LIGHT SCATTERING STUDIES OF THE

Transcription

1 LIGHT SCATTERING STUDIES OF THE RABBIT CORNEA YOSHIZO KIKKAWA* Department of Ophthalmology, School of Medicine, University of Tokyo, Hongo, Tokyo When the light is incident on the transparent cornea, a greater part of it is transmitted without deviation. But it can be expected that a small fraction is absorbed, reflected, scattered or diffracted. To clarify the problem of the transparency of the cornea, the studies on the latter characters are altogether needed. In the previous paper (1) attention was drawn to the diffraction spectra produced by the grating of the corneal fibers. The present paper is concerned with the light scattering properties of the cornea as measured by the use of a photomultiplier tube. The angular intensity distribution of the scattered light is very characteristic, that is, a slight scattering occurs in the forward direction with respect to the incident light even in the transparent cornea; this phenomenon is due partly to the interference of inter-fibrous and partly that of intra-fibrous origin. As the cornea clouds, the scattering increases in the backward and lateral directions, though the greater part of the scattering occurs in the forward direction. This means that the changes in the microscopic as well as submicroscopic lattice in the corneal stroma cause the clouding of the cornea. The transparency and clouding of the cornea can thus be well explained on the basis of the double lattice structure of the fibers and fibrils. METHOD The optical system used to obtain a parallel beam is the same as in the previous paper (1). The light source, a 6 volt lamp, was focussed by the condenser on the vertical slit, 0.1 mm. in width and 1.4 mm. in length; the light was then collimated by the lens (focal length 33 cm.). Thus parallel light was perpendicularly incident on the cornea through a round hole, 2 mm. in diameter. To ensure constant intensity of the beam, the lamp was supplied by batteries. A phototube of multiplier type, R.C.A. 931-A. was used to measure the intensity of the light scattered from the cornea. It was mounted on a turn table which could be rotated about a vertical axis; the angle of rotation was measured with a circular scale. Being held between glass microscope slides, a small piece of cornea was placed at the center of the turn table in such a manner as that the parallel light was incident perpendicularly on the surface Received for publication October 9,

2 LIGHT SCATTERING IN CORNEA 293 of the cornea. By rotating the table, it was possible to measure the intensity of the light scattered at angles between The direction of the scattered light was indicated by the angle (ƒæ) between the direction of propagation of the incident beam and that of the scattered beam. The multiplier phototube was placed in the metal case with a rectangular window, the field of view of which was 5 mm. high and 4 mm. wide. It could catch light scattered over a horizontal angular interval of 2.5 ; the intensity measured represents the total flux within that angular interval. After passing through the cornea and free space comparable to the radius of the turn table, the transmitted beam fell centrally into a matt black tube with suitably placed diaphragms, so as to ensure complete extinction. The bottom of this tube was removable for observing the transmitted light with a telescope focussed for infinity. A clear image of the slit could be observed, when the corneal piece was held sufficiently flat between the glass slides; otherwise, a diffuse surface reflection would disturbe the image severely. When mounted sufficiently flat, even with fairly clouded cornea, a reddish image of the filament of the lamp could be observed. In this way, the corneal piece was checked for its optical smoothness of the surface. Moreover, the light scattering system was placed, as a whole, in a lighttight box. Measurements could not be performed between the angles , being obstructed by the stage on which the microscope slide was placed. Again measurements were not performed at the angles , since the incident beam was interrupted by the tube case of the multiplier. At the position 0, as the transmitted beam reaches the cathode in full, the current would be too large as to fatigue the tube severely, unless the incident beam was not greatly reduced by means of the filter. In the present experiment, two neutral filters were employed to reduce the illumination within the safety limit. The filters were successively removed as the tube was swung out of the direct beam, towards the position of larger scattering angles. The transmission coefficients of the filters were determined by comparing the readings between measurements with and without filter at suitable setting; with two filters the light was reduced in the ratio 1 to 290. In the present experiment, the photomultiplier was run at 90 volts per dynode stage, using battery packs. The potential difference at the ends of a 5 Kƒ resistance which was inserted in the circuit of photocurrent was measured with a precission potentiometer and a high sensible galvanometer. The reading of the potentiometer after the subtraction of the dark current reading was taken as a measure of the scattered intensity. The cornea was excised with particular care against damage. Attention was also paid to avoid contamination of dust in Ringer's solution. RESULTS The scattering from the glass microscope slides without containing a corneal piece was measured as a back-ground. The intensity of the scattered

3 294 Y. KIKKAWA light was expressed in %, taking that of the transmitted light for 100%. The angular intensity distribution of the scattered light is given in fig. 1; the scattering decreases rapidly with increasing ľ. With the sensitivity of the present apparatus, no detectable light was measured at the direction larger than FIG. 1. Scattering curves of the fresh and dried corneas and the glass microscope slides. Ordinate: Intensity in 9,1, Abscissa: in degree. Intensity distribution of the scattered light at the angles over 10 is given at the upper right of the figure with an exaggerated scale. The representation is common to all the figures. F: fresh cornea, D: dried cornea, G: glass microscope slides alone. Fresh cornea Since the measured intensity includes the scattering from the glass microscope slides, subtraction of the latter from the former is necessary to obtain the scattering from the cornea itself. With this procedure, fractions derived from stray light, inclusions of dirt as well as scattering from the microscope slides are eliminated. The light scattering from the cornea itself decreases rapidly with increasing ľ in a similar way as the glass holder alone, except that light is scattered far over 16 in a detectable amount in the cornea as shown in fig. 1. The intensity of the scattered light is represented in %, taking that of the transmitted light through the glass slides without containing the cornea for 100%. Next, it was tested if the scattering characteristics are related to the region of the cornea, or to the direction of the fiber. In the peripheral region, the scattering was slightly augmented in the direction right angles to the radial fiber as compared with that in the parallel direction. In the central region, no directional difference was found in the scattering characteristics. Swollen cornea The excised cornea swells gradually and becomes opaque, when kept in M Ringer's solution. Measurements were made during the course of swelling. As the cornea swells, the backward and lateral scattering increase, though the greater parts of scattering occur in the forward direction (fig. 2). The net increase of scattering could be obtained by subtracting the scattering

4 LIGHT SCATTERING IN CORNEA of the fresh cornea from that of the swollen cornea. The angular distribution of the net increase of scattering is given in fig. 3. At slight swelling, the peak is found at ľ=3. While, as the swelling proceeds, the peak is displaced toward the larger angles. FIG. 2. Scattering curves of the swollen corneas. 2: 2 hours, 1: 1 day, 3: 3 days, 5: 5 days, 7: 7 days, D: dried cornea. FIG. 3. Net increase of scattering. Swollen cornea-2: 2 hours, 1: 1 day, 3: 3 days, 5: 5 days, 7: 7 days. E: injuried epithelium. Injuried epithelium When the epithelium was scratched with the blade of a sharp knife, the clouding occured, instantly when measurements were made. The scattering occured chiefly in the forward direction, and slightly in the lateral and backward directions. The net increase of scattering is plotted in fig. 3. The peak is situated between ľ= It must be noticed that although the cornea swollen for 2 hours (thick solid line) are almost equal to the injuried epithelium (thin solid line) in the intensity of transmitted light, they exhibit quite different behaviors in the diagram showing the net increase of scattering. The fact indicates that the scattering particle in the injuried epithelium is quite different from that in the swollen cornea. Whereas, when the endothelium was scratched, there occured no instant clouding, and the angular intensity distribution of the scattered light was found to be in the range of normal. However, it is well known that the cornea clouds when the endothelium is damaged. The discrepancy may be accounted for as follows: The cornea damaged on the endothelium remains transparent for some time, during which period

5 Y. KIKKAWA the present measurement was done, but it becomes opaque gradually by imbibition of water through the injuried water barrier. Fixed cornea The scattering characteristics of the corneas fixed by 80% acetic acid, 10% formalin, and 95% alcohol were investigated. The scattering occurs chiefly in the forward direction and slightly in the lateral and backward directions as shown in fig. 4. The net increase of scattering is plotted in fig. 5. The maxima are found at the positions of ľ=3-5, 5-7, and 12-14, for the corneas fixed by acetic acid, formalin, and alcohol respectively. FIG. 4. Scattering curves of the fixed corneas. A: alcohol, B: formalin, C: acetic acid. FIG. 5. Net increase of scattering in the fixed corneas. A: alcohol, B: formalin, C: acetic acid. Corneal leucoma As the preliminaries, a drop of 40% sodium hydroxide was instilled in the rabbit eye to cause an intense clouding and inflammation. After three or four months, the cornea was excised and the scattering characteristics were examined (fig. 6). Dried cornea The clouded cornea was dried at room temperature upon the glass microscope slides, covered with Canada balsam, and then the scattering characteristics were investigated. It is of interest that the clouded cornea, whether obtained by swelling or by fixation or by leucoma, all recover completely their trans-

6 LIGHT SCATTERING IN CORNEA FIG. 6. Scattering curves of corneal leucoma, sclera and tendon. N. corneal leucoma, D: dried leucoma, S: sclera, T: tendon, X: scattering curve which is proportional to 1+cos2 ľ. parency on drying. Moreover, the transparency of the fresh cornea is further improved by drying. In conjunction with the clouded cornea, the scattering characteristics of the sclera and tendon of the rabbit were investigated. The scattering occured in the lateral and backward directions as well as in the forward direction (fig. 6). The intensity of light scattered by particles quite small as compared with the wavelength of light is proportional to 1+cos2 ľ, where ľ is the angle between the direction of propagation of the incident beam and that of the scattered beam. The scattering envelope which is proportional to 1+cos2 ľ is shown (X) in fig. 6 for the sake of comparison. POLARIZATION MICROSCOPIC FINDINGS OF THE CLOUDED CORNEA It was shown in the previous paper (2) that the cornea is composed of regularly arranged fibers. In the present experiment, the changes in the fibrous structure on clouding have been investigated by a polarization microscope. The clouded cornea was placed, without being sectioned, upon a slide glass, dried at room temperature, then covered with Canada balsam. Although the clouded cornea became transparent by dehydration, the changes in the fibrous structure remained unaffected. The results obtained by drying variously treated corneas are as follows: The fibrous structure is obscure in the cornea fixed by 80% acetic acid and in the leucoma. In the extremely swollen cornea, the fibers are seen enlarged. A little change is found in the cornea fixed by 10% formalin and in that by 95% alcohol.

7 Y. KIKKAWA DOUBLE LATTICE THEORY AND DISCUSSION On the assumption that the scattering by the whole cornea is the total sum of the scattering of each fiber which is independent of neighbouring fibers, the theoretical calculation leads to the conclusion that the cornea should be opaque. While the explanation of transparency giving the stroma a uniform refractive index does not reconcile with the experimental evidence from the structural stand point. Maurice (3), after giving a good review and criticism of the problem concerning the transparency of cornea, put forward the lattice theory. According to this theory, it is not possible to think that energy scattered by the fibrils which are spaced in a regular order within the lamella would remain independent of one another. Instead, the individual scattered wave would interfere destructively in all directions except that of the incident beam. As the result, only in the direction of the incident beam, the scattered light would be obtained making the cornea appear transparent. The lattice theory attaches a great importance to the interaction between the light and the fibrils. However, the previous work (1) presented the evidence for the presence of interaction between the light and the corneal fiber; the distance between the elements in the corneal grating having been found to be 13 p from the diffraction spectra. The lattice theory of Maurice cannot explain the diffraction spectra of such a magnitude. The corneal lamella is considered to be a two-dimensional lattice of fibers of microscopic order, within which the fibrils are spaced in a regular order forming a one-dimensional lattice of submicroscopic order (fig. 7). Under this condition, the mutual interference between the fibers as well as the internal interference within the fiber should be taken into consideration. In the cornea, the light is scattered at the fibrils, which are disposed in a lattice. When randomness and disorder are absent in the arrangement of the fibrils as in a perfect crystal, destructive interference is complete and no light is scattered. Thus the cornea is transparent. Disorder in the fibril lattice causes the clouding of the cornea, the degree FIG. 7. Diagrammatic representation of the double lattice structure of the cornea. : fiber, œ: fibril. Z of transparency depending upon the spacing of the scattering centers. Strictly speaking, the explanation is not valid. Indeed, in the transparent cornea, the scattering centers may be disposed in a perfect lattice in the timeaverage. However, the thermal movement causes the spacing of the scattering centers to vary continuously. Therefore, the fibrils are never disposed in a perfect lattice at the instant in which the interaction occurs between the light and the fibril, This would result in scattering. Here a brief description will be made on the light scattering phenomena.

8 LIGHT SCATTERING IN CORNEA A beam of light is transmitted through a homogeneous, nonabsorbing medium without alteration of its direction of propagation. If, however, nonabsorbing particles differing from the medium with respect to refractive index are placed in the medium, a disturbace of the wave motion results. According to the theory of light scattering initiated by Debye, the intensity of the light scattered by particles, the dimension of which is quite small as compared with the wavelength of light, is proportional to 1+cos2 ľ, where ľ is the angle between the direction of propagation of the incident beam and that of the scattered beam, providing that the incident beam is unpolarized. Therefore, the angular intensity distribution of the scattered light is symmetrical to the direction right angles to the incident beam (ľ=90 ) (4). On the other hand, when the dimension of the particle exceeds approximately one-twentieth of the wavelength of light, internal interference must be considered; the waves originating from the proximal and distal parts of the particle will interfere. Although this will not significantly affect the forward scattered light, it reduces the backward scattered radiation more appreciably. The phase shifts encountered by rays scattered from different parts of the same particle are very small for the small angles of ľ (ľ being the deviation from the direct path), with the result that there is an increase in the intensity of the forward scattering. The phase shifts increase with increasing of ľ, then destructive interference occurs, resulting in a considerable decrease in the intensity of the backward scattering (fig. 8). The magnitude of interference is a function of the size and shape of the scattering particles (4) (5). FIG. 8. Illustration of the dependence of destructive interference on the scattering angle. The path length difference for light scattered in the backward angle (AB+BC-AC) is larger than in the forward angle (AB+BD-AD). Returning to the main subject, since the dimension of the fiber is very large as compared with the wavelength, the interference within the fiber would result in the forward scattering. Besides, the interference between the fibers would contribute to the forward scattering. Since the diffraction is of Fraunhofer class, the clear spectra appear only in the focal plane of telescope or camera. The spectra will be observed as a diffuse scattering in the forward direction, when no lens is employed as in the present experiment. Thus the both mechanisms of mutual and internal interferences of the fibers would contribute to the forward scattering in the transparent cornea. The above visualization naturally leads to the next anticipation. The more irregular is the spacing of the fibrils within the fiber, the more will be the scattering, resulting in the clouding of the cornea. Since the magnitude of interference depends upon the spacing of the fibries, the curve representing the

9 Y. KIKKAWA net increase of scattering, particularly the maximum, may be regarded as an indicator of the changes in the spacing of the fibrils. Besides, changes would occur in the microscopic lattice in the clouded cornea. They may manifest themselves in the diffraction spectra and the polarization states. By the optical arrangement employed in the previous work (1), the diffraction spectra were observed with polarized ligth. It was found that although alcohol and formalin give rise to little change in the spectra, swelling causes a diminution in the spectra, corresponding to the enlargement of the grating element distance. The distances were 12.3 Đ, 13 Đ, and 20 Đ respectively, for the corneas fixed by alcohol and formalin, and the swollen cornea. The spectra could not be observed in the cornea fixed by acetic acid and in the corneal leucoma, though they became transparent by drying. These modifications in the diffraction spectra are in agreement with the polarization microscopic findings of the clouded cornea. In ordinary rays, the diffraction spectra were obscure, over-lapping with a strong light scattering in the neighbourhood of the image of the slit (1). However, between crossed polaroids, the strong light scattering was extinguished, and the clear spectra could be observed which conditions were exclusively used in the present work for convenience's sake. It is probably permissible to employ polarized light, in the place of ordinary rays, although the transparency and clouding of the cornea are concerned with ordinary rays. On drying the cornea, the transparency was recovered or enhanced. The fact indicates that the fibrils are rearranged in a more perfect lattice by dehydration. The directional difference in the light scattering characteristics gives a further support to the concept that the stroma, in which a directional difference exists in the fiber arrangement (2) plays an important role in the light scattering of the cornea. The stroma constitutes the greater parts of the cornea, while a few flattened cells, and elastic and nerve fibers represent a very small fraction of the volume, which can be ignored for the present consideration. However, the cellular layers which cover the corneal surfaces should not be neglected. The injuried epithelium quite differs from the clouded stroma as shown in fig. 3, indicating that the scattering particle in the clouded epithelium is quite different from that in the stroma. Therefore, transparency and clouding in the cellular layers should be explained from a stand point other than the lattice theory; this point must be the subject of future research. It is considered that the light scattered in an angle to the propagated direction, would be refracted at the interface of the cornea, and some part would be reflected. A slight forward scattering from the glass microscope slides alone may, in some part, be due to such a refraction and reflection at the interfaces of the glass slides. But this contribution from the glass slides has been subtracted from the data. For the refraction and reflection at the interfaces of the cornea itself, any suitable correction has not been found. Maurice (3) explained the opacity of the sclera on the ground that the fibrils are parallel only over limited region, and they are not equal in diameter, ranging mĐ. Accordingly, the scattering elements are considered to be

10 LIGHT SCATTERING IN CORNEA disposed at random in the sclera. This is in agreement with the studies of intensity distribution showing that the scattering is proportional to 1+cos2 ľ in the sclera. Furthermore, the experiment showed that the sclera becomes transparent by drying, and produces the diffraction spectra, though they are not so clear-cut as that of the cornea. Transparency of the cornea has hitherto been explained on the basis of the double lattice structure of collagen fibrils. However, it is well known that the transparency of the cornea is related to its water content; imbibition of water causes the clouding of the cornea. Therefore, the epithelium and endothelium must play important parts in the regulation of the proper fluid balance which keeps the cornea in a state of transparence. It was generally believed until fairly recently that water was continuously drawn out through these layers by means of an osmotic gradient. However, it is most likely, by the recent investigations (6) (7), that the bulk movement of water and salt from the stroma is determined by a secretory process acting across one or both of these layers. The action of this secretion is to transfer salt and water from the stromal fluid by the cellular activities consumming energy. The energy required for the active transport mechanism must be supplied from the metabolic process. Therefore, it may be concluded that the metabolic activities of these layers contribute to the maintenance of the lasting clarity of the cornea. SUMMARY Distribution of the scattered light from the cornea has been determined, using a phototube of multiplier type. A slight scattering occurs in the forward direction even in the transparent cornea. In the peripheral region of the cornea, the scattering in the direction right angles to the radial fibers is greater than that in the parallel direction. In the central region, no directional difference is found in the scattering characteristics. As the cornea clouds, the scattering increases in the backward and lateral directions, though the greater parts of the scattering occur in the forward direction. On drying the cornea, the transparency of the clouded or fresh cornea is recovered or even enhanced. The transparency and clouding of the cornea are explained in terms of interference in the double lattice structure consisting of the fibers and the fibrils. The changes in the microscopic lattice are revealed in the diffraction spectra and the polarization findings. On the other hand, the changes in the submicroscopic lattice are revealed in the angular intensity distribution of the scattered light. The author wishes to express his thanks to Professor H. Hagiwara and Assistant Professor S. Shikano for constant guidance in the course of the work. Thanks are also due to Professor K. Dan of Tokyo Metropolitan University for his criticism, and for reading the manuscript.

11 Y. KIKKAWA REFERENCES 1. KIKKAWA, Y. Diffraction Spectra Produced by the Rabbit Cornea. Jap. J. Physiol. 8: 138, KIKKAWA, Y. Elastic Double System and Selective Permeability to Cations in the Stroma of the Rabbit Cornea. Jap. J. Physiol. 6: 300, MAURICE, D. M. The Structure and Transparency of the Cornea. J. Physiol 136: 263, KOTANI, M. Molecular Physics, p Tokyo: Kyoritsu, ANSON, M. L., EDSALL, J. T. AND BAILEY, K. Advances in Protein Chemistry, Vol. VI, p. 73. New York: Academic Press, LANGHAM, M. E. AND TAYLOR, I. S. Factors Affecting the Hydration of the Cornea in the Excised Eye and the Living Animal. Brit. J. Ophthal. 40: 321, HARRIS, J. E. The Physiologic Control of Corneal Hydration. Am. J. Ophth. 44: 262, 1957.