Scanning electron microscopy in retinal research.

The literature on scanning electron microscopy (SEM) pertaining to the retina has been surveyed and described. The first two papers on SEM and retina appeared in 1969. Most of the earlier studies concentrated on descriptions [by SEM alone, or with light microscopy (LM) and transmission electron microscopy (TEM)] of the appearance of various retinae and retinal cells [fish, newt, primates, rodents and rabbits, bullfrog]; or embryology of the chick retina. Two papers dealt with retinal disease. In all there were 25 papers in SEM/retinal research before and in 1974. Since 1975 there have been 111 papers which have used SEM in retinal research in very many different fields and in diverse ways, viz: the morphology of the retina, specific retinal cells, and the retinal vessels; embryology; labelling of retinal cells with surface markers; naturally occurring and experimentally-induced retinal disease; the effects of light, including lasers and photocoagulation, and other radiation on the retina, and phagocytosis and the retinal pigment epithelium (RPE). There have also been a number of papers concerned with techniques for SEM, applicable to retinal research, and several review articles. SEM has proven to be a very valuable adjunct to TEM in retinal research and it has become an accepted and almost routine way of extending and enhancing reports on retinal research. Properly used, SEM provides a laconic way of presenting information that might otherwise require many serial sections and long explanations.

Scanning electron microscopy of monkey foveal photoreceptors.

Rhesus monkey retina and especially the foveal photoreceptors (PR) were investigated by scanning electron microscopy (SEM). 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 gold-coated. 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 Müller 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. Immediately vitreal to it, the Müller 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. OS break off and remain embedded among the pigment epithelium microvilli (PEM) more often than PEM remain attached to OS distal ends. The foveal OS tapers slightly from its proximal to its distal end. The OS may bear knoblike swellings and convolutions in their more distal regions but not at their tips.

The ultrastructure of monkey foveal photoreceptors, with special reference to the structure, shape, size, and spacing of the foveal cones.

A systematic electron microscopic study was made of the structure of foveal cones of Macaca spp. Transverse sections of inner (IS) and outer segments (OS) were made in sequence, from the pigment epithelial zone (PEZ) to the outer limiting membrane (OLM). The smallest diameters of hundreds of cone sections were measured from electron micrographs with a Zeiss particle-size analyzer, and analyzed statistically. Some details are also included about Cebus photoreceptors. It is claimed in the literature that foveal cones are rod-like (cylindrical) and untapered. Our study shows the foveolar cone to be a tapered structure. There has been some confusion between the foveola, which is rod-free, and the fovea, which has a high concentration of cones, but is not rod-free. Within the fovea, as the ratio of cones to rods falls from infinity to 1, with distance from the central bouquet of cones, the cone center-to-center distances increase, the inner segment diameters increase, and the number of cones/sq mm decreases. The tapered calycal processes are more massive in M. irus than M. mulatta, and the lateral fins are better developed. Lateral fins are not present in the foveola. The cones are arranged in straight lines.

Application of optical transform techniques to laser irradiation of retina.

Novel retinal irradiation techniques for laser damage studies are described; these are straightforward applications of the Fourier transform properties of an optical system. Interposing an appropriate diffracting screen between the laser source and the test eye enables one to simultaneously induce a number of discrete and precisely located retinal exposures with a known gradation of energy. Various kinds of diffracting objects are considered, and it is seen that transparent sinusoidal phase gratings offer the most promise for damage threshold studies. The method provides an objective measure of the ocular focal length at the time of the exposure. The production of arbitrary retinal irradiation patterns for more general research or clinical applications is possible with the extension of the technique by the use of simple holographic methods for forming the appropriate diffracting screens.

High resolution microchemical analysis using soft X-ray lithographic techniques.

High resolution x-ray lithographic studies of cells from chick embryo hearts dried by the CO2 critical point method have been made with soft x-ray radiation of different wavelengths. A marked difference in the relief replica in polymethyl methacrylate (PMMA) resulting from the differential absorption by the dried cells of carbon K alpha radiation at 4.48 nm and broad band synchrotron radiation (SR) with lambda is greater than 1.5 nm demonstrates the potential usefulness of the technique in making high resolution (approximately or equal to 10 nm) chemical identification of the constitutents which make up the various parts of the cell.

Convolution in human rods: an ageing process.

In a morphological survey of 73 human retinae spanning 9 decades, and including 20 retinae which were obtained from eyes enucleated for malignant melanomas, nodular excrescences were noted in the outer segments of rods with an incidence which increased with age. These structures were sectioned in both their vertical and horizontal axes and on electron microscopical examination were seen to result from the localised convolution of affected outer segments. The topographic morphology of such convolutions is described and their modes of formation are discussed.

Scanning electron microscopy of normal and lased rabbit pigment epithelium.

Scanning electron microscopic (SEM) observations of the rabbit retinal pigment epithelium (RPE) are reported both for normal tissue and for tissue subjected to laser radiation. In normal RPE, cells were observed to be closely packed and to have the characteristic hexagonal shape. The microvilli were abundant and of two types. Suprathreshold lesions were produced with 0.4 musec pulses of high-intensity yellow light (585 nm.) from a dye laser. In the lased RPE, cells were denuded of their microvilli, and a number of cells with single discrete holes were observed. Hypertrophied cells were always present. Within 5 days of irradiation groups of small proliferating RPE cells were found in the lesion area.

Fusing human rod outer segments from an eye enucleated for choroidal melanoma.

In the retina from an eye enucleated for choroidal melanoma apposed rod outer segments were seen by transmission electron microscopy to be fusing together in groups by the confluence of their plasma membranes and the formation of cytoplasmic bridges between adjacent outer segments. These fusions were seen in areas distant from the melanoma but where the retina was not normal in appearance.

Scanning electron microscopy of normal and lased rabbit retina.

This study presents the topography as seen by scanning electron microscopy of the rabbit retina in general and the photoreceptors in particular; and of large laser lesions in the retina.