To properly interpret the endothelial photomicrographs obtained clinically, it is helpful to understand the optical principles of the specular microscope.9 Light striking a surface can be reflected, transmitted, or absorbed. Generally, some combination of the three effects occur, with the relative proportions depending on such conditions as the wavelength of the light, the relative transparency of the medium below its surface, and the relative refractive indices on each side of the surface. Of primary importance in clinical specular microscopy is the light that is reflected specularly (i.e. “mirror-like”) where the angle of reflection is equal to the angle of incidence. For the normal transparent cornea, most visible light incident on the epithelial surface is transmitted. As the light passes through the corneal tissue, some of it can be absorbed by the tissue and some can be reflected by nerve fibers, keratocytes, and other refractile objects (i.e. objects having a different index of refraction than the bulk corneal tissue). In the stroma of the normal cornea, most of the incident light is transmitted through the tissue, although a small amount is absorbed and/or scattered (reflected through arbitrary angles) by cellular organelles. With an increase in corneal edema the fraction of scattered light increases and can become the dominant element thus giving rise to a “hazy” cornea. As light strikes the posterior corneal surface, almost all of it is transmitted into the aqueous humor. Because there is a change in index of refraction at the endothelium-aqueous humor interface, about 0.022 per cent of the total incident light is reflected; this reflected light is captured by the clinical specular microscope and forms the endothelial image.
As the illumination beam of the specular microscope passes through the cornea, it encounters a series of interfaces between optically distinct regions. At each of these interfaces, some light is reflected back toward the photomicroscope and some is transmitted deeper into the cornea. The greater the difference in index of refraction between the two regions, the greater the amount (intensity) of the reflected light.The more edematous the tissue, the greater the intensity of scattered light. A portion of the reflected and backscattered light is collected by the objective lens of the specular microscope and forms, at the image plane of the microscope, an image of that part of the cornea on which the instrument is focused.
Figure above shows a narrow slit of light from a specular microscope that is focused onto the posterior corneal surface. The incident light illuminates, in turn, the precorneal tear film or a coupling medium (e.g., artificial tears, methyl cellulose, etc.) between the objective lens and the cornea, the epithelium, Bowman's membrane, the stroma, Descemet's membrane, the endothelium and the aqueous humor. Within each of these regions, in the absence of corneal edema, only a small amount of light is scattered back towards the image plane, while at the major optical interfaces (labeled 1,2,and 3) much more light is specularly reflected back toward the image plane. Using indices of refraction for the objective lens, saline, cornea, and aqueous humor of 1.517, 1,333, 1,376, and 1.336, respectively, the fraction of light reflected from each of these interfaces can be calculated to be 0,36% from the objective lens-saline interface, 0.025% from the saline-corneal epithelium interface, and 0.022% from the corneal endothelium-aqueous humor interface. Intracorneal optical interfaces (e.g., between epithelium and Bowman's membrane or between stroma and Descemet's membrane, etc) also reflect light, but the fraction of reflected light cannot be calculated because the index of refraction of the separate layers of the cornea has never been measured. At the image plane of the specular microscope, light from various corneal regions and interfaces overlaps. Whenever a bright region and a dark region overlap, the dark region is not seen. If a sufficiently narrow slit of incident light is used, one can generally distinguish a bright region (Zone 1), part of the stromal region (Zone 2), the endothelial region (Zone 3), and part of the aqueous humor (Zone 4). Zone 1 is formed by light reflected from the lens-coupling fluid or the coupling fluid-epithelial interfaces or both, depending on the index of refraction of the coupling fluid used. The demarkation line between Zone 3 and Zone 4 that separates the illuminated cornea from the nonilluminated structures located more posteriorly., is called the dark boundary (see figure above)9. One side of the boundary is dark because negligible light is scattered from the aqueous humor. In contrast, the demarkation line between Zone 2 and Zone 3 is called the bright boundary. This boundary separates the endothelial reflection from the overlying illuminated corneal stroma. Since some light is scattered from the stroma, neither side of the boundary is dark. Of clinical importance is the width of the slit of light projected onto the cornea by the specular microscope. If the angle of incidence of the illuminating source is increased, a wider slit can be used and a larger field of endothelial cells can be seen (Figure B). However, the wider slit beam also illuminates more of the corneal tissue anterior to the endothelium, so that the volume of "interfering stroma" increases and more light is scattered back to the image plane of the specular microscope. The net result is a decrease in contrast of the endothelial image and a loss of cellular definition. Although the use of a wide slit gives a larger field of cells it does so at the cost of lower image. This trade-off between the contrast of the photograph and the number of cells within a single frame is not due to an optical limitation of the instrument but rather to light scattering in the tissue overlying the endothelium. At large angles of incidence normal endothelial cells appear shortened in one resulting in a distorted endothelial cell pattern, and this should be compensated for in morphometric analysis. In scanning slit and scanning spot confocal specular microscopes, the slit width (or spot diameter) is made very small to give, at least in theory, an increased image quality and the large field desired is accomplished by moving the illuminated slit (or spot) over the endothelium, stromal nerves, keratocytes, etc. Using a narrow slit four distinct zones are seen; with a wide slit only three zones are apparent. A bright zone, Zone 1, arises from the reflection at the objective lens-epithelial cell interface. Zone 2 arises from light diffusely scattered from corneal stroma and is present only in narrow slit photographs. This zone is darker in clear corneas and brighter in edematous corneas. Zone 3 shows the endothelial cell pattern produced by light specularly reflected from the posterior corneal surface, while Zone 4, the dark zone, is the product of light scattered from the aqueous humor. Since little light is scattered in the aqueous humor and virtually no light from the region normally returns to the collection optics of the microscope, Zone 4 is generally dark. In eyes with considerable debris in the anterior chamber this zone occasionally can be brighter and show some structure, but in most instances it is uniformly dark. |
References: 9. Laing R, Sandstrom M, Leibowitz H. Clinical specular microscopy:.I. Optical principles. Arch Ophthalmol. 1979;97:1714.
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