We describe an imaging scatterometer allowing hemispherical reflectance measurements as a function of the angle of incidence. The heart of the scatterometer is an ellipsoidal reflector, which compresses the hemispherical reflection into a cone-shaped beam that can be imaged by a normal optical system. The instrument’s performance is illustrated by measurements of the scattering profiles of the blue-iridescent dorsal wing scales of the nymphalid Morpho aega and the matte-green ventral wing scales of the lycaenid Callophrys rubi.
©2009 Optical Society of America
The interaction of light with material bodies is generally studied by measuring the reflection and transmission, or more generally their scattering properties. A research area with many unsolved light scattering questions, which has recently witnessed an explosive increase in attention, is that of the photonic crystals. Special attention in that area is taken up by the wing scales of butterflies [1–5]. However, the scale structures are often intricate, and the resulting optical properties accordingly are difficult to analyze.
Butterfly wing scales resemble flattened sacs, a few micrometers thick, and with length and width in the order of 200 and 75 ¼m, respectively [6,7]. The lower lamina of a butterfly wing scale is virtually always more or less smooth, but the upper lamina is commonly highly structured. It consists of parallel, longitudinal ridges, spaced apart by 1 to 2 μm, which are connected by crossribs, with separation 0.5 to 1 μm . The space between upper and lower lamina can be almost empty or it can be highly structured, depending on the butterfly species and the scale type . The scale material can be virtually transparent or it can contain strongly absorbing pigment. The structuring of the scale together with the physical properties of its material components determine how incident light is absorbed and scattered by the scale, and thus the scales determine the coloration of their owners [7,9]. For instance, the scales of pierid butterflies are packed with beads, 100–200 nm sized granules attached to the crossribs, which contain light-absorbing pigments [10,11]. Although the wings of pierids thus are said to have pigmentary colors, the observed light reflections are, of course, due to scattering by the strongly irregularly organized wing scales .
In many butterflies, the scales feature regular structures, resulting in so-called structural colors. The best studied example is the Morpho scale type, where the ridges form multilayers that act as highly effective blue reflectors . Vukusic et al.  studied Morpho scales by focusing monochromatic laser beams at a single, isolated scale. They measured the scattering profile by scanning a detector around the scale. Kinoshita et al. , in a similar approach, applied a broad-band, white light beam, and measured the angular-dependent reflectance with a scanning fiber optic. Additionally, they visualized the scattering pattern on a screen, which had a small hole through which the illuminating light beam passed before hitting the scale. It thus appeared that a normally irradiated Morpho scale creates an approximately line-shaped far-field scattering profile, in the plane perpendicular to the ridges, due to diffraction by the narrow ridges [15–17].
Measuring the scattered beam with a narrow-aperture, scanning detector is very laborious, and quantification of photographs of the scattered light distribution on a screen is a quite insensitive procedure. We therefore have developed as an alternative an imaging scatterometer, which projects the full hemispherical scattering on a digital camera. Here we describe the scatterometer and illustrate its workings on scales of two structurally colored butterflies, the nymphalid Morpho aega and the lycaenid Callophrys rubi.
2. Materials and methods
2.1. Butterfly scales
A small piece, about 2 mm × 2 mm, cut from the wings of a butterfly, was glued to the tip of a glass micropipette. Subsequently, the micropipette was mounted on a micromanipulator, which allowed precise adjustment of a scale in the first focal point of the ellipsoidal reflector of the scatterometer (Fig. 1).
2.2. Imaging scatterometry
The imaging scatterometer, diagrammatically shown in Fig. 1, was inspired by the setup of Kinoshita et al. , where a small diaphragm, illuminated by a xenon lamp, is projected at a butterfly scale via a small hole in a white screen, and the light scattered by the scale is secondarily scattered by the screen. We have replaced the white screen by an ellipsoidal reflector, which has a small, central hole. The study object, for instance a butterfly scale, isolated or attached to a wing fragment, is positioned in the first focal point, F1, of the reflector, M (Fig. 1). The primary beam is supplied by light source S1. It illuminates field diaphragm D1, which is focused by lenses L1 and L2 at the sample in F1. The aperture diaphragm D2 limits the beam to within the hole in the ellipsoid. The approximately axially back-scattered (reflected) light that returns throughout the hole in the reflector is then, via a half-mirror, focused by lenses L2 and L3 at camera C1, which is connected to a binocular viewer.
The secondary beam illuminates diaphragm D3, which is focused by lenses L4 and L5 on diaphragm D5. The aperture diaphragm D4 and the field diaphragm D5 are positioned in the front and back focal planes of lens L5, respectively. The planes of D5 and F2 are conjugated via beam splitter H. The secondary beam reaches the sample via beam splitter H and the ellipsoidal reflector. The angle of incidence of the secondary light beam at the sample is varied by laterally moving D4.
Light that is back-scattered by the sample in off-axis directions is reflected by the ellipsoid and then proceeds through a diaphragm in the plane of the second focal point F2. The sample’s far-field scattering pattern thus is imaged in the back-focal plane I of lens L6, and subsequently lens L7 focuses that plane at camera C2. A spatial filter in plane I, together with the diaphragm in the plane of F2, suppresses the light transmitted by the sample.
The micromanipulator carrying the micropipette with the butterfly wing piece (or with a single scale) allows rotation around its axis. Using the primary beam, this allows measurement of the scattering pattern as a function of the angle of incidence, however only for light incident in a plane perpendicular to the rotation axis. The dependence of the scattering pattern from any angle of incidence can be measured by using the secondary light beam and moving diaphragm D4 laterally, that is vertically and/or horizontally.
We note here that the transmittance characteristics of a scale can be studied by centering D4, on the axial position, so that the sample is axially illuminated, from the reverse side. The transmitted light is then reflected by the ellipsoidal mirror and, after focusing at F2, is then again imaged at camera C2.
The components of the imaging scatterometer as realized in our laboratory are as follows. Light sources S1,2: xenon lamps; lenses L1,3,4: Spindler & Hoyer lenses with focal distances -40, -100, 90 mm, respectively; L2: Zeiss Luminar 16 mm; L5: Mamiya-Sekor 55/1.8; L6: AF Nikor 50/1.8; L7: Wollensak 75/1.9. The beam splitter H is a pellicle. The ellipsoidal reflector, M, symmetric around the major axis, is an aluminum mirror (produced on order by TNO I&T, Delft, the Netherlands). The semi-major and semi-minor axes are a = 100 mm and b = 57.2 mm, respectively, so that, with c = √(a2-b2), the distance between the focal points F1F2 = 2c = 164 mm, and the eccentricity of the reflector ε = c/a = 0.82. The diameter of the reflector is 65.5 mm, so that the focal point is in the plane of the rim of the reflector. In other words, the rim corresponds to θ = 90°, that is, the reflector captures exactly a hemispherical spatial angle. The diameter of the central hole in the mirror is 3.2 mm, so that an angular aperture of 10.2° is lost from the scattering pattern. For collecting the images (by camera C2) we use an Olympus DP70 digital camera (4080×3072 pixels; for RGB color images), or a Photometrics Coolsnap ES monochrome digital camera (1392×1040 pixels). For observing the sample with camera C1, we use a Jenoptik ProgRes C10 or the Olympus DP70.
2.3. Imaging with an ellipsoidal mirror
Consider the ellipsoidal mirror, symmetric around the major axis, where a light ray leaves the first focal point, F1, with an angle θ with respect to the major axis (Fig. 1). After reflection it passes the second focal point, F2, with an angle α given by:
where ε is the eccentricity of the ellipsoid. For the extreme angle θ max = 90°,
With ε = 0.82 it follows that α max = 11.3°, which is well within the 15.5° aperture of lens L6.
If f 6 is the focal length of lens L6, then the distance of the light ray in the back-focal plane I of lens L6 equals r = αf 6 (with α= tana in radian, as α < α max = 0.197 is sufficiently small). The light ray with angle θ hence arrives at a distance p = mr = mαf 6 = Mα from the axis, with m the magnification of the imaging by lens L7, and M = mf 6. For a reflected beam with an extreme angle of θ max = 90°, the pixel distance is p max = Mα max, so that the relative pixel distance is:
2.4. Corrections of the scatterometer images
The scatterometer allows direct visualization of the Bidirectional Reflectance Distribution Function (BRDF; dimension: sr-1) for an incident light beam from any chosen direction (θ i, φ i); see Fig. 2(a). The BRDF is defined as the ratio between the reflected radiance Lr (θ r, φ r) (dimension: W m-2 sr-1) and the incident irradiance E i(θ i, φ i) (dimension: W m-2) :
Light reflected into direction (θ r, φ r) is projected at position (p,φ r) of camera C2 (Fig. 1), where p is the distance from the axial pixel (see Fig. 2(b)). The radial coordinate p is proportional to the angle α: p = Mα (section 2.3), but α is non-linearly related to the angle θ = θ r (Eq. 1a), and therefore the spatial light distribution captured by the camera is not a perfect polar diagram for (θ r,φ r). For obtaining such a diagram the images have to be corrected as follows.
In the spherical coordinate system with origin F1 the radiant intensity I r(θ r,φ r) (dimension: W sr-1) is scattered into the direction (θ r,φ r). I r(θ r,φ r)dω r, the light power scattered by the sample into the spatial angle dω r = sin θ r dθ r, is projected at an area dσ = pdpdφ r of the camera chip (Fig. 2(b), with s = p), and thus it equals the light power E cam(p,φ r)dσ, where E cam(p,φ r) is the irradiance received by the camera. In order to obtain the irradiance in the polar coordinate system with coordinates (θ r,φ r), E cam has to be corrected with the condition E cor θ r dθ r dφ r = E cam pdpdφ r, where E cor(θ r,φ r) is the corrected irradiance. Dropping the suffix r, we obtain (with p = Mα):
where α(θ) is given by Eq. 1a, and the factor K, which corrects the measured irradiance for the non-linear imaging, is
Finally, the radiance L r(θ r,φ r) = I r(θ r, φ r)/A, where A is the scattering (and illuminated) area of the sample. Hence, with E cor θ r dθ r dφ r = I r sin θ r dθ r dφ r, it follows that the radiance can be obtained from the polar diagram of the corrected irradiance E cor(θ r,φ r) via L r(θ r,φ r) = E cor(θ r, φ r)θ r/(Asinθ r) = E cor(θ r,φ r)/(Asinθ r).
2.5. Reflectance spectra
The reflectance spectrum of an object can be measured by taking images with the scatterometer for a series of quasi-monochromatic illuminations, using either a monochromator or interference filters. The latter method is used to determine the reflectance spectrum of scales of Callophrys rubi.
The first step in validating the scatterometer, its calibration, was performed with the primary light beam, reflected by a small mirror positioned in the plane of F1 (Fig. 1). The angular aperture of the primary light beam was 8.5°. The mirror was rotated around a horizontal axis in steps of 5°, resulting in steps of the angle of the reflected beam, θ, of 10°. The reflected light distribution was recorded, and the resulting images were superimposed (Fig. 3(a)). The pixel position at the center of each of the spots was determined, and subsequently their distance, p, to the axial pixel (the pixel corresponding to the system axis: p = 0) was calculated. The distances p were then normalized, yielding the relative pixel distances: p * =p/p max.
These relative pixel distances are plotted in Fig. 3(c) as a function of the angle of the reflected beam, 6. The continuous curve in Fig. 3(c) is the theoretical curve p * = α/α max obtained with Eq. 1a,b and ε = 0.82. The images of Fig. 3(a), obtained with the Olympus camera, yielded p max = 1145, so that M = p max/ α max = 5800 pixels rad-1 = 101 pixels degree-1.
The next calibration step concerned the secondary beam. The small mirror in the plane of F1 was therefore put in a stable, approximately vertical position, and the vertical position of diaphragm D4 was varied in steps of 0.5 mm. Figure 3(b) shows a superposition of every third of the obtained images. The pixel values of the spot centers were determined, and then their distance to the axial pixel (p = 0) was normalized to p max. The position of the diaphragm with respect to the axial position, d, reaches an extreme value, d max, when the angle of incidence θ i = θ max = 90°. The pixel distance of the spot center then is p max.
The plane of diaphragm D4 is via the beam splitter conjugated to plane I, which is imaged at camera C2. Formally, when the angle of the light beam with the secondary axis leaving lens L5 equals α, then d = αf 5 (f 5 is the focal length of lens L5), so that p * = p/p max = α/α max = d/d max = d *, where d *is the relative diaphragm displacement. Indeed, as shown by Fig. 3(d), the pixels receiving the reflections from the successive illuminations are linearly related to the displacement of the diaphragm. The angle of incidence of the secondary beam, θ, thus can be immediately obtained from the diaphragm displacement, d, via the inverse expression of Eq. 1a:
Figure 3(f) presents θ as a function of the relative diaphragm displacement d * = d/d max. In our setup d max = 11.0 mm, in accordance with d max = f 5·tan(α max), where f 5 = 55 mm is the focal length of lens L5.
3.2. Scattering by wing scales of the butterfly Morpho aega
The power of the imaging scatterometer was investigated on a small fragment of the dorsal wings of Morpho aega, a highly iridescent butterfly. The primary beam with aperture 8.5° was focused on one of the scales, resulting in a 40 μm diameter spot (Fig. 4(a)). The light scattered by the scale had a spatial distribution almost restricted to a plane oriented perpendicular to the longitudinal ridges of the scale (Fig. 4(b)). The lateral spread of the light scattering in that plane is wider for light of short wavelengths than for light of long wavelengths. The scattering pattern is somewhat patchy, which is due to the not fully regular organization of the ridges. Minor movements of the scale, so that the focusing spot occurred at slightly different locations, resulted in abrupt changes of the patches.
Widening the area of illumination resulted in a smoother pattern as is illustrated in Fig. 4(c), where the secondary beam was applied. Here the illumination spot was about 130 μm; the diameter of diaphragm D4 was 1.5 mm, meaning an aperture of about 16°, decreasing however with increasing angle of incidence (see Fig. 3(c)). Figure 4(c) presents superimposed scattering diagrams resulting from illuminations with the secondary beam diaphragm D4 at positions d = -6, -4, -2, 0, 2, 4, and 6 mm, respectively. With d max = 11 mm, it follows from Fig. 3(f) (or Eq. 2) that d = 2, 4, 6 mm corresponds to an angle of incidence of θ = 20.5°, 39.9°, and 57.1°, respectively.
3.3. Reflectance of Callophrys rubi scales
The imaging scatterometer allows straightforward measurement of reflectance spectra. As an example, Fig. 5 shows measurements of a scale on the ventral wing of the lycaenid butterfly Callophrys rubi. The scale was illuminated by the primary, narrow beam focused at a 40 μm diameter spot (Fig. 5(a)). The scale appeared to consist of yellow and bluish reflecting microdomains [4,19]. The incident light was scattered into a very wide angle (Fig. 5(b)). Applying monochromatic light and using the reflection from a mirror as reference, the reflectance was calculated by integrating the reflection from spatial areas bounded by cones with apertures 30°, 60°, and 90° (Fig. 5(c)). The resulting spectra are approximately proportional to each other, and thus the scale, although structurally colored, scatters light into a wide angle. (The contributions to the reflectance by the wing substrate and other scales on the wing are minor, as will be described elsewhere.)
The imaging scatterometer described here consists of standard optical elements, except for the ellipsoidal mirror, which had to be custom built. The heart of the design is the property of an ellipsoidal mirror that it compresses a hemispherical space into a small cone, which then allows projection by common lenses. The system has an essential, sensitive point in that the scattering sample has to be positioned quite accurately in the ellipsoid’s first focal point. In practice this is achieved by observing the sample with the binocular viewer to which camera C1 is connected, and furthermore by checking that all reflected light rays are passing a narrow diaphragm surrounding the second focal point, F2 (Fig. 1). The unique power of the instrument is that it visualizes the near-field properties of the sample (with camera C1), and simultaneously visualizes the far-field, hemispherical scattering properties of the study object (with camera C2).
After completion of our instrument we found, not fully to our surprise, that scatterometers with related designs were reported before. The most similar arrangement is that developed for studying aerosols by Chang and co-workers [20,21]. Their ellipsoidal mirror also has a central hole through which the (only) illuminating beam is applied. A single lens images the scattered light directly at a digital camera, and thus a spatial filter for blocking transmitted light cannot be employed. A slightly different scatterometer, also employing an ellipsoidal reflector, was developed by Rodríguez-Herrera et al.  for studying the scattering by rough surfaces. Their laser beam is reflected by a 45° beam splitter, similar to the secondary beam of Fig. 1. The scatterometer described in the present paper appears to combine a number of solutions of the other systems. It has substantial versatility for studying the optics of small scale objects.
Previous studies of the scattering by Morpho wing scales, illuminated more or less perpendicularly, have demonstrated a highly directional reflection in the plane of the scale ridges and a broad diffraction in the perpendicular plane. The more or less line-shaped far-field scattering pattern [14,15,17,23] has been interpreted with an optical model that treats the ridges as a pile of equidistant, reflecting plates . When the direction of the incident light beam deviates from the normal, the shape of the scattering pattern becomes slightly curved (Fig. 4(c); see also Fig. 8.22 of , and ). A quantitative interpretation of this phenomenon will be presented elsewhere (in preparation).
The scattering of the dorsal wing scales of many lycaenids is highly directional, resulting in a spatially limited scattering diagram . However, the scattering pattern of the ventral wing scales of the lycaenid Callophrys rubi covers almost the full hemisphere, causing the matte appearance of the lycaenid in the resting state, when the wings are closed. The green peaking reflectance spectrum (Fig. 5(c)), calculated by integration of the scattering pattern, nicely matches the reflectance spectrum of leaves, thus ensuring excellent camouflage in the natural habitat [4,19].
The reflectance spectrum can be alternatively -and more easily- obtained by using an integrating sphere, but the advantage of the scatterometer is that it allows the much more detailed measurement of the directional reflectance, or, the detailed bidirectional reflectance distribution function. The scatterometer measurements will be helpful to understand the light scattering characteristics of butterfly scales, and hence provide insight into butterfly coloration and its biological function. Of course, the scatterometer can also be used to visualize the scattering patterns of small objects other than butterfly scales. We have successfully explored bird feathers, beetle cuticle, and similar biological tissues with structural coloration.
We thank Drs M. A. Giraldo, P. Vukusic, and S. Yoshioka for critical reading of the manuscript. This research was supported by AFOSR/EOARD grant no. FA8655-08-1-3012.
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