A Multilayer Interference Reflector in the Eye of the Scallop, Pecten Maximus

Highly reflecting surfaces in which metals are not employed have recently been developed industrially. The principle employed is that of the quarter-wavelength film (Text-fig. 1), in which light reflected from the front surface of a transparent thin film is in phase with, and so interferes constructively with, light reflected from the back surface. Light reflected at a low to high refractive index interface (in this case the upper surface of the film) undergoes a phase change of $12$ wavelength. So, for constructive interference, light reflected from the lower interface must be retarded by $12$ wavelength. The optical thickness of the film (refractive index, n, × actual thickness, t) must therefore be $14$ wavelength for maximum constructive interference to occur when light is reflected normally.

The reflectivity of such a film, although approximately four times that of a single interface, is still much less than that of a metallic surface. The reflectivity, however, may be increased by laying down a number of such films, of alternately high and low refractive index (Text-fig. 2). In such a system light reflected at each interface interferes constructively with light reflected from all others, resulting in a very high reflectivity for light of the ‘ideal’ wavelength. If materials with well-spaced refractive indices are chosen, a reflectivity as high as that of a silver mirror (96·6%) can be achieved using as few as seven layers, and with more films surfaces reflecting more than 99 % of the incident light can be produced. The materials commonly used industrially in these ‘all dielectric’ reflectors are vapour-deposited ZnS (n = 2·4) and MgF₂(n = 1·36) (Vašíček,1960).

In a system such as that shown in Text-fig. 2 the reflected light is coloured because constructive interference only occurs for wavelengths close to the ideal wavelength. If the difference between the refractive indices n₁ and n₂ is increased, the ‘bandwidth’ of the reflected light increases, and the number of films required to achieve a given reflectivity decreases, hence for most purposes it is desirable for n₁ and n₂ to be as far apart as possible. Reflectors of this type are used as colour-selective mirrors, heat-reflecting filters, coatings for increasing the reflectivity of the ends of lasers, and in other situations where both high reflectivity and wavelength specificity are required. Such systems are virtually non-absorbing, transmitting all light that is not reflected. Hencein the visible spectrum the light which is transmitted is complementary in colour to the reflected light.

The colours of a number of biological surfaces have been attributed to thin film interference. Mason (1927) produced evidence showing that thin film interference was responsible for the iridescent colours of the wings and bodies of many insects, and Anderson & Richards (1942), in an early electron microscope study, showed that the blue colour of the wing scales of the butterfly Morpho cypris was due to reflexion from an array of appropriately spaced lamellae (see Fox & Vevers (i960) for further references). Only recently, however, has much attention been paid to interference phenomena associated with highly reflecting surfaces. Denton & Nicol (1965) have shown that both the colours and high reflectivity of fish scales are due to multiple thin film reflexion with constructive interference—the thin films in this case being stacks of guanine crystals in the inner surfaces of the scales.

The argentea of the eye of Pecten, with which the present paper is concerned, is a particularly interesting reflector. Unlike the situation in most other eyes, the optical system is in this case based not upon a lens, but upon a reflecting system (Text-fig. 3) (Land, 1965, 1966). The argentea, which is spherical, forms the visual image, much as in a reflecting telescope.

The argentea is optically of very good quality, the law of reflexion being obeyed with little or no scattering of the incident light. A simple examination of the eye shows that the reflectivity of the argentea is comparable with that of a metallic reflector, and also that the reflected light is not spectrally uniform, the apparent colour being a rather unsaturated blue-green. Patten (1886) noticed that the argentea was a multilayered structure, consisting of several layers of thin, square crystals. These facts strongly suggest that, optically, the argentea may be functioning as a multilayer quarter-wavelength reflector.

In the present study this suggestion has been followed up by four methods: light microscopy, electron microscopy, interference microscopy and spectral analysis of the reflected light.

Pecten maximus were obtained from Plymouth and kept in a recirculating sea-water aquarium.

The microscope produces two images, one in the main beam and one in the reference beam. The most consistent results were obtained by matching the brightness of the image of a crystal in the main beam against the background, and then moving the compensator until the image of a crystal in the reference beam matched the background; the amount by which the compensator has to be moved in performing this operation gives the sum of the half path differences of the two crystals, i.e. the mean path difference for the pair. After each series of ten measurements in water, the water was carefully removed and replaced by sucrose, and a further set of measurements was made on the same pair of crystals.

The measurements were made using approximately monochromatic light of wavelength 525 mμ. The microscope has an objective N.A. of 0·9, and was used with an illuminating cone of N.A.0·7.

An eye was removed from the mantle and mounted pupil upwards in a sea-water chamber (Text-fig. 8a). It was illuminated with parallel light from a microscope lamp, whose horizontal beam was directed into the eye by a coverslip inclined at 32·5° to the horizontal (not 45°, to avoid reflections from another coverslip which covered the eye in its chamber). Light reflected from the back of the eye entered a microscope (16 mm. objective, N.A. 0·12) which was focused to give an image of the eye on a screen above the eyepiece. In the centre of this screen was a CdS photocell, which covered the image of the light source produced by reflexion in the eye. The light which entered the microscope was not reflected exactly normally from the argentea, but at an angle of incidence and reflexion of about 9° ±8° (16° cone). The wavelength of the incident light could be varied using a set of seven Balzers interference filters with maximum transmissions between 400 and 710 mμ, and half widths of 40−50 mμ. The resistance of the photocell was measured at each wavelength using a microammeter and a 67 V. battery. The eye was then removed, and replaced by a white opal Perspex plate, and the photocell was calibrated at each wavelength using a set of Wratten neutral density filters. The apparent optical density of the argentea relative to the white plate could thus be found for each wavelength. CdS cells, although sensitive and simple to use, have the disadvantage of taking up to 2 min. before giving a steady reading at low light intensities, and each experiment thus took about 30 min. to complete. The eyes remained in perfect condition over this period, and there was almost no difference between readings made at the beginning and end of each experiment. Each point in Text-fig. 7 is the mean of two measurements, one made with wavelength increasing, and the other decreasing.

In the intact eye the argentea may be examined through the pupil. It is superficially perfectly smooth, the only visible defects being occasional hair-like cracks. By reflected light it is blue-green (λ_max usually about 530 m μ, although the exact maximum varies from eye to eye between 500 and 550 m μ). By transmitted light the reflector appears red, but this colour is mainly due to underlying pigment cells.

The true ‘transmitted light’ colour can be observed by carefully scraping the argentea from an eye on to a slide and removing the pigment cells. This process invariably breaks up most of the structure, but some intact parts remain. These parts retain their high reflectivity and blue-green colour by reflexion, and by transmitted light they continue to appear red (the complementary colour to blue-green). If the organization of the intact piece of tissue is destroyed by pressing on the coverslip, both the colours and the high reflectivity are lost. The colours are thus structural rather than pigmentary.

Examination of the less damaged parts under high-power shows the argentea to be a rather regular mosaic of square crystals. Each crystal has sides 1·1 − 1·3 μ long, and is too thin for the thickness to be measured with the light microscope. Judging from the number of crystals liberated when an intact piece of tissue was broken up, many more crystals were present than could be accounted for by a single layer, indicating a multilayered structure. The surprising ease with which the crystals disperse indicates that they are not embedded in a rigid matrix, but are held loosely in the cytoplasm of the cells containing them. Individual crystals are colourless by transmitted light.

It is a pleasure to thank V. Barber for taking electron micrographs of the argentea. A full account of the ultrastructure of the eye of Pecten, including the argentea, is given in Barber, Evans & Land (1966).

Eyes were removed from the animal, and opened with a small cut to allow penetration of the fixative. They were fixed in phosphate-buffered osmic acid (Millonig, 1961), Araldite embedded after dehydration, sectioned and examined under a Siemens electron microscope. Pl. 1 shows a typical section of the argenteal region, cut at right angles to the surface. In all sections the crystals themselves have dissolved out during preparation of the tissue, leaving electron-transparent spaces. Where these spaces have a consistent parallel-sided appearance, they are considered to be accurate replicas of the crystals. Parts of the sections were clearly worse than others, the spaces being distorted like expanded aluminium mesh.

The argentea contains from 30 − 40 layers of crystals. Each crystal is rather less than 100 m μ thick, and is separated from the next below it by a layer of cytoplasm of slightly greater thickness—about 100 m μ. There are thus five to six ‘repeat units’ per micron, and the thickness of the whole structure is about 6 μ. The gaps between the crystals laterally are very narrow, and the lateral dimension of the crystals, about 1.1 μ, is in close agreement with that obtained from light microscopy. A reconstruction of part of the argentea is shown in Text-fig. 4.

If the argentea is to function in the manner suggested, both the crystals and the spaces between them should have optical thicknesses of $14λmax$⁠., i.e ⁠. The cytoplasmic interstices are unlikely to have a refractive index much different from that of water (1-33), so their optical thickness (n.t. = 133 m μ) is clearly compatible with the theory. So also is the slightly smaller thickness of the crystals, as these might be expected to have a higher refractive index. However, to obtain more exact information about the crystals, an interference microscope was used.

Crystals from a dissected argentea were dispersed in water, and allowed to settle on a microscope slide. The crystals were then examined under an interference microscope : their appearance is shown in Pl. 2. The microscope was used to measure the path difference (p.d.) between individual crystals and two suspending media, water and 50% sucrose. From these p.d.’s the thicknesses (t) and refractive indices (n) of the crystals were calculated. The measurements were made on pairs of crystals (see Methods) and the p.d.’s obtained are the mean p.d.’s of each pair. The results, for five pairs of crystals, are given in Table 1.

The mean value of n.t. from Table 1 is 145 m μ, which is very close to the expected value, ⁠, and the crystals may therefore be regarded as quarterwavelength films.

There is a negative correlation between the values of n and t in Table 1, which is difficult to interpret; it might be caused by individual crystals showing different degrees of swelling and shrinkage, in which case n and t would not be expected to vary independently. If, however, the assumption is made that all crystals are formed of the same material, and have the same refractive index, a better estimate of the variation of thickness between crystals can be made. Taking n as 1·80, the thicknesses can be recalculated from the p.d.’s. The standard deviation of the thicknesses, recalculated from the path differences in water, is only 4·6 m μ, i.e. about 6 % of the mean value (81 m μ). It seems likely that in the animal, crystal thickness is controlled with considerable precision.

λ = 4n₁t₁ = 4n₂t₂. They can be used to calculate the reflectivity of the argentea by taking n₂ as the refractive index of cytoplasm (about 1·34) and as that of the crystals (i-8o), and k as twice the number of crystal layers. Text-fig. 5 shows the results of the calculation.

It can be seen from Text-fig. 5 that with only ten layers of crystals the argentea would be a 99 % efficient reflector for light of the appropriate wavelength. With thirty layers its reflectivity would be undetectably different from 100%. Unfortunately, it has not been possible to measure the absolute reflectivity, as the argentea cannot be removed from the eye intact and laid flat, and in situ methods of measurement involve too many approximations for them to be of any use. There is, however, no reason to suppose that the reflectivity departs far from the theoretical value; it has been shown in the context of interference-filter manufacturer that inaccuracies of up to 10 % in film thickness, such as are likely to occur in the argentea, have a negligible effect on overall reflectivity (Heavens, 1955).

Light reflected from single or multiple quarter-wavelength films is coloured. With single films the colour is very unsaturated; a quarter-wavelength film of refractive index 1·8, in water, reflects between 6 and 9% of the incident light over the whole visible spectrum. However, in a multilayer structure, the variation of reflectivity with wavelength becomes more pronounced with increasing number of layers, the bandwidth becomes narrower and more sharply defined, and the colour correspondingly more intense (Text-fig. 6 a).

The reflectivity of a thin-film system at any wavelength may be calculated using the method given by Vašíček (1960, pp. 236 −7), and other methods are given by Heavens (960). These calculations, however, are laborious to perform by hand. In connexion with the present study, Prof. A. F. Huxley has derived a much more convenient method (Huxley, 1966) which is given below.

It can be seen from Text-fig. 6 that apart from the rapid fluctuations the shape of the reflectivity curve changes very little when k>20, i.e. when the argentea is 10 or more crystal layers thick. If the quarter-wavelength film hypothesis is correct, the actual argentea ought to be a nearly perfect reflector for wavelengths up to 50 mμ on either side of λ_max, and the reflectivity should fall to near zero at either end of the visible spectrum.

Although it is difficult to measure the absolute reflectivity of the argentea, it is comparatively easy to compare the reflectivities at different wavelengths; this can be done with the eye intact. The method of illumination is shown in Text-fig. 8 a. A CdS photocell was used to measure the reflected light, and the wavelength was varied with a set of interference filters (see Methods section). In Text-fig. 7, the ordinate is the relative reflectivity, i.e. reflected light intensity at wavelength A divided by reflected intensity at λ_max. Curves are given for two eyes; three other eyes gave curves very similar to the right-hand curve. The eye which gave the left-hand curve was visibly bluer than the others.

The measured curves are similar in shape to the theoretical curves in Text-fig. 6,b. Like the theoretical curves they show that most of the reflected light lies within a well-defined band of wavelengths, and that outside this band the reflectivity falls more steeply towards the blue end of the spectrum than towards the red. Both curves were almost symmetrical when plotted against ϕ or 1/λ The bandwidth of the measured curves appears to be rather greater than that of the theoretical curves, even after making an allowance for the distorting effect of the broad band filters used in making the measurements, and this larger bandwidth may well be a consequence of ‘inaccuracies’ in crystal thickness or spacing in the argentea itself. It is of interest that interference reflectors with a high (95 %) reflectivity over the whole visible spectrum have been manufactured by deliberately varying the thicknesses of the constituent films (Heavens, 1960). The rapid fluctuations predicted in Text-fig. 6b were sought the light reaching the receptor cells will have a λ_max. appreciably shorter than the figure of 530 mμ given here for angles of incidence close to zero. By estimating the relative amounts of light reflected at each angle of incidence one can form an estimate of λ_max for the cone of light reaching the receptors; λ_max thus obtained is approximately 490 mμ. One would expect the argentea to be ‘timed‖ to reflect best in the part of the spectrum containing the spectral sensitivity curve of the receptors. Cronly-Dillon (1966) found that the animal’s shadow reflex, which is mediated by the receptor cells in question (Land, 1966), has a peak sensitivity in the range 475−480 mμ, which is agreeably close to the maximum reflectivity.

If an eye is placed in distilled water, the colour of the light reflected by the argentea changes from blue-green through yellow to orange, over a period of about 2 min. This process is reversible, the colour returning slowly to blue-green when the eye is replaced in sea water. In hypertonic sucrose the colour becomes bluer. These changes are presumably caused by swelling and shrinking of the spaces between the argenteal crystals, brought about by osmotic entry of water. They give further support to the assumption that the crystals are not held rigidly in position, but are free to move relative to each other. This raises the problem of how the crystals are normally maintained at the ‘correct’ distance apart; at present, no explanation of this spacing mechanism can be offered.

Purines have been identified as the main constituents of reflecting structures in many marine animals. The reflecting material in the scales of fish is guanine; hypoxanthine is also present in some scales. Guanine forms the reflecting tapetum in both teleosts and in crocodiles (see Fox & Vevers, 960). Kleinholz (1959) showed that five different purines and pterins are present in the reflecting layers of the lobster eye, and also that the reflecting material in the eye of Limulus is guanine. Uric acid has been found in the lobster eye, and in iridocytes of the sea anemone Metridium senile.

The argenteal crystals from Pecten have properties compatible with those of purines. They are insoluble in water, but soluble in both N/10 HC1 and N/10 NaOH. They are slightly soluble in alcohol, but not in either acetone or ether. Isolated argenteae gave murexide reactions, although the quantity of material was too small for the colour of the residue to be used to identify the particular purine. The crystals are not uric acid, as they failed to give a reaction with Folin’s reagent.

When crystals from the argentea were dissolved in N/10 NCI, which was then allowed to evaporate, needle-like crystals were formed which were indistinguishable from those of British Drug Houses’ guanine recrystallized in the same way.

The argenteae dissected from two eyes were dissolved in N/10 HC1, and chromatographed using water-saturated n-butanol as the solvent. The standards used were guanine and uric acid (5 μg. each). The chromatograms were developed by the descending method, and when dry were examined under ultraviolet light. The argenteae gave a blue-fluorescent spot, of the same colour, and with the same R_f value as the guanine standard. A green fluorescent spot which had remained on the origin was probably caused by pigment from the cells behind the argentea, as this spot occupied the same area as the red mark on the paper. The argenteal material gave only these two spots, and the crystals are therefore taken to be made of guanine.

It is interesting that the refractive index found by interference microscopy for the crystals of the argentea (1·8o) is close to that found by Denton & Nicol (1965) for the refractive index of guanine crystals from the scales of the herring—1·8 to 1·9. The method they used was an immersion method, crystals being examined in liquids of different refractive index until a match was obtained, the crystals becoming invisible. Similarly, Schmidt (1949), using polarization microscopy and comparison Equids, found that guanine plates from the copepod Sapphirina were strongly biréfringent, having an ordinary refractive index of 1·79 (for rays normal to the plate surface—the index relevant here) and an extraordinary index of 1·55 (for rays parallel to the plate surface).

A ‘good’ material for use in a reflecting system would be one which is insoluble in water, which can be crystallized as parallel-sided plates, and which has a high refractive index in this form. The popularity of purines as reflecting substances may well result from their ability to fulfil these conditions particularly well.

There are two ways, besides thin-film interference, in which highly reflecting non-metallic surfaces may be produced. First, the amount of light reflected at a single interface between two media may be increased by increasing the refractive index difference between the media. Thus when light is incident from air on to a single surface of refractive index (n) 1·5, the reflectivity is 4%; this increases to 11 % when n = 2, 25 % when n = 3, and 36% when n = 4. Transparent materials are known with refractive indices as high as 3 (e.g. Sb₂S₃, n = 3·0), but it is unlikely that the refractive indices of biological materials much exceed 2, which would limit singlesurface reflectivity to 11%.

In Table 2 the reflectivities of a thin-film system (with constructive interference), and a thick-film system using the same materials, are compared.

It can be seen from this table that any thin-film system (k > 1) requires fewer interfaces than a thick-film system for the same reflectivity to be achieved. Nevertheless, thick-film systems using biological materials would be effective reflectors, and they probably do occur in situations where all that is required is a white reflecting patch of indifferent optical quality. A serious drawback of thick-film systems is that, when light is not incident normally, light reflected from every interface follows a separate path: thus one sees multiple reflexions from a pile of microscope slides. This means that as an optical mirror, a thick-film system would be useless. In a thin-film system like the Pecten argentea, where nearly all the light is reflected in the first 2/4 of the structure, this scattering effect would be negligible.

The spectral dependence of the light reflected from thin film systems may limit their applicability as mirrors. However, in the case of Pecten, which lives at depths down to loom., where what light there is lies in the blue-green part of the spectrum, this is clearly no disadvantage.

Reflectors with structures very similar to that of Pecten have been found in the eyes of the cockle, Cardium edule (V. Barber, unpublished) and the median eyes of the crustacean Macrocyclops albidus (Fahrenbach, 1964). In both cases the dimensions of the crystals are compatible with their being quarter-wavelength films. Some vertebrate tapeta are often of good optical quality. The tapetum of the bush-baby Galago crassi-caudatus, for example, is a brilliant reflector, and it, too, has a similar structure to the Pecten argentea (Dartnall et al. 1965). The reflecting crystals in this case are made not of guanine but of riboflavin (Pirie, 1959). The tapetum of the cat contains a system of orientated rod-like structures, arranged in ‘lattice planes’ 450 mμ apart, and Pedler (1963) has given grounds for believing that the green colour and high reflectivity are due to constructive interference.

1. The physical mechanism responsible for the high reflectivity of the argentea of the eye of Pecten maximus has been investigated by light microscopy, electron microscopy, interference microscopy and spectral analysis of the reflected light.

2. The argentea consists of 30−40 layers of high refractive index material (guanine crystals) separated by layers of low refractive index material (cytoplasm). Both high and low refractive index layers have optical thicknesses of approximately one-quarter of the wavelength of the light that the argentea reflects best (blue-green, λ = 530 mμ).

3. The reflecting properties of such a system have been investigated theoretically, and the results compared with the measured reflectivity of the argentea at different wavelengths.

4. The refractive index of the guanine crystals is 1·8o.

My special thanks are due to Prof. A. F. Huxley for detailed advice on optical problems, and in particular for deriving several of the formulae given here. I should also like to thank Dr B. E. C. Banks for chromatographing argenteal material, my supervisor, Prof. J. A. B. Gray, for advice and discussion, and the Medical Research Council for a research training scholarship.

Electron micrograph of a complete vertical section through the argentea. The ‘holes’ are the sites of the guanine crystals. A pigment cell can be seen below the argentea. Stained lead citrate.

Interference micrograph of isolated crystals. Crystals from the image formed by the main beam appear light, those from the comparison beam appear dark. Part of a pigment cell can be seen top left.