- Email: [email protected]
Biological Bifocal Lenses with Image Separation
Current Biology 20, 1482–1486, August 24, 2010 ª2010 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2010.07.012
Report Biological Bifocal Lenses with Image Separation
Annette Stowasser,1 Alexandra Rapaport,1 John E. Layne,1 Randy C. Morgan,2 and Elke K. Buschbeck1,* 1Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221-0006 2Insectarium, Cincinnati Zoo and Botanical Garden, 3400 Vine Street, Cincinnati, OH 45220-1399
Summary Almost all animal eyes follow a few, relatively well-understood functional plans. Only rarely do researchers discover an eye that diverges fundamentally from known types. The principal eye E2 of sunburst diving beetle (Thermonectus marmoratus) larvae clearly falls into the rarer category. On the basis of two different tests, we here report that it has truly bifocal lenses, something that has been previously suggested only for certain trilobites [1]. Our evidence comes from (1) the relative contrast in images of a square wave grating and (2) the refraction of a narrow laser beam projected through the lens. T. marmoratus larvae have two retinas at different depths behind the lens, and these are situated so that each can receive its own focused image. This is consistent with a novel eye organization that possibly comprises ‘‘two eyes in one.’’ Moreover, we find that in contrast to most commercial bifocal lenses, the lens of E2 exhibits asymmetry, which results in separation of the images both dorsoventrally and rostrocaudally within the layered retina. Visual contrast might thus be improved over conventional bifocal lenses because the unfocused version of one image is shifted away from the focused version of the other, an organization which could potentially be exploited in optical engineering. Results and Discussion Sunburst Diving Beetle Larvae Have Tubular Eyes with Two Retinas Almost all animal eyes follow a few, relatively well-understood functional plans. Nevertheless, there is considerable variation among larval eyes (stemmata) of holometabolous insects. Although many of them are quite simple [2], others are highly specialized camera-type eyes [2–4]. On the basis of their anatomy [5] and behavior [6], the eyes of sunburst diving beetle (Thermonectus mamoratus) larvae fall in the latter category. However, their physical organization, and most likely their mode of function, is highly unusual even among specialized forms. T. mamoratus larvae (Figure 1A) are aquatic, visually guided predators native to the southwest United States [7]. The larvae have 12 eyes, 6 on each side of the head. Four of these eyes (E1 and E2 on either side) are tubular and look directly forward (Figure 1B). The larvae scan with these principal eyes by oscillating their heads dorso-ventrally as they approach potential prey [6]. The anatomy of the retinas of these principal eyes is unusual, as has been described in detail for first-instar
*Correspondence: [email protected]
larvae [5]. We here report that a similar organization is also observed in third-instar larvae, although the size and proportions change somewhat. The retinas are divided into distinct distal and proximal portions. Figure 1C schematically illustrates the shape of the two retinas in E2. The horizontal extents of the oval visual fields of the distal and proximal retinas are 40 –50 , whereas the vertical extents are about 14 and 3.5 , respectively. The distal retina consists of at least 12 tiers of photoreceptor cells, which are oriented approximately perpendicular to the light path. The proximal retina lies directly beneath and contains photoreceptor cells oriented parallel to the light path. The pit of the distal retina (Figure 1C) lies 424 mm (610 mm STD, n = 10), and the top surface of the proximal retina lies 493 mm (616 mm STD, n = 10) behind the back surface of the lens. The lens diameter is 228 mm (610 STD, n = 10). The presence of the two anatomically separated retinas raises the question, ‘‘Which of the retinas receives a focused image from the lens?’’ Our present findings suggest that a bifocal lens provides a focused image for each of them. To the best of our knowledge, this is the first demonstration of truly bifocal lenses in the extant animal kingdom.
Two Independent Methods Reveal the Existence of a Bifocal Lens To measure the optics of the lenses, we first used a modified version of the hanging drop method [8], in which a lens produces images of an object at effective infinity, and these images are observed through a microscope (Figure 2A). We consistently observed focused images at two different distances behind the larval lens, and these images were clearly separated by a region where no sharp focus was formed (Figure 2B; Movie S1). For this method, the lens was mounted on a goniometer, and its orientation was adjusted so that one of the resulting images remained approximately stationary to the viewer while the focus of the microscope was changed (see Figure S1C). We objectively established the positions of these two focal planes by computing relative edge sharpness in each of 70–100 images serially photographed approximately along the optical axes of 15 different lenses from E2 (see Experimental Procedures for details). To obtain the actual distance between frames, we corrected the measured distance for the refractive index of insect Ringer’s solution (1.33) [9]. Figure 2C illustrates the results from one lens: peaks occur in edge sharpness around 372 and 499 mm behind the apex of the back surface of the lens. The combined results from E2 lenses (n = 15; Figure 2D) show that edge sharpness significantly decreases between the two peaks as well as 40 mm before the first image plane and 40 mm after the second image plane (p < 0.003). We verified the existence of two focal planes with a second method [10, 11] modified from Kro¨ger et al. [12] and Toh and Okamura [3]. This method visualized the paths of tiny, parallel light rays passing through the lens. Specifically, a narrow light beam was directed at 20 to 50 locations in a line across the face of the lens (so the line passed through the center), and the refracted rays were visualized in a saline solution containing micro beads (Figure 3A). The refracted beams could be classified into two groups according to their point
Biological Bifocal Lenses with Image Separation 1483
are sensitive to longer wavelengths. The layers are spaced so that they can compensate for chromatic aberration [19]. However, this is not the design of the T. marmoratus E2 eye, in which all the layers of the distal retina (closer to the lens) express putative green opsins, whereas those in the proximal retina (farther away) expresses putative UV opsins [20]. Thus, in contrast to jumping spiders, in T. marmoratus larvae opsins are expressed in the ‘‘wrong’’ layers to compensate for chromatic aberration. Probably the most similar previously described lens system is that of schizochroal trilobites [21, 22]. These long-extinct arthropods had compound eyes, each unit of which might have functioned as an image-forming eye [22]. Their corneal lenses are thought to have consisted of an outer unit of calcite [23] and an inner unit possibly composed of organic material [24]. The reconstruction of the optics from lens unit surfaces revealed the possibility of bifocal lenses. This arrangement might have allowed trilobites to see relatively far and near objects simultaneously [1], which is not possible with a rigid monofocal lens. The bifocal lens therefore is thought to have compensated for the absence of the kind of accommodation mechanisms that exist in vertebrate eyes.
Figure 1. Illustration of the Third-Instar Larvae of Thermonectus marmoratus and Its Principal Eyes (A) Picture of the entire animal. (B) Scanning electron micrograph of the larval head, showing the two large lenses of the principal eyes (E1 and E2) on each side of the head. (C) The gross optical and neural organization of E2. Inserts a and b schematically illustrate the eye organization of the two sections indicated in the scanning-electron-micrograph image. White lines show the approximate visual fields of the retinas. Abbreviations are as follows: PR, proximal retina; DR, distal retina; P, pit of distal retina.
of intersection (Figure 3B). We further confirmed this bisectioning of the beams by computing relative beam density (Figure 4B,D). Our findings can only be explained by the presence of a truly bifocal lens. For instance, if it were an astigmatic lens, as described for the ocelli in some bees, wasps, and blowflies [13, 14], it would have two focal planes, but it would not consistently produce two images of a square wave of arbitrary orientation as we observed. Furthermore, point objects resulted in two point images (Figures S1C and S1D) and not the streaks expected from astigmatism. In addition, our results cannot be an example of spherical aberration, which would lead to an extended region of poor focus (caused by a gradual increase of focal power toward the periphery of the lens). In contrast, T. marmoratus lenses produce two distinct and well-focused images (Figure 2, S1D). Likewise, our observations are distinctly different from multifocal lenses found in fish [15, 16] and terrestrial vertebrates [17]. Those lenses have several zones with different focal lengths and are thought to correct for chromatic aberration by focusing all wavelengths onto the same plane in the retina [15–17]. T. marmoratus lenses, in contrast, have two distinct focal planes that are substantially separated from each other. In some ways, the eyes appear to resemble the principal eyes of certain jumping spiders that have retinas with four distinct layers [18]. In the spider retina, the layers closer to the lens have photoreceptors sensitive to shorter wavelengths, whereas those further away
The Bifocal Lenses Might Function as ‘‘Two Eyes in One’’ It is plausible that T. marmoratus larvae also benefit from being able to simultaneously focus far and near objects on individual retinas. In addition, we think that within the principal eyes, separate images of the same object could be focused on each of two retinas, allowing each eye to function as ‘‘two eyes in one.’’ Our optical measurements suggest that the two focal planes of each lens are separated by about 100 mm, which roughly matches the anatomical separation of these two retinas. In E2 the images are separated by 105 mm (610 mm standard error of the mean [SEM], n = 15), and the retinas of their contralateral eyes are separated by 132 mm (65 mm SEM, n = 13) if measured from the upper edge of the distal retina to the upper surface of the proximal retina. Determining the exact locations of the two images will require further investigation because in vivo the image distance depends on the actual distance to a viewed object and is influenced by the refractive index of the tissue behind the lens (which potentially could differ from that of the saline that we used for our measurements). Image Disparity Might Allow Larvae to See Prey Better In contrast to what has been described in trilobites, and what is found in many commercial bifocal lenses (such as contact lenses or cataract replacement lenses), T. marmoratus larval lenses appear to use an interesting optical ‘‘trick.’’ A major problem with comparable bifocal lenses is that their concentric and collinear design causes the contrast in both images to be reduced by the unfocused version of the other image [25–27]. In T. mamoratus, the images are vertically displaced, presumably as a result of asymmetries in the lens. It has been reported that in bifocal systems (such as those used for intraocular lenses), the first image is more ‘‘contaminated’’ by the blurry second image [25] than is the second image by the first. Therefore, we estimated to what extent the observed image disparity improves the contrast of the first sharp image. Specifically, we calculated contrast modulation of the resulting image column by column. This allows the portion of the focused image that overlaps with its unfocused counterpart to be compared with the portion of the image that does not overlap (see Experimental Procedures for details).
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Figure 2. Measurements of Relative Edge Sharpness of Serial Photographs Show that the Lens Forms Two Sharp Images (A) Schematic illustration of the method. (B) Representative series of photographs used for computing edge sharpness. Arabic numerals indicate the distance from the back surface of the lens to the photographed frame (in mm). Roman numerals indicate correspondence between photographs and their edge sharpness in graphs (C) and (D). (C) Measurements of edge sharpness of the lens that produced the images illustrated in (B). Error bars show the standard deviation of ten computations of edge sharpness for each frame. (D) Average edge sharpness of the peak and trough values as indicated in graph (C) from 15 photograph series; standard errors are included. The two peak values (B) were significantly different from the troughs (A) with a p < 0.003.
Figure S1B shows that the image contrast improves at least 3-fold. It is unclear exactly what causes the asymmetries, but they must relate to the precise location of the two optical centers in relation to their respective apertures. The image separation is visible in the 3D reconstruction of a point image (Figure S1D) and of the square-wave image series (Figure 4A; Movie S2), as well as in the ray density plot (Figure 4B). The images are
separated in the vertical plane, and their deviation is not visible if lenses are turned by 90 (Figures 4C and 4D). The divergence of images is probably an advantage for T. marmoratus. Because these larvae use dorso-ventral scanning movements [6], corresponding focused images would reach the two retinas with only a small temporal delay resulting from the small dorsoventral separation between the two images. These larvae normally hunt small objects that would be
Figure 3. Laser Images Confirm the Presence of Two Focal Planes (A) Schematic illustration of the method. (B) Composite of five scan frames reveal two beams that intersect each other nearer to the lens than do the other three. These intersections occur in the two focal planes, the positions of which correspond well to the edge sharpness measurements, as indicated by the two arrows. The position of the lens is indicated schematically by an oval. Grayscale photographs are illustrated with false color.
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Figure 4. Visualization of the Image Disparity (A and C) The three-dimensional reconstruction of an edge-sharpness frame series visualizes a square-wave image that has been projected by the insect lens. Insets show best-focused images, and arrows show their location along the light path. (B and D) illustrate ray density plots. (A) The side view shows that two blur circles diverge within the dorso-ventral plane of the lens. (B) The divergence also is visible in the ray density plot (arrows indicate the approximate directions of blur-circle movement). (C and D) Viewed from the dorsal direction, blur circles are relatively well aligned.
relatively distinct when viewed against a homogenous aquatic background. When some of the photoreceptor cells ‘‘see’’ the focused image of a small prey object, this image is not contaminated by the blurry image of the same object, but rather by a blurry image of the background. A blurry homogenous background would interfere relatively little with the perception of the sharp image. A similar mechanism could be exploited by commercial bi- or multi-focal systems. Experimental Procedures Animals and Lenses T. marmoratus used were offspring of beetles provided by the Insectarium of the Cincinnati Zoo and Botanical Garden or of beetles collected between 2004 and 2008 near Tucson, AZ, USA. After hatching, T. marmoratus larvae were reared at 37 C in separate containers on frozen bloodworms and live mosquito larvae. All data were obtained from third-instar larvae (which have the largest lenses) 24 hr after ecdysis. During this time, larvae hunt very successfully, suggesting that the visual system is fully functional. Schematics are based on histological sections, prepared as described in [5]. To prepare samples for imaging via scanning electron micrograph, we dried whole animals, mounted them on coverslips, and viewed them with an ESEM XL30 (FEI Company) microscope. For optical measurements, larvae were anesthetized via cooling and were decapitated, and a small piece of the head capsule containing lenses was excised. We cleaned the backs of the lenses with a fine brush and mounted them on a pinhole by using wax to attach the exoskeleton surrounding the lenses. Image Contrast Measurements As in the hanging drop method [8], we used a microscope to observe the images formed by the lens. We tested the efficacy of this method by performing it on T. mamoratus adult compound-eye facet lenses, and this resulted in one focal plane (data not shown). The lens was mounted with wax between two coverslips so that images were formed between the lens and upper coverslip (Figure 2A). The space between the coverslips was filled with a 50% dilution of insect Ringer’s solution [28]. In contrast to 100% Ringer’s solution (which resulted in the presence of minor wrinkles) and distilled water (which led to noticeable bloating), this
concentration produced no visible deformation of the lens. The coverslip sandwich was mounted on a goniometer that replaced the microscope stage. The back surface of the lenses faced the microscope objective lens. A square-wave grating (0.353 cycles/mm, USAF 1951 negative test target from Edmund Optics) served as the object and was placed 12.5 cm beneath the microscope stage—effectively infinity for this small lens. The condenser was removed, and the object was aligned with the center of the microscope optics. The square wave was illuminated with monochromatic light (542 nm), and the rays refracted by the lens were photographed with a 3CCD camera (Hitachi HV-f22) with a pixel resolution of 1360 3 1024 and acquired with ImageJ 1.38 (U. S. National Institutes of Health, Bethesda, MD, with plug-in QuickTime_Capture modified for highdefinition image acquisition). The frames were photographed at 5mm intervals from the back surface of the lens to well beyond the focal planes. We evaluated the photographs for the focus of the square-wave image by computing relative edge sharpness. To do so, we first removed shot noise by digitally convolving the frames with a 35 3 35 point 2D Butterworth low-pass filter with a cutoff of ten times the square-wave frequency (Matlab 7.4, The Mathworks, Natick, MA). Then, images were cropped to the region of the square wave (plus some background). Relative edge sharpness of each frame was computed as the grand mean of grayscale intensity value of neighboring pixels, twice differentiated (Figure S1A) along the x, y axes and both diagonal dimensions. This resulted in a single metric for the slope of the change in intensity in the image; higher values indicated a steeper slope (sharper edges). The final relative edge-sharpness value was the average of ten such computations for each frame series, which accounted for variation due to image cropping. This method allowed an automated assessment of image quality of individual frames without involving assumptions about the image. The combined data (Figure 2D) show the average of 15 individuals. Each individual contributed to each of the five bars with three points, as shown in Figure 2C. A Turkey’s test accounted for multiple comparisons. To visualize light rays, we performed 3D reconstructions of image stacks with the Vortex module of Amira, version 5.2.2 (see Figures 4A and 4C; see also Figure S1D and Movie S2). To estimate to what extent the image disparity improves contrast, we calculated the contrast modulation across the images of five lenses (Figure S1B). First, we rotated each image to orient the stripe direction horizontally. Next, we calculated contrast modulation (Equation 1) at four points for each image column (between maximum gray values of the three light stripes and minimum values of the two darker
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inter-stripe areas). To average the five images, we aligned them at the peak. Modulationh
Imax 2 Imin Imax + Imin
6. (1)
7. Laser Measurements The pinhole, with the lenses, was mounted vertically with wax on one side of a glass container filled with 50% concentrated insect Ringer’s solution [28] and a dilute suspension of microbeads (0.1 mm, Fluka, from Sigma-Aldrich) (Figure 3A). To ensure a flat refracting surface, we filled the container to the top and covered it with a coverslip. A horizontal laser beam (commercial 50 mW high-powered green laser pointer, 530 nm) was projected through the lens, and the rays refracted by the lens and scattered by the microbeads were photographed through a microscope with a MagnaFire camera (a 1300 3 1030 pixel digital camera from Optronics, Goleta, CA). In order to achieve a narrow beam of no more than 10–20 mm diameter, we focused the laser at the front surface of the insect lens with a glass lens (f = 30 mm). If we assume a beam of Gaussian intensity distribution, this resulted in approximately parallel light for several 100 mm around the focus of the beam (the initial beam diameter was w1 mm; thus, the Rayleigh range was w600 mm). For each lens, 20–50 photographs/lens were taken as the laser was moved with a motorized micromanipulator in steps of 10 mm, so that each lens was scanned from one edge through its center to the opposite edge, approximately horizontally or vertically relative to the animal. The image in Figure 3B is a composite of five representative frames from the scan. To further establish the presence of two focal points, we computed the relative local ray density of all images in each scan. To do so, we converted the paths of the laser beams in all photographs to linear equations and solved their y coordinates at 1 pixel (1.1 mm) intervals along the x axis. The relative local density of all such x, y coordinates from a laser scan was computed by 3 pixel kernel density estimation (MATLAB toolbox Version 1.0 09/13/05, developed by Joern Diedrichsen). We chose this method over the method developed by Malkki and Kro¨ger [16] to avoid making assumptions about the aperture(s) and optical center(s) of the lens.
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Supplemental Information 19. The supplemental Information includes one figure and two movies and can be found with this article online at doi:10.1016/j.cub.2010.07.012. Acknowledgments We thank Shannon Werner, Doug J. Kohls, and Necati Kaval for providing technical assistance and the Cincinnati Zoo and Botanical Garden for providing the original population of sunburst diving beetles. Moreover, we are grateful to Shannon Werner for taking care of our beetle population and helping with histology, Peter Mu¨ller for helpful tips regarding the laser method, and Thomas Cronin for generously lending us his goniometer. Three anonymous reviewers provided valuable comments that greatly helped to improve the manuscript. This material is based upon work supported by the National Science Foundation under Grant No. 0545978.
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Received: May 12, 2010 Revised: July 3, 2010 Accepted: July 5, 2010 Published online: August 5, 2010
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References
26.
1. Ga´l, J., Horva´th, G., Clarkson, E.N.K., and Haiman, O. (2000). Image formation by bifocal lenses in a trilobite eye? Vision Res. 40, 843–853. 2. Gilbert, C. (1994). Form and function of stemmata in larvae of holometabolous insects. Annu. Rev. Entomol. 39, 323–349. 3. Toh, Y., and Okamura, J.-Y. (2007). Morphological and optical properties of the corneal lens and retinal structure in the posterior large stemma of the tiger beetle larva. Vision Res. 47, 1756–1768. 4. Toh, Y., and Okamura, J.-Y. (2001). Behavioural responses of the tiger beetle larva to moving objects: role of binocular and monocular vision. J. Exp. Biol. 204, 615–625. 5. Mandapaka, K., Morgan, R.C., and Buschbeck, E.K. (2006). Twentyeight retinas but only twelve eyes: an anatomical analysis of the larval
27.
28.
visual system of the diving beetle Thermonectus marmoratus (Coleoptera: Dytiscidae). J. Comp. Neurol. 497, 166–181. Buschbeck, E.K., Sbita, S.J., and Morgan, R.C. (2007). Scanning behavior by larvae of the predacious diving beetle, Thermonectus marmoratus (Coleoptera: Dytiscidae) enlarges visual field prior to prey capture. J. Comp. Physiol. [A] 193, 973–982. Larson, D.J., Alarie, Y., and Roughley, R.E. (2000). Predaceous Diving Beetles (Coleoptera: Dytiscidae) of the Nearctic Region, with emphasis on the fauna of Canada and Alaska (Ottawa: National Research Council of Canada Research Press). Homann, H. (1924). Zum Problem der Ocellenfunktion bei den Insekten. Z. Vgl. Physiol. 1, 541–578. Galbraith, W. (1955). The optical measurement of depth. Q. J. Microsc. Sci. 96, 285–288. Campbell, M.C.W., and Hughes, A. (1981). An analytic, gradient index schematic lens and eye for the rat which predicts aberrations for finite pupils. Vision Res. 21, 1129–1148. Sivak, J.G. (1982). Optical properties of a cephalopod eye (the short finned squid, Illex illecebrosus). J. Comp. Physiol. 147, 323–327. Kro¨ger, R.H.H., Fritsches, K.A., and Warrant, E.J. (2009). Lens optical properties in the eyes of large marine predatory teleosts. J. Comp. Physiol. [A] 195, 175–182. Warrant, E.J., Kelber, A., Walle´n, R., and Wcislo, W.T. (2006). Ocellar optics in nocturnal and diurnal bees and wasps. Arthropod Struct. Dev. 35, 293–305. Schuppe, H., and Hengstenberg, R. (1993). Optical properties of the ocelli of Calliphora erythrocephala and their role in the dorsal light response. J. Comp. Physiol. [A] 173, 143–149. Kro¨ger, R.H.H., Campbell, M.C.W., Fernald, R.D., and Wagner, H.-J. (1999). Multifocal lenses compensate for chromatic defocus in vertebrate eyes. J. Comp. Physiol. [A] 184, 361–369. Malkki, P.E., and Kro¨ger, R.H.H. (2005). Visualization of chromatic correction of fish lenses by multiple focal lengths. Journal of Optics A: Pure Applied Optics. 7, 691–700. Malmstro¨m, T., and Kro¨ger, R.H.H. (2006). Pupil shapes and lens optics in the eyes of terrestrial vertebrates. J. Exp. Biol. 209, 18–25. Land, M.F. (1969). Structure of the retinae of the principal eyes of jumping spiders (Salticidae: dendryphantinae) in relation to visual optics. J. Exp. Biol. 51, 443–470. Blest, A.D., Hardie, R.C., McIntyre, P., and Williams, D.S. (1981). The spectral sensitivities of identified receptors and the function of retinal tiering in the principal eyes of a jumping spider. J. Comp. Physiol. 145, 227–239. Maksimovic, S., Cook, T.A., and Buschbeck, E.K. (2009). Spatial distribution of opsin-encoding mRNAs in the tiered larval retinas of the sunburst diving beetle Thermonectus marmoratus (Coleoptera: Dytiscidae). J. Exp. Biol. 212, 3781–3794. Clarkson, E., Levi-Setti, R., and Horva´th, G. (2006). The eyes of trilobites: The oldest preserved visual system. Arthropod Struct. Dev. 35, 247–259. Fordyce, D., and Cronin, T.W. (1993). Trilobite vision: A comparison of schizochroal and holochroal eyes with the compound eyes of modern arthropods. Paleobiology 19, 288–303. Towe, K.M. (1973). Trilobite eyes: calcified lenses in vivo. Science 179, 1007–1009. Horva´th, G. (1989). Geometric optics of trilobite eyes: a theoretical study of the shape of the aspherical interface in the cornea of schizochroal eyes of phacopid trilobites. Math. Biosci. 96, 79–94. Monte´s-Mico´, R., Espan˜a, E., Bueno, I., Charman, W.N., and Menezo, J.L. (2004). Visual performance with multifocal intraocular lenses: Mesopic contrast sensitivity under distance and near conditions. Ophthalmology 111, 85–96. Navarro, R., Ferro, M., Artal, P., and Miranda, I. (1993). Modulation transfer functions of eyes implanted with intraocular lenses. Appl. Opt. 32, 6359–6367. Artal, P., Marcos, S., Navarro, R., Miranda, I., and Ferro, M. (1995). Through focus image quality of eyes implanted with monofocal and multifocal intraocular lenses. Opt. Eng. 34, 772–779. O’Shea, M., and Adams, M.E. (1981). Pentapeptide (proctolin) associated with an identified neuron. Science 213, 567–569.
DOI 10.1016/j.cub.2010.07.012
Report Biological Bifocal Lenses with Image Separation
Annette Stowasser,1 Alexandra Rapaport,1 John E. Layne,1 Randy C. Morgan,2 and Elke K. Buschbeck1,* 1Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221-0006 2Insectarium, Cincinnati Zoo and Botanical Garden, 3400 Vine Street, Cincinnati, OH 45220-1399
Summary Almost all animal eyes follow a few, relatively well-understood functional plans. Only rarely do researchers discover an eye that diverges fundamentally from known types. The principal eye E2 of sunburst diving beetle (Thermonectus marmoratus) larvae clearly falls into the rarer category. On the basis of two different tests, we here report that it has truly bifocal lenses, something that has been previously suggested only for certain trilobites [1]. Our evidence comes from (1) the relative contrast in images of a square wave grating and (2) the refraction of a narrow laser beam projected through the lens. T. marmoratus larvae have two retinas at different depths behind the lens, and these are situated so that each can receive its own focused image. This is consistent with a novel eye organization that possibly comprises ‘‘two eyes in one.’’ Moreover, we find that in contrast to most commercial bifocal lenses, the lens of E2 exhibits asymmetry, which results in separation of the images both dorsoventrally and rostrocaudally within the layered retina. Visual contrast might thus be improved over conventional bifocal lenses because the unfocused version of one image is shifted away from the focused version of the other, an organization which could potentially be exploited in optical engineering. Results and Discussion Sunburst Diving Beetle Larvae Have Tubular Eyes with Two Retinas Almost all animal eyes follow a few, relatively well-understood functional plans. Nevertheless, there is considerable variation among larval eyes (stemmata) of holometabolous insects. Although many of them are quite simple [2], others are highly specialized camera-type eyes [2–4]. On the basis of their anatomy [5] and behavior [6], the eyes of sunburst diving beetle (Thermonectus mamoratus) larvae fall in the latter category. However, their physical organization, and most likely their mode of function, is highly unusual even among specialized forms. T. mamoratus larvae (Figure 1A) are aquatic, visually guided predators native to the southwest United States [7]. The larvae have 12 eyes, 6 on each side of the head. Four of these eyes (E1 and E2 on either side) are tubular and look directly forward (Figure 1B). The larvae scan with these principal eyes by oscillating their heads dorso-ventrally as they approach potential prey [6]. The anatomy of the retinas of these principal eyes is unusual, as has been described in detail for first-instar
*Correspondence: [email protected]
larvae [5]. We here report that a similar organization is also observed in third-instar larvae, although the size and proportions change somewhat. The retinas are divided into distinct distal and proximal portions. Figure 1C schematically illustrates the shape of the two retinas in E2. The horizontal extents of the oval visual fields of the distal and proximal retinas are 40 –50 , whereas the vertical extents are about 14 and 3.5 , respectively. The distal retina consists of at least 12 tiers of photoreceptor cells, which are oriented approximately perpendicular to the light path. The proximal retina lies directly beneath and contains photoreceptor cells oriented parallel to the light path. The pit of the distal retina (Figure 1C) lies 424 mm (610 mm STD, n = 10), and the top surface of the proximal retina lies 493 mm (616 mm STD, n = 10) behind the back surface of the lens. The lens diameter is 228 mm (610 STD, n = 10). The presence of the two anatomically separated retinas raises the question, ‘‘Which of the retinas receives a focused image from the lens?’’ Our present findings suggest that a bifocal lens provides a focused image for each of them. To the best of our knowledge, this is the first demonstration of truly bifocal lenses in the extant animal kingdom.
Two Independent Methods Reveal the Existence of a Bifocal Lens To measure the optics of the lenses, we first used a modified version of the hanging drop method [8], in which a lens produces images of an object at effective infinity, and these images are observed through a microscope (Figure 2A). We consistently observed focused images at two different distances behind the larval lens, and these images were clearly separated by a region where no sharp focus was formed (Figure 2B; Movie S1). For this method, the lens was mounted on a goniometer, and its orientation was adjusted so that one of the resulting images remained approximately stationary to the viewer while the focus of the microscope was changed (see Figure S1C). We objectively established the positions of these two focal planes by computing relative edge sharpness in each of 70–100 images serially photographed approximately along the optical axes of 15 different lenses from E2 (see Experimental Procedures for details). To obtain the actual distance between frames, we corrected the measured distance for the refractive index of insect Ringer’s solution (1.33) [9]. Figure 2C illustrates the results from one lens: peaks occur in edge sharpness around 372 and 499 mm behind the apex of the back surface of the lens. The combined results from E2 lenses (n = 15; Figure 2D) show that edge sharpness significantly decreases between the two peaks as well as 40 mm before the first image plane and 40 mm after the second image plane (p < 0.003). We verified the existence of two focal planes with a second method [10, 11] modified from Kro¨ger et al. [12] and Toh and Okamura [3]. This method visualized the paths of tiny, parallel light rays passing through the lens. Specifically, a narrow light beam was directed at 20 to 50 locations in a line across the face of the lens (so the line passed through the center), and the refracted rays were visualized in a saline solution containing micro beads (Figure 3A). The refracted beams could be classified into two groups according to their point
Biological Bifocal Lenses with Image Separation 1483
are sensitive to longer wavelengths. The layers are spaced so that they can compensate for chromatic aberration [19]. However, this is not the design of the T. marmoratus E2 eye, in which all the layers of the distal retina (closer to the lens) express putative green opsins, whereas those in the proximal retina (farther away) expresses putative UV opsins [20]. Thus, in contrast to jumping spiders, in T. marmoratus larvae opsins are expressed in the ‘‘wrong’’ layers to compensate for chromatic aberration. Probably the most similar previously described lens system is that of schizochroal trilobites [21, 22]. These long-extinct arthropods had compound eyes, each unit of which might have functioned as an image-forming eye [22]. Their corneal lenses are thought to have consisted of an outer unit of calcite [23] and an inner unit possibly composed of organic material [24]. The reconstruction of the optics from lens unit surfaces revealed the possibility of bifocal lenses. This arrangement might have allowed trilobites to see relatively far and near objects simultaneously [1], which is not possible with a rigid monofocal lens. The bifocal lens therefore is thought to have compensated for the absence of the kind of accommodation mechanisms that exist in vertebrate eyes.
Figure 1. Illustration of the Third-Instar Larvae of Thermonectus marmoratus and Its Principal Eyes (A) Picture of the entire animal. (B) Scanning electron micrograph of the larval head, showing the two large lenses of the principal eyes (E1 and E2) on each side of the head. (C) The gross optical and neural organization of E2. Inserts a and b schematically illustrate the eye organization of the two sections indicated in the scanning-electron-micrograph image. White lines show the approximate visual fields of the retinas. Abbreviations are as follows: PR, proximal retina; DR, distal retina; P, pit of distal retina.
of intersection (Figure 3B). We further confirmed this bisectioning of the beams by computing relative beam density (Figure 4B,D). Our findings can only be explained by the presence of a truly bifocal lens. For instance, if it were an astigmatic lens, as described for the ocelli in some bees, wasps, and blowflies [13, 14], it would have two focal planes, but it would not consistently produce two images of a square wave of arbitrary orientation as we observed. Furthermore, point objects resulted in two point images (Figures S1C and S1D) and not the streaks expected from astigmatism. In addition, our results cannot be an example of spherical aberration, which would lead to an extended region of poor focus (caused by a gradual increase of focal power toward the periphery of the lens). In contrast, T. marmoratus lenses produce two distinct and well-focused images (Figure 2, S1D). Likewise, our observations are distinctly different from multifocal lenses found in fish [15, 16] and terrestrial vertebrates [17]. Those lenses have several zones with different focal lengths and are thought to correct for chromatic aberration by focusing all wavelengths onto the same plane in the retina [15–17]. T. marmoratus lenses, in contrast, have two distinct focal planes that are substantially separated from each other. In some ways, the eyes appear to resemble the principal eyes of certain jumping spiders that have retinas with four distinct layers [18]. In the spider retina, the layers closer to the lens have photoreceptors sensitive to shorter wavelengths, whereas those further away
The Bifocal Lenses Might Function as ‘‘Two Eyes in One’’ It is plausible that T. marmoratus larvae also benefit from being able to simultaneously focus far and near objects on individual retinas. In addition, we think that within the principal eyes, separate images of the same object could be focused on each of two retinas, allowing each eye to function as ‘‘two eyes in one.’’ Our optical measurements suggest that the two focal planes of each lens are separated by about 100 mm, which roughly matches the anatomical separation of these two retinas. In E2 the images are separated by 105 mm (610 mm standard error of the mean [SEM], n = 15), and the retinas of their contralateral eyes are separated by 132 mm (65 mm SEM, n = 13) if measured from the upper edge of the distal retina to the upper surface of the proximal retina. Determining the exact locations of the two images will require further investigation because in vivo the image distance depends on the actual distance to a viewed object and is influenced by the refractive index of the tissue behind the lens (which potentially could differ from that of the saline that we used for our measurements). Image Disparity Might Allow Larvae to See Prey Better In contrast to what has been described in trilobites, and what is found in many commercial bifocal lenses (such as contact lenses or cataract replacement lenses), T. marmoratus larval lenses appear to use an interesting optical ‘‘trick.’’ A major problem with comparable bifocal lenses is that their concentric and collinear design causes the contrast in both images to be reduced by the unfocused version of the other image [25–27]. In T. mamoratus, the images are vertically displaced, presumably as a result of asymmetries in the lens. It has been reported that in bifocal systems (such as those used for intraocular lenses), the first image is more ‘‘contaminated’’ by the blurry second image [25] than is the second image by the first. Therefore, we estimated to what extent the observed image disparity improves the contrast of the first sharp image. Specifically, we calculated contrast modulation of the resulting image column by column. This allows the portion of the focused image that overlaps with its unfocused counterpart to be compared with the portion of the image that does not overlap (see Experimental Procedures for details).
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Figure 2. Measurements of Relative Edge Sharpness of Serial Photographs Show that the Lens Forms Two Sharp Images (A) Schematic illustration of the method. (B) Representative series of photographs used for computing edge sharpness. Arabic numerals indicate the distance from the back surface of the lens to the photographed frame (in mm). Roman numerals indicate correspondence between photographs and their edge sharpness in graphs (C) and (D). (C) Measurements of edge sharpness of the lens that produced the images illustrated in (B). Error bars show the standard deviation of ten computations of edge sharpness for each frame. (D) Average edge sharpness of the peak and trough values as indicated in graph (C) from 15 photograph series; standard errors are included. The two peak values (B) were significantly different from the troughs (A) with a p < 0.003.
Figure S1B shows that the image contrast improves at least 3-fold. It is unclear exactly what causes the asymmetries, but they must relate to the precise location of the two optical centers in relation to their respective apertures. The image separation is visible in the 3D reconstruction of a point image (Figure S1D) and of the square-wave image series (Figure 4A; Movie S2), as well as in the ray density plot (Figure 4B). The images are
separated in the vertical plane, and their deviation is not visible if lenses are turned by 90 (Figures 4C and 4D). The divergence of images is probably an advantage for T. marmoratus. Because these larvae use dorso-ventral scanning movements [6], corresponding focused images would reach the two retinas with only a small temporal delay resulting from the small dorsoventral separation between the two images. These larvae normally hunt small objects that would be
Figure 3. Laser Images Confirm the Presence of Two Focal Planes (A) Schematic illustration of the method. (B) Composite of five scan frames reveal two beams that intersect each other nearer to the lens than do the other three. These intersections occur in the two focal planes, the positions of which correspond well to the edge sharpness measurements, as indicated by the two arrows. The position of the lens is indicated schematically by an oval. Grayscale photographs are illustrated with false color.
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Figure 4. Visualization of the Image Disparity (A and C) The three-dimensional reconstruction of an edge-sharpness frame series visualizes a square-wave image that has been projected by the insect lens. Insets show best-focused images, and arrows show their location along the light path. (B and D) illustrate ray density plots. (A) The side view shows that two blur circles diverge within the dorso-ventral plane of the lens. (B) The divergence also is visible in the ray density plot (arrows indicate the approximate directions of blur-circle movement). (C and D) Viewed from the dorsal direction, blur circles are relatively well aligned.
relatively distinct when viewed against a homogenous aquatic background. When some of the photoreceptor cells ‘‘see’’ the focused image of a small prey object, this image is not contaminated by the blurry image of the same object, but rather by a blurry image of the background. A blurry homogenous background would interfere relatively little with the perception of the sharp image. A similar mechanism could be exploited by commercial bi- or multi-focal systems. Experimental Procedures Animals and Lenses T. marmoratus used were offspring of beetles provided by the Insectarium of the Cincinnati Zoo and Botanical Garden or of beetles collected between 2004 and 2008 near Tucson, AZ, USA. After hatching, T. marmoratus larvae were reared at 37 C in separate containers on frozen bloodworms and live mosquito larvae. All data were obtained from third-instar larvae (which have the largest lenses) 24 hr after ecdysis. During this time, larvae hunt very successfully, suggesting that the visual system is fully functional. Schematics are based on histological sections, prepared as described in [5]. To prepare samples for imaging via scanning electron micrograph, we dried whole animals, mounted them on coverslips, and viewed them with an ESEM XL30 (FEI Company) microscope. For optical measurements, larvae were anesthetized via cooling and were decapitated, and a small piece of the head capsule containing lenses was excised. We cleaned the backs of the lenses with a fine brush and mounted them on a pinhole by using wax to attach the exoskeleton surrounding the lenses. Image Contrast Measurements As in the hanging drop method [8], we used a microscope to observe the images formed by the lens. We tested the efficacy of this method by performing it on T. mamoratus adult compound-eye facet lenses, and this resulted in one focal plane (data not shown). The lens was mounted with wax between two coverslips so that images were formed between the lens and upper coverslip (Figure 2A). The space between the coverslips was filled with a 50% dilution of insect Ringer’s solution [28]. In contrast to 100% Ringer’s solution (which resulted in the presence of minor wrinkles) and distilled water (which led to noticeable bloating), this
concentration produced no visible deformation of the lens. The coverslip sandwich was mounted on a goniometer that replaced the microscope stage. The back surface of the lenses faced the microscope objective lens. A square-wave grating (0.353 cycles/mm, USAF 1951 negative test target from Edmund Optics) served as the object and was placed 12.5 cm beneath the microscope stage—effectively infinity for this small lens. The condenser was removed, and the object was aligned with the center of the microscope optics. The square wave was illuminated with monochromatic light (542 nm), and the rays refracted by the lens were photographed with a 3CCD camera (Hitachi HV-f22) with a pixel resolution of 1360 3 1024 and acquired with ImageJ 1.38 (U. S. National Institutes of Health, Bethesda, MD, with plug-in QuickTime_Capture modified for highdefinition image acquisition). The frames were photographed at 5mm intervals from the back surface of the lens to well beyond the focal planes. We evaluated the photographs for the focus of the square-wave image by computing relative edge sharpness. To do so, we first removed shot noise by digitally convolving the frames with a 35 3 35 point 2D Butterworth low-pass filter with a cutoff of ten times the square-wave frequency (Matlab 7.4, The Mathworks, Natick, MA). Then, images were cropped to the region of the square wave (plus some background). Relative edge sharpness of each frame was computed as the grand mean of grayscale intensity value of neighboring pixels, twice differentiated (Figure S1A) along the x, y axes and both diagonal dimensions. This resulted in a single metric for the slope of the change in intensity in the image; higher values indicated a steeper slope (sharper edges). The final relative edge-sharpness value was the average of ten such computations for each frame series, which accounted for variation due to image cropping. This method allowed an automated assessment of image quality of individual frames without involving assumptions about the image. The combined data (Figure 2D) show the average of 15 individuals. Each individual contributed to each of the five bars with three points, as shown in Figure 2C. A Turkey’s test accounted for multiple comparisons. To visualize light rays, we performed 3D reconstructions of image stacks with the Vortex module of Amira, version 5.2.2 (see Figures 4A and 4C; see also Figure S1D and Movie S2). To estimate to what extent the image disparity improves contrast, we calculated the contrast modulation across the images of five lenses (Figure S1B). First, we rotated each image to orient the stripe direction horizontally. Next, we calculated contrast modulation (Equation 1) at four points for each image column (between maximum gray values of the three light stripes and minimum values of the two darker
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inter-stripe areas). To average the five images, we aligned them at the peak. Modulationh
Imax 2 Imin Imax + Imin
6. (1)
7. Laser Measurements The pinhole, with the lenses, was mounted vertically with wax on one side of a glass container filled with 50% concentrated insect Ringer’s solution [28] and a dilute suspension of microbeads (0.1 mm, Fluka, from Sigma-Aldrich) (Figure 3A). To ensure a flat refracting surface, we filled the container to the top and covered it with a coverslip. A horizontal laser beam (commercial 50 mW high-powered green laser pointer, 530 nm) was projected through the lens, and the rays refracted by the lens and scattered by the microbeads were photographed through a microscope with a MagnaFire camera (a 1300 3 1030 pixel digital camera from Optronics, Goleta, CA). In order to achieve a narrow beam of no more than 10–20 mm diameter, we focused the laser at the front surface of the insect lens with a glass lens (f = 30 mm). If we assume a beam of Gaussian intensity distribution, this resulted in approximately parallel light for several 100 mm around the focus of the beam (the initial beam diameter was w1 mm; thus, the Rayleigh range was w600 mm). For each lens, 20–50 photographs/lens were taken as the laser was moved with a motorized micromanipulator in steps of 10 mm, so that each lens was scanned from one edge through its center to the opposite edge, approximately horizontally or vertically relative to the animal. The image in Figure 3B is a composite of five representative frames from the scan. To further establish the presence of two focal points, we computed the relative local ray density of all images in each scan. To do so, we converted the paths of the laser beams in all photographs to linear equations and solved their y coordinates at 1 pixel (1.1 mm) intervals along the x axis. The relative local density of all such x, y coordinates from a laser scan was computed by 3 pixel kernel density estimation (MATLAB toolbox Version 1.0 09/13/05, developed by Joern Diedrichsen). We chose this method over the method developed by Malkki and Kro¨ger [16] to avoid making assumptions about the aperture(s) and optical center(s) of the lens.
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Supplemental Information 19. The supplemental Information includes one figure and two movies and can be found with this article online at doi:10.1016/j.cub.2010.07.012. Acknowledgments We thank Shannon Werner, Doug J. Kohls, and Necati Kaval for providing technical assistance and the Cincinnati Zoo and Botanical Garden for providing the original population of sunburst diving beetles. Moreover, we are grateful to Shannon Werner for taking care of our beetle population and helping with histology, Peter Mu¨ller for helpful tips regarding the laser method, and Thomas Cronin for generously lending us his goniometer. Three anonymous reviewers provided valuable comments that greatly helped to improve the manuscript. This material is based upon work supported by the National Science Foundation under Grant No. 0545978.
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Received: May 12, 2010 Revised: July 3, 2010 Accepted: July 5, 2010 Published online: August 5, 2010
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References
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1. Ga´l, J., Horva´th, G., Clarkson, E.N.K., and Haiman, O. (2000). Image formation by bifocal lenses in a trilobite eye? Vision Res. 40, 843–853. 2. Gilbert, C. (1994). Form and function of stemmata in larvae of holometabolous insects. Annu. Rev. Entomol. 39, 323–349. 3. Toh, Y., and Okamura, J.-Y. (2007). Morphological and optical properties of the corneal lens and retinal structure in the posterior large stemma of the tiger beetle larva. Vision Res. 47, 1756–1768. 4. Toh, Y., and Okamura, J.-Y. (2001). Behavioural responses of the tiger beetle larva to moving objects: role of binocular and monocular vision. J. Exp. Biol. 204, 615–625. 5. Mandapaka, K., Morgan, R.C., and Buschbeck, E.K. (2006). Twentyeight retinas but only twelve eyes: an anatomical analysis of the larval
27.
28.
visual system of the diving beetle Thermonectus marmoratus (Coleoptera: Dytiscidae). J. Comp. Neurol. 497, 166–181. Buschbeck, E.K., Sbita, S.J., and Morgan, R.C. (2007). Scanning behavior by larvae of the predacious diving beetle, Thermonectus marmoratus (Coleoptera: Dytiscidae) enlarges visual field prior to prey capture. J. Comp. Physiol. [A] 193, 973–982. Larson, D.J., Alarie, Y., and Roughley, R.E. (2000). Predaceous Diving Beetles (Coleoptera: Dytiscidae) of the Nearctic Region, with emphasis on the fauna of Canada and Alaska (Ottawa: National Research Council of Canada Research Press). Homann, H. (1924). Zum Problem der Ocellenfunktion bei den Insekten. Z. Vgl. Physiol. 1, 541–578. Galbraith, W. (1955). The optical measurement of depth. Q. J. Microsc. Sci. 96, 285–288. Campbell, M.C.W., and Hughes, A. (1981). An analytic, gradient index schematic lens and eye for the rat which predicts aberrations for finite pupils. Vision Res. 21, 1129–1148. Sivak, J.G. (1982). Optical properties of a cephalopod eye (the short finned squid, Illex illecebrosus). J. Comp. Physiol. 147, 323–327. Kro¨ger, R.H.H., Fritsches, K.A., and Warrant, E.J. (2009). Lens optical properties in the eyes of large marine predatory teleosts. J. Comp. Physiol. [A] 195, 175–182. Warrant, E.J., Kelber, A., Walle´n, R., and Wcislo, W.T. (2006). Ocellar optics in nocturnal and diurnal bees and wasps. Arthropod Struct. Dev. 35, 293–305. Schuppe, H., and Hengstenberg, R. (1993). Optical properties of the ocelli of Calliphora erythrocephala and their role in the dorsal light response. J. Comp. Physiol. [A] 173, 143–149. Kro¨ger, R.H.H., Campbell, M.C.W., Fernald, R.D., and Wagner, H.-J. (1999). Multifocal lenses compensate for chromatic defocus in vertebrate eyes. J. Comp. Physiol. [A] 184, 361–369. Malkki, P.E., and Kro¨ger, R.H.H. (2005). Visualization of chromatic correction of fish lenses by multiple focal lengths. Journal of Optics A: Pure Applied Optics. 7, 691–700. Malmstro¨m, T., and Kro¨ger, R.H.H. (2006). Pupil shapes and lens optics in the eyes of terrestrial vertebrates. J. Exp. Biol. 209, 18–25. Land, M.F. (1969). Structure of the retinae of the principal eyes of jumping spiders (Salticidae: dendryphantinae) in relation to visual optics. J. Exp. Biol. 51, 443–470. Blest, A.D., Hardie, R.C., McIntyre, P., and Williams, D.S. (1981). The spectral sensitivities of identified receptors and the function of retinal tiering in the principal eyes of a jumping spider. J. Comp. Physiol. 145, 227–239. Maksimovic, S., Cook, T.A., and Buschbeck, E.K. (2009). Spatial distribution of opsin-encoding mRNAs in the tiered larval retinas of the sunburst diving beetle Thermonectus marmoratus (Coleoptera: Dytiscidae). J. Exp. Biol. 212, 3781–3794. Clarkson, E., Levi-Setti, R., and Horva´th, G. (2006). The eyes of trilobites: The oldest preserved visual system. Arthropod Struct. Dev. 35, 247–259. Fordyce, D., and Cronin, T.W. (1993). Trilobite vision: A comparison of schizochroal and holochroal eyes with the compound eyes of modern arthropods. Paleobiology 19, 288–303. Towe, K.M. (1973). Trilobite eyes: calcified lenses in vivo. Science 179, 1007–1009. Horva´th, G. (1989). Geometric optics of trilobite eyes: a theoretical study of the shape of the aspherical interface in the cornea of schizochroal eyes of phacopid trilobites. Math. Biosci. 96, 79–94. Monte´s-Mico´, R., Espan˜a, E., Bueno, I., Charman, W.N., and Menezo, J.L. (2004). Visual performance with multifocal intraocular lenses: Mesopic contrast sensitivity under distance and near conditions. Ophthalmology 111, 85–96. Navarro, R., Ferro, M., Artal, P., and Miranda, I. (1993). Modulation transfer functions of eyes implanted with intraocular lenses. Appl. Opt. 32, 6359–6367. Artal, P., Marcos, S., Navarro, R., Miranda, I., and Ferro, M. (1995). Through focus image quality of eyes implanted with monofocal and multifocal intraocular lenses. Opt. Eng. 34, 772–779. O’Shea, M., and Adams, M.E. (1981). Pentapeptide (proctolin) associated with an identified neuron. Science 213, 567–569.