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1 Reports A scanning slit optical microscope. DAVID M. MAURICE. A transparent tissue is illuminated with a narrow slit of light which is formed by light transmitted down one-half of a microscope objective, and the tissue is observed through the other half. The tissue is moved slowly across the light, and a photographic film is moved at a corresponding rate over a parfocal slit at the eyepiece level. In this way, thin optical sections are reproduced on the film, free from light scattered in the bulk of the tissue. Various comeal layers are illustrated, and a system of alternating light and dark zones in the stroma are revealed. An adaptation of the optical microscope has been previously described 1 which allows the corneal endothelium of the intact eye to be examined by means of the light reflected from its surface, so-called specular microscopy. In this system, light was passed down one-half of a 40x water immersion objective and, after reflection, was observed down the other half. It was necessary to use a relatively narrow slit of light focused on the endothelial surface in order to avoid detail in the image being obscured by light scattered from the overlying connective tissue, the stroma. Structures within the stroma can also be made out as the result of light returned in a combination of the dark-field and specular modes, but the image of the slit suffers worse degradation in this case as a result of interference from the tissue on both sides (Fig. 1). The narrower the slit the clearer the detail, and even in the case of the endothelium the maximum useful width is about 50 fim, which requires that a photographic montage has to be constructed if an extended view is to be obtained. This disadvantage can be overcome if a narrow slit is used and the specimen is slowly displaced across it, while at the same time a film is moved across a corresponding slit at the level of the eyepiece. If tv rate and directions of movement of the film correspond to the magnification of the eyepiece, details of the image remain in register on it during its exposure. A microscope has been adapted for this purpose in which the stage is shifted by means of a micrometer screw rotated by a synchronous motor (Fig. 2). An identical motor advances the 35 mm. film in the specially constructed camera by means of rollers which engage it along its edges. Pressure on the area of the emulsion used to record the image was found to produce artifacts. The microscope is focused by means of observation through a hole punched in the film, and the jaws of the camera Observation Illumination \ $mm>-j \ksiiimj Fig. 1. Diagram illustrating the principle of the instrument. Illumination of the specimen takes place through one-half of the objective and observation through the other, both fields being defined by parfocal slits perpendicular to the plane of the figure. When the slit is wide (right diagram), the details of the image are obscured by out-of-focus light scatter from the surrounding tissue. When a narrow slit is used (left diagram), the scatter is very much reduced, but the field of view is inconveniently small. A wider field is obtained by slowly moving the observed object through the focal slit and recording the image on a film which is undergoing a corresponding movement i v X \

2 1034 Reports lnoestigatioe Ophthdmdogy December 1974 Fig. 2. Diagram illustrating construction of the instrument. The specimen holder and the film advance rollers were turned by identical synchronous motors. A hole could be punched in the film through which the microscope was focused, and the jaws of the slit in the camera could be adjusted to correspond to those in the illuminating system. The take-up chamber can be detached and replaced so that film can be developed without having to readjust the camera. slit can be adjusted to correspond to those of the illun~ination while focusing on a reflecting surface under these conditions. Clutch mechanisms are provided for the rapid advance of the film and for the readjustment of the stage micrometer. A variety of photographs of the corneal tissues have been obtained in this way. The pictures of the endothelium in the specular mode are not only more extensive but improved in quality over those with the stationary slit; the marks on the surface do not appear to cast shadows but are sharply delineated (Fig. 3). Descemet's membrane can be optically isolated, and the existence of fibrous structures is revealed (Fig. 4). In the stroma the structure of the keratocytes can be seen (Fig. 5), which corresponds to that seen by staining." An unexpected discovery was the appearance of alternnting bands of greater and less scattering in the tissue which frequently form a straight and regular pattern (Fig. 6). When viewed in the specular mode, the epithelial surface also shows a variety of features that may be compared with its appearance in scanning electron microscopy (Fig. 7).3 This system is easy to adjust and use with a little experience. The illustrations were made with a scanning slit 3 pm wide and a specimen advance rate of 127 pm per minute. Tri-X film was used and was developed for 5 minutes in Acufine. The Zeiss 40x water immersion objective has a numerical aperture of 0.75, and geometry indicates that the depth of the optical section should be about equivalent to the slit width. This has been checked by serial photographs of a suspension of 1 pm diameter polystyrene spheres embedded in gel as the microscope was focused down in 1 pm steps. A sphere appeared only in three successive photographs, in general. It would be surprising if a similar instrument had not been thought of previously, but I am aware of no photographs of similar quality in the literature. Another group has been working on similar systems4~ using spot scanning through the objective system. A close examination of the principles on which they are constructed shows

3 Volume 13 Number 12 Reports 1035 m Fig. 3. Specular reflection from surface of comeal endothelium of fresh isolated rabbit corneax450. Fig. 4. Optical section through Descemet's membrane of fresh isolated rabbit cornea. The presence of fibrous structures will be noted. Since no trace of the endothelium can be seen, these structures must lie within the membrane or on its stromal surface. x450.

4 1036 Reports Investigative Ophthalmology December 1974 Fig. 5. Optical section through normal stroma of fresh isolated rabbit cornea. x450. Fig. 6. Optical section through swollen stroma of fresh isolated rabbit cornea. x500.

5 Volume 13 Number 12 Reports 1037 Fig. 7. Specular reflection from outer epithelial surface of fresh isolated rabbit cornea. x500. that they would not evade the problem of scattering from neighboring tissue layers, as illustrated in Fig. 1. Search of their cited references led to a recent patent by Baer.' : This instrument operates on the principle of the present instrument and is superior in conception, since, if enough light could be made available, it would allow the optical section of the tissue to be directly observed as well as photographed. No photographs taken with this instrument have been published to my knowledge, however. Goldmann 7 made use of a similar technique to that described here, at a different order of magnification, in his method of photographing the anterior chamber. I wish to thank Mr. Gunther Kuhn for engineering and constructing the modifications to the microscope, and Miss Betty. Cassiman for taking the photographs. From the Division of Ophthalmology, Stanford University Medical Center, Stanford, Calif This project was supported by National Institutes of Health Grant EY Submitted for publication June 7, Key words: scanning microscopy, corneal structure. REFERENCES 1. Maurice, D. M.: Cellular membrane activity in the corneal endothelium of the intact eye, Experimentia 24: 1094, Maurice, D. M.: The cornea and sclera, in: The Eye, Davson, H., editor. Ed. 2. London, 1969, Academic Press, vol Pfister, R. R.: The normal surface of corneal epithelium: a scanning electron microscopic study, INVEST. OPHTHALMOL. 12, 654, Davidovits, P., and Egger, M. D.: Scanning laser microscope for biological investigations, Appl. Optics 10: 1615, Petran, M., Hadravsky, M., Eager, M. D., et al.: Tandem-scanning reflected-light microscope, J. Opt. Soc. Am. 58: 661, Baer, S. C: United States Patent 3, 547 : Goldmann, H.: Spaltlampenphotographie und -photometrie, Ophthalmologica 98: 257, Histopathology of keratopathy in the tyrosine-fed rat. MARGARET E. BEARD, ROBERT P. EDWIN SQUIRES. BURNS, LARRY F. RICH, AND Young rats fed a diet containing excess l-tyrosine develop a reproducible and reliable keratopathy. This keratopathy clears spontaneously, although the initiating dietary stitnidus is maintained. In less than 24 hours, edema of the central corneal epithelium develops, first in the basal cells and then in focal full-thickness areas. These areas of epithelial disease enlarge to form fullthickness "snowflake" opacities with cellular sepa~

Lecture 2 Slit lamp Biomicroscope

Lecture 2 Slit lamp Biomicroscope 1 Slit lamp is an instrument which allows magnified inspection of interior aspect of patient s eyes Features Illumination system Magnification via binocular microscope