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Vision Research 44 (2004) 2129–2134 www.elsevier.com/locate/visres

Compensation for longitudinal chromatic aberration in the of the firefly , Watasenia scintillans

Ronald H.H. Kroger€ *, Anna Gislen Department of Cell and Organism Biology, Lund Vision Group, Zoology Building, Lund University, Helgonava€gen 3, 22362 Lund, Sweden Received 31 October 2003; received in revised form 26 March 2004

Abstract The of fishes and have come forth by convergent . In a variety of capable of color vision, longitudinal chromatic aberration (LCA) of the optical system is corrected for by the exactly tuned longitudinal spherical aberration (LSA) of the crystalline . The LSA leads to multiple focal lengths, such that several wavelengths can be focused on the . We investigated whether that is also the case in the firefly squid (Watasenia scintillans), a species that is likely to have color vision. It was found that the lens of W. scintillans is virtually free of LSA and uncorrected for LCA. However, the eye does not suffer from LCA because of a banked retina. Photoreceptors sensitive to short and long wavelengths are located at appropriate distances from the lens, such that they receive well-focused images. Such a design is an excellent solution for the firefly squid because a large area of the retina is monochromatically organized and it allows for double use of the surface area in the dichromatically organized part of the retina. However, it is not a universal solution since compensation for LCA by a banked retina requires that eye size and/or spectral separation between photopigments is small. Ó 2004 Elsevier Ltd. All rights reserved.

Keywords: Crystalline lens; Spherical aberration; Chromatic aberration; Banked retina; Evolution

1. Introduction The typical fish lens is spherical (Fig. 1B). If a lens is spherical or nearly so, asymmetrical aberrations such as The eyes of fishes and cephalopods (, cuttle- coma and astigmatism are absent or negligible. In ani- fishes, and ) are a stunning example of con- mals using vision under low-light conditions, such as vergent evolution. Both phyla have eyes of the camera most aquatic species including fishes and cephalopods, type, in contrast to the complex, facetted type of eye the pupil is large relative to the focal length of the eye, present in arthropods (Land & Nilsson, 2002). At first i.e. the f -number is small. In consequence, light-gath- sight, one can easily mistake a cephalopod eye for a fish ering ability is high, depth of focus is short, and the disc eye, and vice versa (Fig. 1). In most aquatic eyes of the of diffraction (Airy’s disc) is small (Born & Wolf, 1999; camera type, the entire refractive power of the optical Land & Nilsson, 2002). Longitudinal spherical aberra- system resides in the crystalline lens. The usually tion (LSA) is the dominating factor influencing mono- has negligible refractive power in water because it is thin chromatic image quality. LSA is compensated for by and on both sides surrounded by media of similar and internal gradients of refractive index in most high refractive index, i.e. water and aqueous humor lenses (Land & Nilsson, 2002) and fish lenses are the (Matthiessen, 1886). Some cephalopods––including most extensively studied biological gradient index lenses Watasenia scintillans, the firefly squid of the Japanese (e.g. Jagger, 1992; Kroger,€ Campbell, Munger, & Fer- Sea––do not even have a cornea (Fig. 1A). Iris and lens nald, 1994; Matthiessen, 1893; Maxwell, 1854). are in direct contact with the surrounding sea water. Color vision complicates eye design considerably, since it is far from trivial to create well-focused color images. In simple optical systems, different wavelengths * Corresponding author. Tel.: +46-46-2220596; fax: +46-46- are focused at different distances from the lens (longi- 2224425. tudinal chromatic aberration, LCA) (Born & Wolf, E-mail address: [email protected] (R.H.H. Kroger).€ 1999). If an animal is capable of color vision and depth

0042-6989/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.visres.2004.04.004 2130 R.H.H. Kro€ger, A. Gislen / Vision Research 44 (2004) 2129–2134

Fig. 1. Eyes of the firefly squid (A) and the South American cichlid fish Aequidens pulcher (B). Note that both eyes are similar in general design. The main difference is the lack of a cornea in the eye of the firefly squid. For the optics of the eye in water, however, the cornea is of little relevance. Both pictures were taken from the faces of sectioning blocks containing freeze-embedded eyes. The black and grey lines in (A) illustrate measured intraocular dimensions (see Section 2). The retina is banked in the ventral (left) part of the eye of the firefly squid. The original color of the proximal layer (light-gray) is yellow, while the distal layer (dark-gray to almost black) is orange-red. Scale bars are 1 mm. of focus is shorter than LCA, chromatic defocus is a spherical, about as powerful as fish lenses, and also have problem. In a number of vertebrates active under low- gradients of refractive index that compensate for light conditions, natural evolution has found an elegant spherical aberration (Sivak, 1991). Just as in fishes, solution to that problem: multifocal lenses create well- chromatic defocus should be a problem for cephalopods focused images at the wavelengths of maximum absor- capable of color vision, such as the firefly squid. We bance of the cone photoreceptor cells in the retinae wondered whether the of verte- (Kroger,€ Campbell, Fernald, & Wagner, 1999). Con- brate and cephalopod eyes also includes similar solu- centric zones of the lens have different focal lengths and tions for the problem of chromatic defocus. each focal length creates well-focused retinal images at a different wavelength (see Fig. 5 for illustration). At least in Astatotilapia (formerly Haplochromis) burtoni,an African cichlid fish, those wavelengths are identical or 2. Material and methods close to the wavelengths of maximum cone absorbances. It is the exact tuning of the gradient of refractive index, The experiments were performed during the spawning and thus spherical aberration, that bestows the lenses of season of the firefly squid (April 2001), when those fishes and a variety of other vertebrates with several aggregate in large numbers in the Toyama Bay, focal lengths (Kroger€ et al., 1999). Animals having Sea of Japan (Michinomae et al., 1994). Living animals multifocal lenses usually have immobile irises or slit were brought to the laboratory (Toyama University, pupils, such that the entire diameter of the lens, and thus Department of Biology) and kept in darkness in cold sea all zones of different focal lengths, can be used irre- water for a maximum of 8 h. spective of the level of ambient light (Kroger€ et al., 1999). Most cephalopods have only one visual pigment and 2.1. Measurements of ocular dimensions are color blind (Messenger, 1981; Williamson, 1995). W. scintillans is an exception and has three visual pigments. Whole animals were rapidly frozen by immersion in Two of the pigments have wavelengths of maximum liquid nitrogen. The were removed and the absorption (k max) of 470 and 500 nm, respectively. head separated from the body. The specimen was freeze- Those pigments are present in the ventral retina in dif- mounted and cryo-sectioned in a dorso-ventral plane ferent types of . The third pigment perpendicular to the longitudinal axis of the animal. absorbs maximally at 484 nm and is the only visual From several sections before and after the center of each pigment present in all other parts of the retina (Seidou eye, photographs of the face of the sectioning block were et al., 1990). Because of the dichromatic organization of taken with a digital camera (SONY DSC-F707). The the ventral retina, it is likely that the animals can dis- sectioning plane included the ventral part of the retina criminate between wavelengths of light in the dorsal where the 470 and 500 nm (k max) visual pigments are visual field (Michinomae, Masuda, Seidou, & Kito, located. A metric scale was placed in the sectioning 1994). plane and photographed for calibration. The photo- Cephalopod and fish lenses develop in different ways graph showing an axial section of the eye, indicated by and differ in cellular structure (Sivak, West, & Camp- the largest apparent diameter of the lens, was selected bell, 1994; West, Sivak, & Moccia, 1994). Optically, for measurements. Eye dimensions were determined however, they are similar. Cephalopod lenses are almost using ScionImage 4.02b software (Scion Corp.). R.H.H. Kro€ger, A. Gislen / Vision Research 44 (2004) 2129–2134 2131

2.2. Measurements of spherical aberration BCD as a function of BEP was symmetrical around it. The BCDs from both halves of the lens were combined Squids were sacrificed by rapid destruction of the and smoothed with a windowed average over 5 data brain. One eye at a time was enucleated and the lens points. As a measure of LSA, BCDs were extracted by isolated. Pigmented tissue adhering to the lens was interpolation between smoothed data points in steps of carefully removed. For measurements we used a por- 1% Re from 0% to 100% Re (101 data points) (Kroger€ table laser scanning device similar to the apparatus used et al., 1994). The LSAs of all lenses were averaged and by Kroger€ et al. (1994). The lens was immersed in sea the 95% confidence interval was calculated (Sokal & water and placed on a holder made of black modeling Rohlf, 1995). clay. Orientation was such that the optical axis was parallel to the beam of a red (655 nm) diode laser. The 2.3. Model calculations laser was mounted on a linear translator and its beam focused on the squid lens with an f ¼ 50 mm lens We determined the focusing properties of the lens of (Melles Griot) to reduce beam diameter. The laser beam the firefly squid for monochromatic light by the same was scanned through a meridional plane of the squid method as used on fish lenses (Kroger€ et al., 1999). lens. A small amount of milk had been added to the sea Using the mean LSA, rays of light were traced to the water used as immersion medium such that some light retinal plane. Each exit ray was weighted by the lateral was scattered from the laser beam. Beam paths were distance to the optical axis of the corresponding en- recorded with a digital video camera (SONY DCR-TRV trance ray to determine the exit ray’s contribution to 620E PAL) mounted above the immersion bath. retinal irradiance. The contributions of rays reaching the From each scan, 125 frames were captured using retina were summed in a central circular area around the Adobe Premiere 6.0 software. Equatorial and axial radii optical axis of about the diameter of the disc of dif- of the lens were determined from a composite image fraction (0.0015 Re) and concentric annuli of the same obtained by averaging all captured frames (see Fig. 2). width. The pattern of retinal irradiance was determined Beam paths before and after deflection by the squid lens for a section including the optical axis. The distance were semi-automatically determined and analyzed with between lens and retina was varied from 2.26 to 2.44 Re, a program written in IDL 5.0 (Research Systems, Inc.). which included the position of best focus, in steps of The program determined position and direction of each 0.002 Re. beam and calculated the intercepts between entrance Measuring LCA in W. scintillans lenses was prohib- and exit beams as well as between exit beams and the ited by the amount and quality of the equipment optical axis. Back center distance (BCD; the axial dis- (Kroger€ & Campbell, 1996) that would have to be tance between the center of the lens and the intercept of transported to Toyama to do such measurements. We the exit beam with the optical axis) was recorded as a therefore estimated LCA from measurements performed function of beam entrance position (BEP; the lateral on other species. Dispersion––and thus LCA––is distance between an entrance beam and the optical axis). dependent on the molecular structure of any material. The data were normalized to the equatorial radius of the Both fish and squid lenses consist of cells containing lens (Re) to remove size differences between lenses. The more or less concentrated solutions of proteins, such position of the optical axis was manually adjusted until that we assumed that the lens of the firefly squid has the same LCA (relative to focal length) as the lens of the fish A. burtoni, from which exact data on LCA are available (Kroger€ & Campbell, 1996). We also made a calculation using data on LCA in Eledone cirrhosa, an (Sroczynski & Muntz, 1985). Most of the energy of light reaching the lens at en- trance positions higher than 95% R is lost by reflection (Sroczynski, 1976). The analysis was therefore limited to light entering the lens at entrance positions up to 95% Re.

3. Results

Fig. 2. Composite image showing the lens of a firefly squid, illumi- 3.1. Intraocular dimensions nated in a meridional plane by 125 thin laser beams. There is little structure in the cone of exit beams and the light is concentrated in a narrow focal area. This indicates that the lens is of high optical quality The lenses used for optical investigations were slightly and well-corrected for spherical aberration. elliptical. The ratio between axial and equatorial radius 2132 R.H.H. Kro€ger, A. Gislen / Vision Research 44 (2004) 2129–2134 was 0.95 ± 0.01 (standard deviation, n ¼ 21). Average equatorial radius was 2.07 ± 0.07 mm (standard devia- tion). Most frozen eyes (n ¼ 10) were severely deformed, probably because squid eyes are very pliable and loss of intraocular pressure upon death of the animal. Mea- surements were therefore taken from only one eye that appeared to be particularly well-preserved (Fig. 1A). The equatorial diameter of the lens (black line in Fig. 1A) was 4.2 mm. The distance between the center of the lens and the surface of the ventral retina (grey line in Fig. 1A), was 4.8 mm or 2.3 Re.

3.2. Optics

LSAs were determined for 21 lenses from as many animals. The lenses of firefly squids are well-corrected for LSA (Fig. 2). The slope of the mean LSA did not significantly deviate from zero over most of the aper- ture. Deviations from zero occurred only for beam en- Fig. 4. Result of a model calculation showing that the lens of W. trance positions exceeding 95% Re (Fig. 3). Variance in scintillans focuses light of 655 nm onto a narrow area at a distance of data points close to optical axis is large because exit 2.35 Re from the lens center. The sharpness of the peak indicates good beams in this region are almost parallel to the optical correction of longitudinal spherical aberration. axis. Consequently, even small errors in exit beam po- sition and angle lead to large variance in back center lated from the focal length at 655 nm and the LCA as a distance. However, this uncertainty is irrelevant for function of wavelength in A. burtoni (Kroger€ et al., estimates of image quality because (i) the central region 1999), were 2.289 Re (484 nm), 2.278 Re (470 nm), and of the lens contributes little to the image and (ii) rays of 2.300 Re (500 nm). The difference between the most light almost parallel and close to the optical axis gen- relevant wavelengths (470 and 500 nm) is 0.022 Re.Ina erate little blur. lens of an equatorial radius of 2.1 mm this corresponds As expected from the flat LSA curve, results from to a difference in focal length of 460 lm. If one uses the model calculations using the mean LSA show a single, estimate of LCA in L. cirrhosa (Sroczynski & Muntz, well-defined focal area (Fig. 4). Focal length measured 1985), the difference in focal length is 380 lm. at 655 nm was 2.35 Re. Focal lengths at the wavelengths of maximum absorbance of the photopigments, calcu- 4. Discussion 2.6 The lenses of firefly squids are well-corrected for ) e 2.5 LSA. High optical quality of the lens, several visual pigments, and a number of bioluminescent organs 2.4 (Tsuji, 1983) indicate that visual information is of high importance for W. scintillans. The lenses of other 2.3 cephalopod species studied so far are less well-corrected for LSA (Sivak, 1991; Sivak et al., 1994). The fish lenses 2.2 for which LSA is known in sufficient detail also have

Back center distance (R distance center Back considerable amounts of LSA (Kroger€ et al., 1994; Sroczynski, 1975a, 1975b, 1976, 1977, 1978, 1979). This 2.1 0 0.2 0.4 0.6 0.8 1 LSA corrects for LCA in A. burtoni, and most probably € Beam entrance position (Re) also in many other fish species (Kroger et al., 1999). In the eye of the firefly squid, however, such a mechanism Fig. 3. Mean longitudinal spherical aberration (thick solid line; is clearly absent. n ¼ 21) and the 95% confidence interval (thin solid lines). The dashed The convergent evolution of fish and cephalopod eyes line has a slope of zero, which would indicate perfect correction of longitudinal spherical aberration. The measured data do not signifi- has not brought forth similar mechanisms to correct for cantly deviate from this line for beam entrance positions between 0% LCA. The question, how chromatic defocus is handled and 95% Re (equatorial radius of the lens). in the eye of W. scintillans, seems to be unanswered. R.H.H. Kro€ger, A. Gislen / Vision Research 44 (2004) 2129–2134 2133

However, we actually can provide a likely answer. ments are co-localized. For the remaining, monochro- Firstly, spectral separation between the k max of the matically organized part of the retina a monofocal lens pigments in the ventral retina is small (30 nm) such that is the best solution. A disadvantage of using a banked there is only a moderate difference in focal length be- retina is that it limits eye size. In large eyes, the differ- tween the most relevant wavelengths. Secondly, photo- ences in radial photoreceptor positions would also have receptor rhabdoms containing different visual pigments to be large, such that the transmission of nervous signals occur in banks in the retina of W. scintillans. In fixated as graded potentials within the retina would be time- retinae, the proximal tips of rhabdoms containing the consuming and unreliable. Spectral separation of visual 470 nm pigment are about 400 lm closer to the lens than pigments is limited for the same reason, because wide the tips of rhabdoms containing the 500 nm pigment spectral separation of pigments would also require wide (Michinomae et al., 1994). If one takes into account radial separation of photoreceptor types. shrinkage of the tissue during fixation, this agrees well The banked retina of W. scintillans may have evolved with the difference in focal length of 460 lm between 470 for more reasons than just compensation for chromatic and 500 nm calculated from LCA in A. burtoni. It is also defocus. If located in separate banks, different spectral close to the estimate of 380 lm from data on LCA in E. types of photoreceptor do not have to be interspersed, cirrhosa. We therefore conclude that the problem of such that the animal can make double use of its retinal chromatic defocus is solved in the eye of W. scintillans surface area. Such a design is advantageous for maxi- by a banked retina, with photosensitive elements con- mization of sensitivity and resolution, both being of taining spectrally different visual pigments at appropri- importance for an animal relying on faint and small ate radial distances from the monofocal lens (Fig. 5). bioluminescent sources. Furthermore, more proximal Compensation for chromatic defocus by a banked layers of photoreceptor act as spectral filters for more retina is an excellent solution for W. scintillans because distal layers. This can be used to sharpen spectral sen- such compensation is only necessary locally, i.e. in the sitivities of photoreceptors (Cronin & Marshall, 1989). ventral retina where two spectrally different visual pig- Such advantages may be the reason for the convergent evolution of banked retinae in the firefly squid and tiers of cones in some fish species. The differences in radial position between spectrally different types of cone are small in fishes, usually in the range of 10 lm, and therefore insufficient for compensation of chromatic defocus in all but very small eyes (lens radius a few hundreds of microns) (Kroger,€ 2000). There is no need for wide radial separation of photoreceptor types, however, for the eye to take advantage of multiple uses of retinal surface area and spectral filtering.

5. Conclusions

The lenses of W. scintillans are well-corrected for longitudinal spherical aberration. Compensation for longitudinal chromatic aberration is achieved by having the rhabdoms of photoreceptors containing spectrally different visual pigments at appropriate distances from the lens in a banked retina. This design has further advantages, such as allowing for double use of retinal surface area and sharpening of the spectral responses of photoreceptors.

Acknowledgements Fig. 5. Schematic illustration of the compensation for longitudinal chromatic aberration in fish and squid eyes. Fishes have multifocal We are deeply indebted to Dr. Kinya Narita, Toyama lenses that focus different wavelengths of light on the retina and all University, Japan, for assistance in obtaining firefly cones are located in about the same plane. The firefly squid has a monofocal lens and photoreceptors of different spectral sensitivities are squids and the use of laboratory facilities. We thank Dr. located in different planes in a banked retina. There may be more than Almut Kelber for useful suggestions and Carina Ras- two zones of different focal lengths in fish lenses. mussen for skillful technical assistance. 2134 R.H.H. Kro€ger, A. Gislen / Vision Research 44 (2004) 2129–2134

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