Do M¨Uller Cells Act As Optical Fibers in the Primate Retina?

Retina Do Muller¨ Cells Act as Optical Fibers in the Primate Retina?

Kuno Kirschfeld Max-Planck-Institute for Biological Cybernetics, Tubingen,¨ Germany

Correspondence: Kuno Kirschfeld, PURPOSE. To examine whether Muller¨ cells in the primate retina act as optical light guides. Max-Planck-Institute for Biological METHOD. In the literature, it has been suggested that Muller¨ cells in the primate retina act as Cybernetics, Tubingen,¨ Germany; optical ﬁbers. I have conducted a survey of the literature for papers in support of or refuting [email protected]. this assumption.

Submitted: October 4, 2018 RESULTS. I show that neither histology, nor the direct observation of photoreceptors, nor the Accepted: December 17, 2018 entoptic observation of the retina agree with the assumption that Muller¨ cells in the human retina act as optical waveguides. Citation: Kirschfeld K. Do Muller¨ cells act as optical fibers in the primate CONCLUSIONS. I confirm the classic view that the inner/outer segments act together as optical retina? Invest Ophthalmol Vis Sci. waveguides and that they are the origin of the Stiles-Crawford effect of the first kind. 2019;60:345–348. https://doi.org/ Keywords: primate retina, Muller¨ cells, optical fibers 10.1167/iovs.18-25831

hotoreceptor inner and outer segments have a higher and higher primates....; here the photoreceptor cells are not P refractive index than their surrounding tissue. Sandwiched obscured by any inner retinal layers at all.’’ The question is together between the external limiting membrane and the whether Muller¨ cells also form a fiberoptic plate-like structure pigment epithelium, they act as dielectric light guides. Because outside the fovea. The results of several different approaches do their diameters are of the order of the wavelength of light, light not support this view. intensity is not distributed homogeneously over their cross section but is guided by so-called ‘‘modes.’’ In this case, light guides are called ‘‘waveguides.’’ RESULTS During the second half of the last century, waveguide properties of photoreceptors were identified in the area called Do Whole Müller Cells Act As Optical Fibers? ‘‘photoreceptor optics.’’ The main results were summarized in 1 Histology. To be able to work as fiberoptic plates, Muller¨ the monography ‘‘Photoreceptor Optics.’’ cells need to be straight (Fig. 1A); they cannot be curved or Answers could be given to questions such as the following: arranged in a zig-zag fashion, as light would then escape the What kinds of modes are possible in vertebrate inner/outer guide due to the bending.3 Around the primate fovea, Muller¨ segments? What explanations do we have for the two Stiles- cells are not straight but ‘‘Z-shaped.’’4 To ascertain the course Crawford effects? What is the smallest possible diameter of an taken by the Muller¨ cells, we only need to trace the course of outer segment before optical cross-talk between neighboring the photoreceptor axons, that is, the Henle fibers as well as the photoreceptors increases to such an extent that resolution is bipolar cells that follow. This suffices because Muller¨ cells are impaired? intertwined with the axons of the photoreceptors.4 Drasdo and During the past decade, it was suggested that, in addition to coworkers5 calculated the length of the Henle fibers by the photoreceptor inner/outer segment waveguides, a second comparing the lateral displacement of ganglion cells and type of retinal cell in the mammal retina, the Muller¨ cell, can act photoreceptors. They observed a displacement close to the as an optical fiber. Because some of the conclusions drawn in fovea of some 100 lm, which increased up to 500 to 600 lmat these papers are at variance with the results of photoreceptor 61 mm in the horizontal meridian, decaying to 100 lmata optics, I have compared the methods and results of the two distance of 63 mm. A displacement of 100 lm means that, at approaches. this location, Muller¨ cells are still Z-shaped. On the retina, 3 mm In their seminal article, Franze and coworkers2 report that correspond to approximately 10 degrees. And therefore, due to Muller¨ cells extend from the inner to the outer limiting their Z-shape, Muller¨ cells seem to be unable to form a membrane, thereby spanning the entire retinal thickness. fiberoptic plate-like structure in front of the photoreceptor Because they can be seen to have higher refractive indices layer in the most important field of our view of a diameter of 20 than the surrounding tissue, they can guide light (Fig. 1A). degrees. Because they are directed along the direction propagating light, In fact, the structure of the retina provides proof, at least in these cells are believed to provide a low scattering passage of the foveal region, that Muller¨ cells do not guide light: Foveal light from the retinal surface (inner limiting membrane) to the cones are connected to Z-shaped Muller¨ cells. If they were to photoreceptor cells (external limiting membrane). According guide light to the photoreceptors, this would, for obvious to this view, Muller¨ cells form what is known as a fiberoptic reasons, be detrimental for foveal resolution. And so we are left plate-like structure in front of the photoreceptor layer. with the question, if we proceed from the fovea toward the Franze and coworkers2 conclude that the ‘‘fiber optic plate- periphery of the retina: at which point could Muller¨ cells begin like structure is especially characteristic for the retina of all to guide light? A theoretical analysis cannot solve the problem mammals with the exception of the fovea centralis of humans because we know neither the refractive indices of the relevant

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comparing rod and cone spacing with equivalent measures from histology. Meanwhile, the cone and rod mosaic in the human eye has been observed over large retinal areas. In the article by Marcos and coworkers,10 for example, these areas span from nasal 30 degrees over the fovea to 30 degrees temporal. Such observations with resolution of individual cones and rods are not compatible with the idea that a fiberoptic plate-like structure, as formed by Muller¨ cells, lies in front of the photoreceptor layer. The fact that photoreceptors can be observed directly without a fiberoptic plate-like structure in-between comes as no surprise, given that the Henle fibers and Muller¨ cells form a weakly reflecting, dark band in optical coherence tomography (OCT).11 This indicates that Muller¨ cells do not backscatter or reflect the incident light significantly, meaning that these layers are optically transparent, at least at the wavelength of light used in OCT recording. Entoptic Image of the Retina. By illuminating the eye FIGURE 1. Muller¨ cell shapes. (A) Schematic illustration of the Muller¨ side-on, it is possible to observe one’s own retina. In this case, 2 cell of a guinea pig in situ, according to Franze and coworkers. Light light enters the eye behind the lens (‘‘retrolental illumination’’ enters from top at the inner limiting membrane of the retina. The [RLI], Fig. 2A) and the retinal vessels cast a shadow onto the diameters of the cell at different locations are indicated, as well as the photoreceptors so that the subject is granted a wonderful view waveguide parameter V at different locations for the two wavelengths of his/her own vessels, known as the entoptic image of the of light, 500 nm (top) and 700 nm (bottom). (B) Shape of a human 12 foveolar Muller¨ cell according to Tschulakow and coworkers.17 The retina. This phenomenon was first described by Purkinje. It is cells are generally shaped by a plateau zone, where they assumed the elicited by focusing light from the sun or from a penlight onto light enters (arrow). the sclera. The phenomenon also can be easily generated by a light-emitting diode (LED), which is found in many laser pointers in addition to the laser output.13 structures nor the structural details necessary for such an When RLI is used, the fovea is seen as a grainy spot in the analysis. area without vessels. These grains prompted v. Helmholtz14 to Besides the problem of light guiding in Z-shaped Muller¨ describe this area as having the appearance of ‘‘shagreen cells, the histology of the primate retina shows other structural leather.’’ By estimating the grain size, Ehrich15 came to the details that disqualify out Muller cells as light-guiding ¨ conclusion that each grain could correspond to the activity of a structures. Neighboring receptors are almost parallel. And single cone. If the LED-lamp is moved, the grainy area can be their long axes, throughout the retina, are not oriented seen to move in relation to the vessels. This conspicuous perpendicularly to the sclera and pointing to the center of 6 phenomenon, which is a necessary consequence of the fact the eye. Instead they point to the center of the exit pupil. The that vessels and receptors are not located on the same plane of consequence is that receptors near the posterior pole lie the retina, had already been observed by v. Helmholtz.14 As perpendicular to the sclera, whereas those located more and illustrated in Figure 2B, when the direction of incidence of the more anteriorly (i.e., closer to the ora serrata), form light is altered, the image of the vessels is displaced in relation increasingly acute angles with pigment epithelium and sclera. to that of the photoreceptors (Fig. 2B, double arrowheads). This arrangement is interpreted as optimizing the acceptance Given the anatomical dimensions of the retina, a displacement of light rays coming from the pupil. If light was accepted by of the light incidence of 610 degrees leads to a relative Muller¨ cells, and not photoreceptors, it would not be necessary displacement between fovea and vessels of approximately a to adjust the photoreceptors in the way described. Instead, quarter of a degree, corresponding to the observation.13 This such an arrangement would be expected for Muller¨ cells, albeit can occur only if the Muller¨ cells do not act as a fiberoptic it has not been observed to date. plate-like structure. In this case, no displacement would be The following finding excludes a light-guiding function of expected (Fig. 2B, single arrowhead). We can therefore also Muller¨ cells even further: If a subject wears a contact lens conclude from this experiment that Muller¨ cells cannot act as containing an artificial displaced pupil, one can show that the optical fibers in the periphery of the fovea. peak of the Stiles-Crawford effect of the first kind (SC1),7 indicative of the orientation of the light-receiving elements, Are Müller Cells Wavelength-dependent Wave moves to the center of the displaced pupil within 5 days. After removing the artificial pupil, the SC1 peak reverts again after Guides in the Human Retina? 8 approximately 5 days to its original location. This finding is Labin and coworkers16 proposed that Muller¨ cells function not interpreted as follows: An active mechanism arranges the only as optical fibers in the retina, but also as wavelength- orientation of photoreceptors such as to optimize light dependent light guides, concentrating the green-red part of the acceptance. Even if it is not yet clear as to how this mechanism visible spectrum onto cones and allowing the blue-purple part is realized, the following is a necessary condition: the elements to leak onto nearby rods. These conclusions were drawn from that receive light must be capable of detecting the efficiency of computational analyses that have been confirmed by experi- light acceptance. This information is available to photorecep- ments in the guinea pig retina. For their computation, they tors, but not to Muller¨ cells: when guiding light, it would make assumed that the Muller¨ cells in the nerve fiber layer have a no difference to the Muller¨ cells whether they guide strong or proximal cup-like funnel followed by a rod-like structure that weak light, because there is no light-absorbing pigment. extends to the outer limiting membrane. Such a structure may Direct Observation of Photoreceptors in the Living be adequate to describe the situation in the guinea pig, but it is Eye. Using scanning laser ophthalmoscopes combined with not appropriate for the human retina. Here, as discussed adaptive optics, it is possible to resolve individual rods and above, Muller¨ cells are Z-shaped. Therefore the conclusions cones in the living eye.9 The results have been confirmed by drawn by Labin and coworkers16 may well be relevant in the

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They furthermore investigated wholemount preparations from human retinae. The condenser of the microscope was replaced by a light beam, the angle of incidence of which could be modified on the sample. Light entering the fovea center at an angle of 0 degrees caused a very bright spot observable by the microscope after passing through this area. However, when the angle of the light beam was changed to 10 degrees, less light was observed or measured after passing through the retina: the foveolar center became darker and a Stiles-Crawford effect-like phenomenon became directly visible. Tschulakow and coworkers17 ascribed this phenomenon to properties of Muller¨ cells, because the effect persisted after they carefully brushed the outer segments away. Several findings speak against this interpretation: 1. As can be observed in Figure 1B, the diameter of that part of the Muller¨ cell believed to be an element of a fiberoptic plate-like structure in the human foveola is of the order of 10 lm. The resolution in the human fovea corresponds approximately to 1 arcmin. This corresponds to a mosaic of scanning elements of the order of 3 lm.18 This mosaic, in turn, corresponds to the distance of cones in the human fovea. An optic plate-like structure, formed from elements of a diameter of approximately 10 lm in front of the foveal cones, was not compatible with the measured human acuity and would be detrimental to acuity. FIGURE 2. Generation of the entoptic image of the retina. (A): Retrolental illumination of the retina. A small LED lamp, which is used 2. It is possible to observe cones and rods in healthy to illuminate the retina from behind the lens, needs to be moved subjects. In the fovea, the cones have center-to-center (double arrow) to overcome fading of the observed image due to distances of the order of 3 arcmin.19 Using specific stabilized image conditions. (B): Cross section through the human bleaching procedures, the three cone types L, M, and S20 retina with light rays coming from different directions. The black line can be distinguished from each other. It turned out that at the bottom is the pigment epithelium. The rods orthogonal to the the reflectance of individual cones changes over time, pigment epithelium are the inner and outer segments of the that these changes are independent from cone to cone, photoreceptors, which work together as light guides (for clarity, only a small percentage was drawn). The vitreous body is shown in gray. and that they are present in all cone classes. This change Different retinal layers between pigment epithelium and vitreous body in reflectance is explained by the ‘‘shedding’’ of discs, are indicated. The black circles with white centers indicate two blood but could not be explained by any property of Muller¨ vessels located at the border between retina and vitreous body. Light cells. rays entering from two different directions due to movement of the LED lamp are indicated as white lines. In the right half of the retina, It also transpired that all three types of cones reflect more two different cases are indicated. In the left case,Muller¨ cells are not light to a point close to the center of the pupil than toward the supposed to form a fiberoptic plate. In this case, when the LED lamp is pupil margin. Such behavior is closely related to the SC1 effect, moved, the shadow of the blood vessels can be seen to fall on different that is, the SC1 effect can be explained by the collective photoreceptors (double arrowheads), causing it to be shifted relative directional properties of the cones themselves, and no Muller¨ to the foveal receptors, which remain stable. In the right case, the cells are required. Muller¨ cells are supposed to act as light guides. Light from the position of the blood vessel therefore always appears at the location of identical 3. In their paper, ‘‘The Stiles–Crawford effect: a theoretical photoreceptors and so no movement between fovea and blood vessels revisit,’’ McIntyre and Pask21 introduced some refine- 13 is expected. However, as described in Kirschfeld, this is not what we ments into the original theory, in an aim to explain the see in this experiment. SC1 effect with the optical waveguide theory22 and considered new experimental results. Theory and retina of the guinea pig, but cannot be realized in the human experimental results were in such good agreement that retina. the authors concluded that, with some minor adjust- ments, the waveguide theory constitutes the theoretical Do Portions of Müller Cells Act as Optical Fibers? basis for the comprehension of the SC1 effect. In fact, it was recently proposed that waveguiding may not In an anatomical reinvestigation of the foveola of monkeys and be as fundamental for vision as commonly assumed, because a humans, Tschulakow and coworkers17 came to several number of observations do not seem to be compatible with unexpected conclusions. They found a unique morphology this concept.23 A ‘‘Volumetric integration model of the Stiles- to describe photoreceptors and Muller¨ cells in the fovea. They Crawford effect of the first kind and its experimental ascribe optical fiber-like properties to Muller¨ cells, concluding verification’’ was therefore developed.24 However, in this that an effect equivalent to the so-called SC1 effect7 occurs model too, the outer segments, and not the Muller¨ cells, are solely due to the properties of Muller¨ cells. responsible for the generation of the SC1 effect. They came to this conclusion following two observations: They found that outer segments do not run parallel to the incident light as generally assumed, but that they are curved or DISCUSSION even coiled. Due to this finding, they conclude that no convincing hypothesis for the origin of the SC1 effect can be The evidence for the conclusion that Muller¨ cells form living proposed on the basis of these properties of foveolar cones. optical fibers emanates from theoretical considerations and

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