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Muller cells are living optical fibers ¨ in the vertebrate retina
Kristian Franze*, Jens Grosche*, Serguei N. Skatchkov, Stefan Schinkinger§, Christian Foja¶, Detlev Schild , Ortrud Uckermann*, Kort Travis§, Andreas Reichenbach*,**, and Jochen Guck§
*Paul Flechsig Institute of Brain Research, Universitat Leipzig, Jahnallee 59, 04109 Leipzig, Germany; Interdisciplinary Center of Clinical Research, ¨ Inselstrasse 22, 04103 Leipzig, Germany; Center for Molecular Biology and Neuroscience, Department of Biochemistry, School of Medicine, ¨ Universidad Central de Caribe, Bayamon, Puerto Rico 00960; §Division of Soft Matter Physics, Department of Physics, Universitat Leipzig, ¨ Linnestrasse 5, 04103 Leipzig, Germany; ¶Department of Ophthalmology and Eye Clinic, Universitat Leipzig, 04103 Leipzig,Germany; ´ Deutsche Forschungsgemeinschaft Molecular Physiology of the Brain Research Center and Department of Neurophysiology and Cellular Biophysics, Universita Gottingen, 37073 Gottingen, Germany; and Department of Physics, University ¨t ¨ ¨ of Cambridge, JJ Thomson Avenue, Cambridge CB3 0HE, United Kingdom Edited by Luke Lee, University of California, Berkeley, CA, and accepted by the Editorial Board March 27, 2007 (received for review December 15, 2006)

Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact retinal tissue and individual Muller cells, which are radial glial cells ¨ spanning the entire retinal thickness. Muller cells have an extended ¨ funnel shape, a higher refractive index than their surrounding tissue, and are oriented along the direction of light propagation. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo showed that these cells provide a low-scattering passage for light from the retinal surface to the photoreceptor cells. Using a modified dual-beam laser trap we could also demonstrate that individual Muller cells act as optical fibers. Further¨ more, their parallel array in the retina is reminiscent of fiberoptic plates used for low-distortion image transfer. Thus, Muller cells ¨ seem to mediate the image transfer through the vertebrate retina with minimal distortion and low loss. This finding elucidates a fundamental feature of the inverted retina as an optical system and ascribes a new function to glial cells.
fiberoptic plate glial cells refractive index light guides optical trap

B

iological cells and tissues are usually fairly transparent due to the lack of strong intrinsic chromophores in the v isible part of the spectr um and especially in the near-inf rared. This transparenc y is ex ploited, for example, in multiphoton microsc opy, where this low absorption of excit ation light leads to relatively large penetration depths. A lthough such biological objects do not modulate the amplitude of a passing electromagnetic wave, they impart a phase shif t due to ref ractive index variations. This propert y was rec ogn ized by Zern ike and used for the c ontrast enhancement of indiv idual cells in phase-c ontrast microsc opy (1). However, when light passes through multiple layers of cells, as in tissues, images rapidly deteriorate due to scattering events caused by optical and geometrical inhomogeneities w ith length scales on the order of the wavelength of v isible light (2). Consequently, nature has implemented ingen ious solutions in the properties and the arrangement of str uctures and cell assemblies that light has to pass for nor mal physiological function ing. The lens body in vertebrate eyes, for inst ance, c onsists of elongated fiber cells. These cells do not only display a ver y regular oval or hexagonal cross-section, a smooth sur face, and a regular distribution, they even lose most of their organelles during dif ferentiation, including the cell nucleus (3). In the

vertebrate retina, the inner and outer segments of photoreceptor cells are c onsidered natural optical fibers, supported by their highly specialized shape and optical properties (4). Other natural optical fibers oc cur in deep-sea glass sponges or in the c ompound eye of insects, whose biomimetic c opies have even found their way into techn ical c omponents (5, 6). What these examples have in c ommon is a relatively regular geometr y of the light-guiding str uctures and, in the case of liv ing cells, a sophisticated specialization for this ver y function. Considering these facts, it seems surprising that the retina in the vertebrate eye is inverted and that images projected onto the retina have to pass several layers of randomly oriented and irregularly shaped cells w ith intrinsic scatterers before they reach the light-detecting photoreceptor cells (7, 8). This situation seems to be ``equivalent to placing a thin dif fusing screen directly over the film in your camera'' (9). However, this ``screen'' c ont ains a regular pattern of cells, which are arranged in parallel to each other and span the entire thick ness of the retina ( 150 m). These cells, Muller cells, are radial glial cells in the inner ¨ vertebrate retina, which have a c ylindrical, fiber-like shape (their original name was ``radial fibers of Muller'') (10). They fulfill a ¨ w ide range of physiological functions to support the function ing and sur v ival of retinal neurons (11). For this purpose Muller cells ¨ are, unlike the natural optical fibers mentioned above, endowed w ith many c omplex side branches, which ensheath neuronal c ompartments, such as synapses (12). On the other hand, they put atively oc cupy a strategic position in the path of light through the retina f rom the v itreous, where light enters the tissue, to the outer limiting membrane, where the inner segments of the photoreceptor cells receive the incident light. Therefore, it is intriguing to investigate whether they c ould play a role in the transfer of light through the inner retina. Results As a first step to characterize the retina as a phase object, we investigated f reshly dissected guinea pig eyes by using modified transmission microsc opy (Fig. 1 a and b). Physiological illumination was simulated by insertion of an optical fiber as a light source into the eye cup. Images were obt ained by scann ing a plane close to the outer plex ifor m layer, c orresponding to the
Author contributions: K.F. and J. Grosche contributed equally to this work; K.F., S.N.S., S.S., D.S., K.T., A.R., and J. Guck designed research; K.F., J. Grosche, S.N.S., S.S., and O.U. performed research; C.F. contributed new reagents/analytic tools; K.F., J. Grosche, S.N.S., and K.T. analyzed data; and K.F., D.S., A.R., and J. Guck wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. L.L. is a guest editor invited by the Editorial Board. **To whom correspondence should be addressed. E-mail: reia@medizin.uni-leipzig.de. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0611180104/DC1. © 2007 by The National Academy of Sciences of the USA

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Fig. 1. Light transmission and reflection in the inner retina. (a) Experimental design to study light transmission through the inner retina. (Inset) Light emanating from a multimode optical fiber inserted into a freshly dissected eye simulates physiological illumination of the retina. The eye is opened at the posterior side, and all outer structures, including photoreceptor cells, are surgically removed. The laser light ( 543 nm) that is transmitted through the inner retina (NFL, nerve fiber layer; IPL, inner plexiform layer; INL, inner nuclear layer) is captured at the end of the prephotoreceptor light path with a confocal microscope. ONL, outer nuclear layer; ROS, photoreceptor outer segments. (b) Confocal transmission image of a living unstained retina. The brighter the signal, the more light is relayed to the corresponding area of the tissue. (c) Light reflection in the inner retina. Laser light is delivered via the microscope objective of an upright confocal microscope, and light scattered back from inner retinal layers is detected. (d) Confocal reflection image at the level of the IPL. The brighter the signal, the more light is reflected by the corresponding area. (Scale bar, 10 m; also applies to b.)

end of the ``prephotoreceptor'' light path. Remark ably, these images showed a high degree of inhomogeneit y, revealing an almost regular pattern of bright spots alternating w ith areas of lower transmitt ance (Fig. 1b). This pattern showed that some retinal str uctures relayed light better than others. Interpreting the dark areas in Fig. 1b as areas of higher scattering, laser scann ing measurements in ref lection mode (Fig. 1 c and d) should approx imately yield the negative of the above image. Follow ing this hypothesis, we took series of 50 ­ 60 c onsecutive optical sections f rom f lat-mounted retinae. The v itreous body did not ref lect any light, c onsistent w ith its lack of phase variations. In c ontrast, the almost un ifor m ref lect ance throughout much of the retinal thick ness was interr upted by a fairly regular pattern of dark, less ref lective spots (Fig. 1d). The spots had diameters of 2­3 m and were spaced 5­ 6 m apart, c orresponding well to diameter and spacing of the bright spots in Fig. 1b [see also supporting infor mation (SI) Fig. 5]. The same ref lection patterns were also obser ved in retinae of rabbits (dat a not shown) and humans (SI Fig. 6). To show that the obser ved phenomenon is relevant in physiological c onditions, these experiments were suc cessfully repeated w ith retinae of liv ing guinea pigs in situ (SI Fig. 7d). Import antly, rec onstr uction along the z ax is (Fig. 2a and SI Figs. 6 and 7) showed that the dark spots were c ontiguous in adjacent horizont al sections and for med tubes that c orresponded to distinct optical pathways. At the inner most retinal layer, closest to the v itreous body, these tubes w idened to funnel-like str uctures, which together for med a 15- m-thick c ontinuous low-ref lecting zone only interr upted by axon bundles (Fig. 2a). The amount of back-scattering f rom the retina has prev iously been measured to be 1­5% of the incident light (7, 8). Because biological tissues are t ypically strongly for ward-scattering (13), the tot al amount of scattering in the retina is most likely at least a factor of 2 larger. The distribution of this scattering w ithin the retina is shown in Fig. 2a. Sign ificant back-scattering oc curred in
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all retinal layers prox imal to the photoreceptors w ith the exception of the tubes. The main locations of light scattering are both plex ifor m layers and the axon bundles (Fig. 2b), which c ont ain numerous light-scattering objects w ith sizes on the order of the wavelength of v isible light (14 ­16) such as ``synaptosomes,'' bundles of neurofilaments, and neurotubules. In c ombination, our transmission and ref lection measurements demonstrate the presence of tubular str uctures in the retina that transmit sign ificantly more light than their surrounding tissue. The obser ved spatial pattern of these tubes c orresponded well to the spacing and diameters of the c olumnar Muller cells (Fig. ¨ 2b) (17, 18). Further more, the funnel-like str uctures obser ved in ref lection-mode were remin iscent of the densely packed c obblestone pattern of the Muller cell endfeet at the inner retinal ¨ sur face (19). Indeed, the tubular str uctures c ould be unambiguously identified as Muller cells. They were capable of selective ¨ upt ake of v it al dyes (Fig. 2 b­g) (20, 21) and c ould be c ounterst ained w ith an antibody directed against v imentin (Fig. 2 f and g). In the retina, v imentin is a protein specific to Muller cells (17, ¨ 22). Hence, it is the Muller cells that prov ide a passage for light ¨ through the retina to the photoreceptor cells. These dat a, together w ith their c ylindrical geometr y, suggested a mechan ism of light transport similar to optical fibers. In classical optical fibers, light is c onfined in the transverse direction by an elevated ref ractive index of the c ore c ompared w ith its cladding. Thus, we analyzed the ref ractive indices of enz y matically dissociated v it al retinal cells by using quantit ative phase microsc opy (Fig. 3) (23, 24). The somat a of various retinal neurons (ganglion, amacrine, and bipolar cells) displayed similar ref ractive indices (n 1.358 0.005; mean SD) (Fig. 3a) close to earlier estimates for the tot al retina (25­27). In c ontrast, the mean ref ractive index of Muller cell st alks was sign ificantly ¨ higher (n 1.380 0.021) (Fig. 3a). Toward the so-called endfoot, the funnel-shaped ter mination of the Muller cell facing ¨ the v itreous body w ith n 1.335 (26), the ref ractive index
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a

b

e

c f

g d
Fig. 2. Structures of low reflection are Muller cells. (a) Z-line reconstruction of reflection images of a living retina. The main scattering elements (bright) are ¨ the axon bundles and both plexiform layers. Low-reflecting tubular structures span the entire retina. (b) Living retinal slice preparation, visualizing Muller cells ¨ with the vital dye CellTracker orange (green) and synaptic elements in both plexiform layers (IPL and OPL) with the activity-dependent dye FM1­ 43 (red) (20). The levels of the inner and outer plexiform layers (IPL and OPL, respectively) and nerve fiber layer (NFL) are the same as in a. The asterisks indicate axon bundles in the NFL. (c and d) Overlay of light detected in reflection mode (purple) and the green fluorescence of the vital dye CellTracker green. (c) Z-line reconstruction of a confocal image stack. (d) Oblique optical section at the level of the red horizontal line in c. The dye-filled irregularly shaped Muller cell somata of the inner ¨ nuclear layer (INL) are visible in the left upper part. The central area shows Muller cell cross-sections in the IPL. In the lower right part, the Muller cell endfeet ¨ ¨ are visible, which enclose the ganglion cell somata in the ganglion cell layer (GCL). The lack of merging of the two colors, which would result in white areas, demonstrates that the dye filled exclusively those structures that showed low light reflection. (e­g) Confocal image at the IPL of a retinal whole mount fixed in 4% paraformaldehyde after exposure to the green vital dye and immunocytochemical labeling of vimentin (red), which in the retina is specific to Muller cells ¨ (17, 22). (e) Fluorescence of the vital dye. ( f) Vimentin immunofluorescence. (g) Overlay of e and f. Colocalization of the red and green dyes results in yellow labeling. The observed complete colocalization means that the vital dye-filled and the immunoreactive cells are identical and thus identifies the low-reflecting tubular structures as Muller cells. [Scale bars: b, 10 m (also applies to a); c­g, 25 m.] ¨

decreased to n 1.359 0.003. Such a local decrease of the ref ractive index c ould ser ve to min imize ref lection at the inter face bet ween v itreous and retina. Similar results were c onsistently found in Muller cells f rom four dif ferent vertebrate ¨ species (SI Table 1). Both the obser ved dif ferences bet ween the ref ractive indices of Muller cells and their surroundings as well as the fiber-like cell ¨

shape are remin iscent of the basic requirements of optical fibers. However, Muller cells display a c omplex morpholog y (Fig. 3), ¨ and their radius is c omparable to the wavelength of light so that the t ypical tot al-internal-ref lection model of light guidance is not applicable. In a waveguide, light propagates in cert ain patterns, or modes, deter mined by boundar y c onditions follow ing electromagnetic theor y (28). Light guidance only oc curs if propagating modes ex ist. The key parameter most w idely used in optical engineering to evaluate the presence of propagating modes is the waveguide characteristic f requenc y, or V parameter, V d n
2 1

n 2, 2

Fig. 3. Muller cell shape, refractive properties, and light-guiding capability. ¨ (a) Nomarski differential interference contrast microscopy image of a dissociated guinea pig Muller cell with several adherent photoreceptor cells, ¨ including their outer segments (ROS) and a dissociated retinal neuron (bipolar cell) to the left. The refractive indices of the different cell sections are given. (b) Schematic illustration of a Muller cell in situ. The lighter the coloring of the ¨ Muller cell, the lower the refractive index. Typical diameters and the calcu¨ lated V parameters for 700 nm (red) and 500 nm (blue) are indicated at the endfoot, the inner process, and the outer process. Although diameters and refractive indices change along the cell, its light-guiding capability remains fairly constant. (Scale bar, 25 m.)

where is the f ree-space wavelength of the v isible light, d is the diameter of the waveguide, and n1 and n2 are the ref ractive indices of the waveguide and the surrounding material, respectively (28, 29). For a c onser vative estimate, it is suf ficient to calculate V at the longest v isible wavelength (700 nm) and the smallest diameter, which oc curs at the inner process (d 2.8 m) (21). The largest possible value for the extracellular ref ractive index is that of the adjacent neurons w ith n2 1.358. The calculated V 2.6 ­2.9 for the dif ferent parts along the Muller ¨ cell (Fig. 3b) is suf ficiently high to allow low-loss propagation of a few modes in the str ucture even at 700 nm (28). At a wavelength of 500 nm, the V parameter increases to V 3.6 ­ 4.0. A lthough the ref ractive index and diameter of the Muller cells ¨ both change along their length, the V parameter and, thus, the light-guiding capabilit y st ay nearly c onst ant (Fig. 3b). In c ontrast to the smooth c ylindrical shape of artificial or other biological optical fibers (6, 30, 31), each cell possesses c omplex sidebranching processes import ant to its interactions w ith neurons (12). Their inclusion through an ``ef fective'' ref ractive index gradient actually increases the V parameter of the Muller cell ¨ (32). Consequently, despite their c omplex morpholog y, Muller ¨ cells c ould thus function as waveguides for v isible light.
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trapped object, this setup could be used to directly test the ax ial light transmission through individual cells. WithaMuller cell present in the trap surrounded by media with ¨ refractive indices up to 1.36 (to mimic the surrounding in the retina), light transmission into the output fiber was comparable to the situation where both fibers were in contact, as long as the direction of the light propagation was the same as in the retina. When the Muller cell endfoot was pointing away from the input ¨ fiber, significantly less light arrived at the output fiber, most likely because of a less efficient light coupling into the outer cell process. When the cell was removed, the power measured dropped considerably due to the numerical aperture of the input fiber and the resulting divergence of the laser beam (Fig. 4d and SI Figs. 8 and 9). This effect was elucidated when the light path was directly visualized by using a f luorescent vital dye (MitoTracker orange) that was both present in solution and taken up by the cell. The dye was excited by the visible light emanating from the input fiber. Although the laser beam diverged as expected without the cell, the light remained confined to the Muller cell when present in the trap ¨ (SI Fig. 8). Both experiments clearly showed that Muller cells ¨ capture the visible light, prevent it from diverging, and guide it to their distal end. To further demonstrate the light collection and guidance power of Muller cells, the optical fibers were then ¨ intentionally misaligned, so that without a cell almost no light was detected (SI Fig. 9). Even in this case, the Muller cells were still able ¨ to capture and guide the light. The relative guiding efficiency, Pwith cell/Pwithout cell, increased up to a factor of 9, depending on the angle between the fibers. In combination, our single-cell experiments, the theoretical considerations, and the transmission and ref lection measurements strongly suggest that Muller cells are, and ¨ function as, optical fibers in the retina, relaying light from the inner sur face to the layer of the photoreceptors while bypassing scattering structures present. Discussion These results provide insight into the optical properties of the retina. Most structures in the retina, especially those in the ner ve fiber layer and both plex iform layers, are phase objects that necessarily cause light scattering (Fig. 2 and SI Figs. 6 and 7) (14, 36, 37). In contrast, the optical properties and geometr y of Mu ler ¨l cells are consistent with those of optical fibers so that they ser ve as low-scattering conduits for light through the retina. The low scattering is likely due to their peculiar ultrastructure because highly scattering objects, such as mitochondria, are rare, or even absent (38), whereas abundant long thin filaments are oriented along the cell ax is (12), thereby setting a dielectric anisotropy as typically seen in photonic cr ystal fibers. The endfeet of Muller cells cover the ¨ entire inner retinal sur face and have a low refractive index, allowing a highly efficient entr y of light from the vitreous into the Muller ¨ cells (Figs. 2 and 3). At the same time, the increasing refractive index together with their funnel shape at nearly constant lightguiding capability (Fig. 3) make them ingeniously designed light collectors (31). These findings along with their general orientation along the light path might well explain the low absolute backscattering in the retina of only 1­5% reported previously (7, 8). The collective parallel arrangement of Muller cells in the retina ¨ resembles that of optical fibers in fiberoptic plates, which are used to transfer images between spatially separate planes with low loss and low distortion. The structural similarity suggests an analogous function of the Muller cell array in situ (SI Fig. 5). The basic ¨ fiberoptic plate-like structure is especially characteristic for the retinae of all mammals with the exception of the fovea centralis of humans and higher primates, the region of our retina that is responsible for sharp vision; here, the photoreceptor cells are not obscured by any inner retinal layers at all. On average, ever y mammalian Muller cell is c oupled to one ¨ c one photoreceptor cell (17) (responsible for sharp seeing under daylight c onditions, i.e., photopic v ision) plus a species-specific
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Fig. 4. Demonstration of light guidance by individual Muller cells measured ¨ in a modified dual-beam laser trap. (a) A cell is floating freely between the ends of two optical fibers, which are aligned against a backstop visible at top. (b) The Muller cell is trapped, aligned, and stretched out by two counter¨ propagating near-infrared laser beams diverging from the optical fibers (42). (c) The fibers are brought in contact with the cell. Visible light ( 514 nm) emerges from the left (input) fiber and is collected and guided by the cell to the right (output) fiber. The fraction of visible light reentering the core of the output fiber is measured by a power meter, and the near-infrared light is blocked by an appropriate short-pass filter. (Scale bar, 50 m.) (d) Typical time course of the power of visible light measured. When the cell is removed from the trap, it no longer prevents the light from diverging, and the measured power drops considerably. The ratio Pwith cell/Pwithout cell defines the relative guiding efficiency.

To test this hypothesis, we investigated light propagation through individual, enzymatically dissociated living Muller cells by using a ¨ fiberoptical dual-beam laser trap (Fig. 4) (33­35). The optically induced forces in the trap allowed the gentle capture of individual cells from suspension (Fig. 4b). The forces also aligned the cells along the optical ax is without any mechanical contact (Fig. 4 b and c). In addition to the infrared trapping laser beams, visible light was coupled into one of the fibers (input fiber), and the light power coupled back into the opposing (output) fiber was measured (Fig. 4 c and d). Because the light reentering the output fiber depends on the distance from the input fiber and on the optical properties of the
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number of rod photoreceptor cells (17) ( 10 in both man and guinea pig), ser v ing low light level (sc otopic) v ision. Thus, in the case of photopic v ision, the parallel array of Muller cells may ¨ preser ve the in itial image resolution by guiding the light directly to their respective c one photoreceptor cell, min imizing image distortion. This array might also ser ve to improve image c ontrast by increasing the signal-to-noise ratio (39). In sc otopic v ision, Muller cells c ould reduce loss of intensit y by min imizing light ¨ ref lection, particularly at the inner retinal sur face. In summar y, Muller cells in the retina assume the role of optical fibers and ¨ reliably transfer light w ith low scattering f rom the retinal sur face to the photoreceptor cell layer. At the same time, their funnelshape leaves 80% of the retinal volume for other cells and the neuronal c onnectiv it y (SI Fig. 5) and might thus spatially dec ouple light transport f rom neuronal signal processing. The function of glial cells that we describe here ex plains a fundament al feature of the inverted retina as an optical system. Materials and Methods Experimental Animals. Adult guinea pigs of either sex (300 ­500 g) were used throughout the study if not mentioned other w ise. A n imals were deeply anesthetized and k illed by an overdose of 2 g/kg urethane admin istered i.p. A ll ex periments were carried out in ac c ordance w ith applicable Ger man laws of an imal protection and w ith the Association for Research in Vision and Ophthalmolog y St atement for the Use of A n imals in Ophthalmic and Vision Research. The protoc ol of this study was approved by the local ethical c ommittee and adhered to the tenets of the Declaration of Helsink i for ex periments involv ing human tissue.
Confocal Microscopy. Transmission measurements. Freshly dissected

7.4 by using 1 M Tris. In the case of frog retinae, NaCl was reduced from 136 to 115 to maintain physiological osmolarity.
Refractometry. Cells were isolated f rom retinae of humans (clinical samples), cats, guinea pigs, and f rogs (Rana pipiens) as described above. The cells were then immersed in a chamber filled w ith PSS on the st age of an upright phase microsc ope (MBIN-4; LOMO, St. Petersburg, Russia) and allowed to settle to its bottom. Computer-aided phase microsc opy was used to obt ain quantit ative infor mation about the ref ractive index distribution w ithin the cells as described in refs. 23 and 24. The use of a water-immersion lens ( 40, N.A. 0.65) yielded a dif f raction-limited resolution of 0.4 m. Imaging light was filtered by a monochromatic band-pass inter ference filter ( 550 5 nm), polarized, and used to measure dif ferences in ref ractive index bet ween a cellular c ompartment and the surrounding PSS solution w ith k nown ref ractive index. Defined samples of other salt solutions as well as rod outer segments were measured as c ontrols. The latter yielded ref ractive indices of 1.407 0.009 (f rog) and 1.409 0.025 (guinea pig), which are close to an average 1.41 published prev iously (31). Modified Dual-Beam Laser Trap Experiments. Indiv idual acutely isolated guinea pig Muller cells were trapped and aligned in a ¨ dual-beam laser trap (trapping power 0.1 W in each beam) as prev iously described (42). The output of a near-inf rared fiber laser ( 1,064 nm; YLD-10-1064; IPG Photon ics, Burbach, Ger many) was fed into t wo single-mode fibers (PureMode HI 1060; Corn ing, Berlin, Ger many), which were aligned against a backstop opposing each other on the st age of an inverted microsc ope (DMIL; Leica, Wetzlar, Ger many). A n additional laser w ith a wavelength in the v isible range (argon ion laser; 514 nm) was c oupled into one of the fibers, and a power meter (LM2; Coherent Deutschland, Dieburg, Ger many) measured the intensit y of v isible light that c oupled back into the opposing fiber. A short-pass filter was used to exclude the inf rared trapping light f rom detection. In a series of ex periments, the ref ractive index n of the solution was increased to that of the natural surrounding of Muller cells ¨ (n 1.36) (25­27) by adding BSA (P-0834; Sigma). The ref ractive index of that solution was deter mined w ith a ref ractometer (Abbe-Ref raktometer A R 4; A. K r uss Optron ic, Hamburg, ¨ Ger many). In a further set of ex periments, the fibers' backstop was modified in a way such that the fibers were intentionally misaligned by an angle of 2­3°. We thank Dr. L. Cvetkova and Mrs. N. Mozhaiskaja for technical help; Drs. H. Wolburg and A. L¨mmel for contributions to preliminar y stages of the a experiments; Drs. V. Govardovski, M. J. Eaton, and R. Novakovski for critical discussions of earlier versions of the manuscript; Drs. J. Ka and P. ¨s Wiedemann for their continued support and critical discussions; Mr. M. Weinacht at Edmund Optics (Karlsruhe, Germany) for the generous provision of a fiberoptic plate; and the librar y of the Universit¨t Leipzig for a making microfiches available for this project. This work was supported by the Interdisziplina s Zentrum fur Klinische Forschung Leipzig (Faculty of ¨re ¨ Medicine, Universit¨t Leipzig) (A.R., K.F., J. Grosche, K.T., and O.U.), the a Sa ¨chsisches Staatsministerium fur Wissenschaft und Kunst (A.R.), Deut¨ sche Forschungsgemeinschaft Research Training School ``InterNeuro'' Grant GRK 1097, and individual grants to A.R. (R E 849/10-2) and D.S. [DFG-CMPB (FZT 103)]. S.N.S. was supported by National Institutes of Health Grants RCMI-G12RR03035 and MBRS-SO6-GM50695, National Institute of Neurological Disorders and Stroke and National Center for Research Resources Grant SNRP-NS39408, and National Institute of Neurological Disorders and Stroke/Center for Neurological Studies Grants S11-NS48201 and A A BR EP-20-RR-16470.
5. Lee LP, Szema R (2005) Science 310:1148 ­1150. 6. Sundar VC, Yablon AD, Grazul JL, Ilan M, Aizenberg J (2003) Nature 424:899 ­900. 7. Hammer M, Roggan A, Schweitzer D, Muller G (1995) Phys Med Biol 40:963­978. 8. Vos JJ, Bouman M A (1964) J Opt Soc Am 54:95­100.

guinea pig eyes were gently opened at opposite areas. Cornea and lens were removed for insertion of a multimode optical fiber. Sclera, choroid, pigment epithelium, and the photoreceptor layers were locally cut away to allow direct optical access to the end of the prephotoreceptor light path. The transmitted light emanating from the optical fiber was captured through the objective ( 40, N.A. 0.75, water immersion) of a confocal microscope (LSM 510 Meta; Zeiss, Oberkochen, Germany). Reflection measurements. Freshly isolated retinal whole-mounts of several species, including man, were placed on a confocal microscope with the inner sur face pointing toward the objective ( 40, N.A. 0.75, water immersion) and obser ved in ref lection mode. In vivo measurements. A n imals were anesthetized by i.m. application of 50 mg/kg ket amine (Ratiophar m, Ulm, Ger many) and 5 mg/kg x ylazine (BayerVit al, Leverkusen, Ger many). Eyes were gently opened in situ f rom rostral, c ornea and lens were removed, an imals were placed on a c onfocal microsc ope (LSM 510 Met a), and the objective ( 20, N.A. 0.5, water immersion) was inserted into the eye. Images were t aken in ref lection mode.
Cell Isolation. Fresh retinal pieces were incubated in Ca2 - and

Mg2 -free PBS containing 0.03­ 0.1 mg/ml Papain (Boehringer, Mannheim, Germany) for 30 min at 37°C. After washing with PBS containing 200 units/ml DNase I (Sigma, Deisenhofen, Germany), the tissue pieces were gently triturated by a wide-pore pipette to obtain suspensions of isolated cells (40, 41). The supernatant was collected, and PBS was replaced by a physiological salt solution (PSS; 136 mM NaCl/3 mM KCl/1 mM MgCl2/2 mM CaCl2/10 mM Hepes/10 mM D-glucose). The pH of the solution was adjusted to
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