity of the surfaces. “In spite of shrinkage, however, the proportions, topography and integrity seem to have been maintained extremely well” (Lewis et al., 1969) compared with most of the techniques used for preparation of materials for SEM. All the animals were treated according to the protocol specified in “Guide for Care and Use of Laboratory Animals” (DHEW Publication No. NIH 78-23, 1978). MATERIALS AND METHODS Mature rhesus monkeys were first sedated with Sernylan and then killed by injection of Nembutal. The enucleated eyes were incised at the ora serrata and immersed in cold fixative (2.5% glutaraldehyde + 0.5% formaldehyde in 0.1 M Sorensen’s or cacodylate buffer, pH 7.4). As fixation proceeded, the cornea, lens, and vitreous were dissected away, in three successive stages, separated by 10-minute intervals. When the tissues were hardened (about 30 minutes) the fovea was removed with a trephine. Total fixation time in aldehydes was 1%hours at room temperature. The samples were rinsed in buffer, postfixed in 1% buffered Os04 for 1% hours, washed in several changes of distilled water, immersed in thiocarbohydrazide for 15 minutes (modified from Malick et al., 19751, washed again several times, postfixed in 1%Os04 for 1 hour, washed again and dehydrated in graded alcohols, critical point dried, coated with gold (20 nm) in a Technics Hummer sputter coater, and viewed in a Philips SEM 501 or a n Hitachi HHS-2R SEM a t 20 kV. Photographs were taken on Kodak plus-X film. A considerable degree of shrinkage occurs in the preparation of tissue for SEM, estimated by Lewis et al. (1969) and Ali and Wagner (1976) as about 30% greater than for plastic-embedded material. Hansson (1970~) tried several fixation methods and found that he got the most reproducible results with buffered glutaraldehyde and formaldehyde but good results were also obtained with 1% buffered osmium. I have surveyed the methods reported for SEM work and although airdrying was used (Antal, 1977; Borwein and OBSERVATIONS The fovea appears as a sharply defined pit with steep slopes. The floor of the pit is covered by matted material and it differs in appearance from that of the rest of the vitreal surface of the retina, where there is a pattern of striations radiating from the foveal slope (Fig. 1).The narrowing of the retina is clearly displayed where the inner layers thin out in the fovea. The region of the “central bouquet of cones” (Rochon-Duvigneaud, 19431, the foveola, is seen a t the thinnest part of the retina in the center of the fovea (Figs. 1, 2). The apical surface of the pigment epithelium is covered by a dense mat of microvillous processes Figs. 1, 3). The retina easily detaches from the pigment epithelium during tissue preparation and many outer segments break off and remain attached to the pigment epithelium, embedded among the microvillous processes (Fig. 3). Bruch’s membrane appears as a narrow, relatively homogeneous layer lying immediately below the pigment epithelium on its scleral side (Figs. 2, 3). The choroid has a spongelike appearance (Figs. 2 , 3 ) and the broad sclera appears fibrous with the fibers running approximately parallel to the vitreal surface (Fig. 2). The nuclei of the foveal cones are seen to be arranged in seven layers in Figure 4.Each nucleus is closely surrounded by a “nuclear nest,” which is known to be formed by Muller cell processes (Borwein et al., 1980). Occasionally, when a nucleus is lost during tissue preparation, it leaves a discrete, empty “nuclear nest,” with the inner and outer cone fibers intact and in place (Fig. 4). SEM OF MONKEY FOVEAL PHOTORECEPTORS The long, inner cone fibers, which form the Henle fiber layer of the fovea, turn at a very sharp angle from the inner and outer segments (Fig. 4).The external limiting membrane (ELM) (formed by a series of junctional complexes, zonulae adhaerentes, between photoreceptors and Muller cells) appears as a clear, thin line (Fig. 41,while the ciliary connectives are aligned and appear as a wider, shadowy zone extending across the photoreceptors at their inner-outer segment junctions, more or less parallel to the ELM (Figs. 4-6). This zone is made more apparent also because the inner segment tapers abruptly at this level, where the cilium is apparent, and there is thus more extracellular material present. Microvillous processes of Muller cells are of variable lengths and they project beyond the ELM to surround the most proximal (vitreal) parts of the inner segments of both rods and cones. They can be seen faintly in Figure 4 and are well displayed in Figure 5. The inner segment occupies the region between the external limiting membrane and the ciliary connective. The cell is constricted sharply at the region of the cilium, more markedly so away from the foveal center, the foveola (compare Figs. 5 and 7). The cone inner segment is wider in the parafoveal areas (Fig. 5) and it lengthens and narrows toward the foveola (foveal center) where cones only are present (Fig. 7). The rod inner segment and the foveolar cone inner segment change comparatively little in shape and size from the ELM to the ciliary zones (Figs. 5,7). In the very center of the fovea, in the foveola, the outer segments and inner segments are in a straight line and they are parallel to each other. On either side of these most central cones, the outer segments tilt slightly away from their inner segments in the direction away from the central cones (Fig. 6). The surface of the inner segment is longitudinally ridged and grooved (Figs. 5, 8, 10) and some of these ridges bear small knobs (Figs. 8, 10, 11).The ridges are most prominent a t the scleral ends of the inner segments and here they are seen to be continuous with the freestanding calycal processes which appear to arise from the ridges (Figs. 8, 10). The calycal processes surround the vitreal ends of the outer segments. They are broad where they originate, and they taper. They vary slightly in length (Figs. 810).They are prominent and long in the cones but extend for only a relatively short part of the total cone outer segment length (Figs. 9, 365 11).One or two (more rarely, three) calycal processes usually arise from one inner segment ridge (Figs. 8, 10). The ciliary connectives are aligned in the photoreceptors to form a shadowlike zone extending across the photoreceptors a t the cone inner-outer segment junctions, where the cells narrow (Figs. 3, 6, 7). The cilium has a very irregular surface outline. Two or three very short, stublike calycal processes are associated with it (Figs. 8,lO). In both rods and cones the cilium extends to become the ciliary backbone which lies alongside the discs on one side of the outer segment. There seems to be no particular pattern in the arrangement of the photoreceptors with regard to the positions of the cilia and ciliary backbones in their relation to the discs. The orientation seems random (Figs. 8, 10). There are no calycal processes around the ciliary backbone (Figs. 8 , l O ) . When the outer segment breaks off from the inner segment it does so above the region of the ciliary connective whereas the calycal processes often remain attached to the inner segment and project above the plane of the break (Fig. 11). Often the cone outer segments are seen to be irregular in outline, bearing nodules, or they are folded on themselves, mainly in their midregions and more distal parts. The nodules and folds are found scleral to the region of the calycal processes, but the distal tip itself is never involved (Figs. 3,7,9, 11). The long outer segments of the foveolar cones are slightly narrower a t their tips than they are immediately proximal to the cilium (Figs. 7, 11).The foveal cones are elongated, very closely packed together, and their inner and outer segments are of approximately equal lengths. There is a slight taper in the outer segments (Figs. 7, 11).When the inner and outer segments are considered together then the cell tapers more markedly (Figs. 3, 6, 7). Within the foveola there are cones only (Figs. 6, 7). Rods are present on the foveal slopes (Fig. 5) and increase in number toward the foveal periphery. At the foveal margin, more rods than cones are present (Fig. 5). Where rods and cones appear together, the rod inner segment is much narrower than the cone inner segment (Fig. 5). On the surface of the outer segment there can be seen imprints formed by the discs (or saccules) they contain (Figs. 8,9,12). When retinal detachment occurs during preparation of tissue, some outer segments break off. Most of the broken outer segments 366 B. BORWEIN SEM OF MONKEY FOVEAL PHOTORECEF’TORS remain embedded in the pigment epithelium microvillous processes, but some microvillous processes of the pigment epithelium break off and remain adhering closely to the distal outer segment tip (Fig. 12). DISCUSSION The basic pattern of organization of photoreceptor cells is remarkably similar in all classes of the vertebrate kingdom, but there is nonetheless a wide range of variations in these structures. SEM studies display these A bbrewations Bruch’s membrane ciliary backbone cone choroid ciliary connective or cilium calycal procesdes) discs external limiting membrane Henle fibers Miiller cell microvilli outer segment pigment epithelium rod sclera Fig. 1. A portion of the retina showing the foveal slope and pit. The detached pigment epithelium lies free below the outer segments. Note the thinning of the retina towards the foveal center. There is a pattern of radiating striations on the vitreal surface of the retina, starting on the foveal slopes, but not affecting the floor of the pit, the foveola. x70. Fig. 2. A portion of the fundus viewed in vertical section. There is an artefactual detachment in the foveal region. The inner retinal layers are thinned. The choroid (Ch) has a spongy appearance; the sclera (S)is fibrous. Bruch’s membrane (B) appears as a narrow structureless zone. ~ 1 7 0 . Fig. 3. A view of the inner and outer segments of the elongated foveal photoreceptors, seen in a vertical section through the retina. The cilia are aligned and appear as a shadowy, wavy zone (Ci). The outer segments (0s) bear nodules. Outer segments (arrow)can be seen on the detached pigment epithelium (P),which is separated by Bruch’s membrane from the spongy choroid (Ch). ~ 7 0 0 . Fig. 4. The region of the foveal photoreceptor nuclei. Empty nuclear nests (arrows), sharply angled Henle fibers (HI, the external limiting membrane (E), and the ciliary zone (arrowhead) are clearly displayed. The microvilli of the Miiller cells, immediately scleral to the external limiting membrane, are faintly discernible. x 1,400. 367 variations in size, shape, and surface morphology very well (Borwein and Hollenberg, 1973; Breipohl et al., 1973; Dickson and Hollenberg, 1971; Hansson, 1970c; Lewis et al., 1969; Pietzsch-Rohrschneider, 1976; Steinberg, 1973). Photoreceptors, pigment epithelium, and retina in general have been described by SEM more extensively in vertebrates below the Primates, but the sampling of the vertebrates i s rather limited. These studies fall into several categories: (1)studies of retinal and photoreceptor development of chick embryos (Breipohl et al., 1973, 1974a,b; Meller and Tetzlaff, 1976; Olson, 1975, 1977, 1979) and newborn albino rats (Galbavy and Olson, 1979; Garcia-Porrero and Ojeda, 1979); (2) studies of adult mammalian retina-normal and also in various experimental conditions-of albino rats (Hansson, 1970a-e; Leuenberger, 1971; Puzzola et al., 1978; Puzzola and de Simone, 1979), rabbits (Aoki, 1974; Antal, 1977; Borwein et al., 1976, 1977a; Leuenberger, 1971; Miki et al., 1976; Newton et al., 1980), mouse (Smith, 19731, and cattle (Molday, 1976); (3) descriptions of amphibian retinas of bullfrog (Steinberg, 19731, Necturus (mudpuppy) (Lewis et al., 19691, and newt (Dickson and Hollenberg, 1971); (4)studies of dark- and light-adapted retinas of fish (Ali and Wagner, 1976; Borwein and Hollenberg, 1973; Pietzch-Rohrschneider, 1976);( 5 ) studies partly or wholly describing the pigment epithelium of albino rats (Hansson, 1970a; Leuenberger, 1971; Puzzola et al., 1978; Puzzola and de Simone, 19791, chickens (Breipohl et al., 1973), rabbit (Borwein et al., 1977a; Leuenberger, 1971; Miki et al., 19761, bullfkog (Steinberg, 19731, and man (Goldbaum and Madden, 1982). There have been only a few SEM studies of primate photoreceptors. None is concerned specifically with the fovea, a n area of particular interest since it subserves normal color vision and photopic acuity. Knowledge of the detailed morphology of the foveal photoreceptors is of major importance to a n understanding of their function. Borwein et al. (1980) showed that both the foveal and foveolar cone cells taper slightly in their outer segments, and also when the inner and outer segments are considered together. The dimensions were derived from measurements taken of cross sections of foveal cone outer segments seen by TEM, a t various levels, from their proximal bases to their distal tips among the pigment epithe- 368 B. BORWEIN SEM OF MONKEY FOVEAL PHOTORECEPTORS 369 They become particularly prominent, in both rods and cones, as the junction of the inner and outer segments is approached. It has been suggested that the ridges are the outlines of the elongated inner segment mitochondria made apparent a s a result of tissue shrinkage. This is very unlikely. In Primates, the mitochondria are frequently absent from the cone inner segment apex, precisely the location where the ridges are most pronounced. In TEM micrographs the inner segment often shows a scalloped margin, but this is not always so. The ridges are always seen in SEM preparations and regularly present the same appearance at the IS0s (inner segment-outer segment) junction. The ridges may represent zones of a microtubular skeletal organization which may become more apparent and protrude on the surface when the rest of the inner segment shrinks during tissue processing. Engstrom (1963),in LM and TEM studies on fish, saw that the calycal processes “do not really begin at the outer tip of the ellipsoid but run like mouldings along the surface of the entire ellipsoid.” The ridges are within the inner segments but the calycal processes, though continuous with the longitudinal ridges, are Fig. 5. A view of the foveal photoreceptors toward the freestanding, and they surround the proximargin of the fovea, showing slender rod (R) and plump cone (C) inner segments, bearing longitudinal surface mal part of the outer segement in both rods ridges. The narrowing of the inner segment toward the and cones (Borwein et al., 1980; Brown et al., cilium is especially marked in the cones. Miiller cell 1963; Cohen, 1963).The calycal processes remicrovilli (MI surround the inner segments immediately main attached to the inner segment if the scleral to the external limiting membrane. ~ 2 , 0 0 0 . outer segment breaks off, demonstrating Fig. 6. A view of the central foveolar photoreceptors their continuity with the inner segments. In showing the external limiting membrane (E), the shadmost animals, both the calycal processes and owy line of the ciliary connectives (Ci), the nuclei 0, the inner segment ridges are larger and more and Henle fibers (H). Many cone outer segments can be seen embedded among the microvillous processes of the prominent in the cones than in the rods, as detached pigment epithelium (P). The central cone outer shown in this study. and inner segments are in a straight line. The outer By SEM it can be seen that calycal prosegments on either side of these tilt away from these cesses surround the outer segment except for central outer segments. ~ 4 0 0 . the part around the ciliary backbone, as had Fig. 7. The outer segments of the central cones seen been seen in cross sections of monkey photoin Figure 6 are magnified to demonstrate their knobs receptors viewed by TEM by Borwein et al. and folds, their generally irregular outlines, and their (1980), and by Reme and Young (1977) in slight taper from their proximal to their most distal ends. The external limiting membrane (E) and the ciliground squirrels. Calycal processes surround ary region (Ci) are clearly displayed. X 1,400. fish cones also but are not seen around its associated accessory-outer segment (Borwein Fig. 8. The foveal cones are seen at the region of the and Hollenberg, 1973; Fig. 9, Braekevelt, junction of the inner and outer segments. The calycal processes (CP) arise from the longitudinal surface ridges 1975). This comparison prompts the queson the inner segments and become free-standing struction: Is the accessory-outer segment homolotures which surround the proximal ends of the outer gous with the ciliary backbone? The SEM segments, except for the area around the ciliary backalso shows clearly that only very short calybone. The calycal processes vary slightly in length and are tapered. The discs (D) are clearly outlined on the cal processes are associated with the cilium surface of the outer segments. The cilium (Ci) has a very area, and this was demonstrated also in TEM uneven surface outline. A few very short calycal prostudies of cross sections of monkey photorecesses (arrowheads) and a ciliary backbone (CB) can be ceptors (Borwein et al., 1980). seen associated with the cilium. X 10,800. lial cell processes; and of cone inner segments from near the ELM and also a t the ellipsoid. It had become widely accepted that foveolar and foveal cones are rodlike. While a taper is very difficult to detect in longitudinal section, nevertheless a few workers had previously suggested that such a taper exists (for discussion on this see Borwein et al., 1980). The SEM pictures confirm that there is a slight taper in this very elongated cell. The longitudinal surface ridges of the inner segments have been described in many papers, both by LM and TEM (reviewed by Borwein, 1981). The inner segment ridges reported in this paper and by Borwein et al. (1980) in monkey can also be seen in SEM photomicrographs of the human cone cell (Kuwabara, 19701, monkey cones (Smith and Finke, 1972), mudpuppy cones (Lewis et al., 19691, newt cones (Dickson and Hollenberg, 19711, and in teleost rods and even more markedly so in the cones (Borwein and Hollenberg, 1973; Pietzsch-Rohrschneider, 1976). These longitudinal ridges start in the myoid. 370 B. BORWEIN SEM OF MONKEY FOVEAL PHOTORECEPTORS Ueck et al. (1978) described and illustrated by SEM “parallel oriented filamentous processes” (similar to calycal processes) surrounding the outer segment of pinealocytes of a teleost fish, projecting from the apical border of the inner segment and extending to the tip of the rather squat outer segment, and bearing small protrusions. This is the only other reference to protrusions such as we saw on the ridges near the calycal processes and the ciliary backbone (Figs. 9,lO). The present study and prior studies demonstrate that primate outer segments of both rod and cones are not always uniform but present “bulbous swellings and other deformities” (Polyak, 1957). They are often displayed in light micrographs (e.g., p. 34, Steinberg and Wood, 1979). Bonvein et al. (197723) reported what appeared to be fused human rod outer segments in cross sections, by TEM. These findings were later reinterpreted by Marshall et al. (1979), who described “nodular excrescences” and “convolutions,’ in human rod outer segments, with increasing incidence with age. These were seen in both longitudinal and cross sections, by LM and TEM. The nodular excrescences or knobs are areas of intact discs which are dislocated and rearranged within one outer segment, while the convolutions are corrugations in the long axis so that curves are formed by folds of ascending and descending Fig. 9. Calycal processes (CP) can be seen a t the proximal ends of the outer segments. Outer segment discs are apparent. Some of the outer segments are convoluted or folded (arrowheads) on themselves. Others bear nodular excrescences. ~ 4 , 0 0 0 . Fig. 10. A group of foveolar cones and one rod outer segment (R). Note the small knoblike extensions from the calycal processes, cilium, and inner segment surface ridges (arrows). The ciliary backbone (CB) has no overlying calycal processes. The cilium (Ci) has a very irregular surface and with it are associated a few very short calycal processes (CP). The long calycal processes show a distinct taper. x 10,800. Fig. 11. A group of foveolar photoreceptors several of which show the outer segments folding on themselves in their midregions (broad arrows). In the left-hand (upper) corner there are two photoreceptors, the outer segments of which have broken off, and calycal processes can be seen extending beyond the fracture plane (thin arrow). ~2,700. Fig. 12. Broken-off pigment epithelial cell microvillous processes are seen lying appressed to the distal tips of foveal cones. The discs of the outer segments are apparent. x 16,200. 371 portions of the outer segment. These convolutions and excrescences often contain normal-looking discs and were seen in otherwise well-fixed material; their distribution was discontinuous, and they appeared first in the paramacula in the fourth decade (Marshall et al., 1979). Borwein et al. (1980) reported “knobs” and convolutions also in monkey foveal cones, seen by TEM, and they are here displayed by SEM, in both rods and cones. The observations suggest that shedding and phagocytosis may not be well synchronized with disc renewal in older outer segments. These convolutions and knobs appear to be restricted to the midregion of the outer segment, beyond the calycal processes. The calycal processes may possibly function to keep the outer segment shape and orientation intact close to the base where the new discs are constantly being formed. SEM is a n ideal tool for investigations of the calycal processes, the Muller cell microvilli, and the overall cell shape because the full lengths and widths can be seen simultaneously. This SEM study has shown with special clarity several features of the calycal processes: That they taper, except for those two or three which are very short and stublike in the immediate vicinity of the ciliary connective; that there are no calycal processes around the ciliary backbone; that the calycal processes seem to be continuous with the longitudinal ridges of the inner segment; that if the outer segment breaks off near the inner segment-outer segment junction the calycal processes remain intact and attached to the inner segment. The study also shows the slight taper of the foveal cone outer segments, and it displays the convolutions and bulges of the outer segments which were shown by Marshall et al. (1979) to increase in frequency with age. While TEM yields rich information on the morphology of the cell interior and cellular associations, SEM is particularly valuable as an adjunct to TEM studies, in its emphasis on cell shape and surface features. ACKNOWLEDGMENTS I wish to thank Mrs. Artee Karkhanis and Mr. Stephen Smith for their excellent technical assistance. This research was supported by the U S . Army Medical Research and Development Command (No. DAMD 17-80-G-9466)and the Medical Research Council of Canada. The views and opinions herein do not necessarily 372 B. BORWEIN reflect the positions or decisions of the Army and no official endorsement should be inferred. LITERATURE CITED Ali, M.A., and H.-J. Wagner (1976) Scanning electron microsopy of four teleostean retinas. Rev. Can. Biol., 35: 199-210. Antal, M. (1977) Scanning electron microscopy of photoreceptors. Ophthalmologica, 174:280-284. Aoki, A. (1974) Experimental studies on ruby laser photocoagulation in the retina of pigmented rabbits. Part 4. Scanning electron microscopic findings in the retina immediately after photocoagulation. Nippon Ganka Gakkai Zasshi. 10t780-791. Borwein, B. (1981) The retinal receptor: A description. 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