1. Introduction

The iridescent effect of colors, known as “orient”, caused by interference and/or diffraction is a well-established phenomenon in nature. This has been the subject of various specific studies in seashells, plants, insects, birds, and clouds [1–9]. Iridescent colors in seashells and pearls are usually attributed to diffraction effect caused by the evenly grooved surface microstructure similar to that of a diffraction grating [10,11]. Moreover there is evidence that interference of light in multiple-layered microstructure just below the surface causes iridescent colors [11,12]. Studies on pearls have shown that iridescence is caused by a combination of both diffraction and interference [11]. Diffraction and interference studies on the iridescent colors of abalone shell are still limited. Recently, an x-ray diffraction experiment was carried out to study the microstructure of the mollusk Haliotis rufescens [13].

In this work, we studied the outside of the polished shell of a mollusk Haliotis glabra from the Philippines, commonly known as abalone, which showed very strong iridescent colors (Fig.1.) This iridescent effect is similar to that observed for pearls and mother-of-pearl materials. The colors of the shell under white light vary with changes in the angle of observation. The causes of the strong iridescent colors were examined by studying the microstructure of the surface and cross-section of the shell using a scanning electron microscope (SEM). A laser diffraction experiment shows that diffraction plays a major role in generating iridescent colors. The uniform stacks of nacreous layers just below the surface of the abalone shell suggest that the iridescent colors can be also attributed to multilayer interference. The SEM observations of the crystalline structure and absorption peaks using infrared spectroscopy confirm the aragonite structure of the nacreous layers of the shell.

 

Fig. 1. The polished shell of the mollusk Haliotis glabra has beautiful iridescent colors.

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2. Materials and experimental methods

A He-Ne laser (λ = 632.8 nm) was used to investigate the diffraction effect of the shell. The laser beam was directly incident on the shell and the reflected and diffracted beams were captured on a screen. Measurements of fringe separation on the diffraction pattern were done on a white screen, 19 – 40 cm away, with a scale of ± 1 mm.

Scanning electron microscope (SEM) observations were carried out to study the microstructure of the shell sample. Before every experiment, the surface of the shell was cleaned using ethanol solution in order to remove any contaminant present due to handling. Samples of about 5 mm × 5 mm area were cut from the shell. Observations of up to 1600× were made on the surface and the cross-section of the samples. All the samples were coated with a thin layer of gold before the SEM experiments. SEM work was carried out using the JEOL JSM - 5600LV SEM/EDX microscope with a working voltage of 15 or 20 kV.

To confirm the crystalline structure of the nacreous layers of the shell, Fourier transform infrared (FTIR) spectroscopy was used. The Perkin Elmer (Model Spectrum One) Fourier transform infrared spectrometer was used to record the spectra of the shell in the wavenumber range of 450 to 4000 cm^-1. The accuracy of the absorption peaks was ± 1 cm^-1. Since pure shell material is basically opaque to infrared radiation, a diluted sample in an infrared-transparent matrix of potassium bromide (KBr) is needed for FTIR work. The shell material was ground to a fine powder, mixed with KBr, and pressed with a 2-tonne force to make flat circular pellets. These pellets are translucent and allow infrared light to pass through it and be absorbed. The infrared transmission spectra were recorded to give absorption peaks for identification.

3. Results and discussion

3.1 Microstructure of the shell

The surfaces and the cross-sections at various locations of the shell were studied using SEM. Figure 2 shows the typical groove structures on the surface, one with fine closely-spaced grooves of about 2–8 μm width and the other with a wider spacing of about 30–50 μm. The direction of the wider groove microstructures appears to be perpendicular to that of the fine grooves. It can be seen from Fig. 2 that the fine grooves are uneven and wavy, with a tile-like structures all over the shell surface. These microstructures are not as regular as those observed for pearls [14] and seashells [15].

The cross-section of the abalone shell shows a layered microstructure consisting of stacks of platelets, as shown in Fig. 3. These uniform nacreous layers are actually those of aragonite platelets, separated by a thin layer of conchiolin, similar to those observed for pearls [16]. The nacreous structure of crystalline layers in organic matrix has been described in detail by Barnes [17]. The thickness of each nacreous composite layer is about 0.5 μm. Similar microstructure of aragonite platelets was observed for the mollusk Haliotis laevigata [18] using SEM and atomic force microscopy (AFM) and for red abalone Haliotis rufescens [13] using synchrotron x-ray microbeam diffraction. Another point of observation is that the shell thickness is in the range of 1–3 mm and appears to be translucent to visible light. The presence of such regular stacks of thin layers below the surface of the shell strongly suggests that interference of light plays an important role in generating iridescent colors.

 

Fig. 2. The typical groove structures on the shell surface, one with fine closely-spaced grooves of about 2–8 μm width and the other with a wider spacing of about 30–50 μm.

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Fig. 3. The cross-section of the abalone shell showing a layered microstructure composed of aragonite platelets, separated by a thin layer of conchiolin. The thickness of each nacreous layer is about 0.5 μm.

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3.2 Diffraction pattern of shell using laser

A diffraction experiment on the surface of the shell using a He-Ne laser (λ = 632.8 nm) was carried out. The laser beam was incident directly onto the shell at an incident angle of about 40° giving a spot size of about 3 mm on the shell. The reflected beam shows a typical diffraction pattern on a screen with several orders. The typical diffraction patterns as shown in Fig. 4 can be explained by the reflection grating structure of the shell surface. The bright fringes are diffused and not sharp since the groove microstructures are observed to be uneven. Figure 4(a) shows 4 bright spots with about equal spacing y. The groove spacing d₁ of the grating can be calculated using the relation d₁ = λD/y where D is the distance between the point of reflection on the shell surface and the screen. The diffracted angle is assumed to be small. Diffraction experiments for the measurements of d₁ were repeated at 5 different areas of the shell. The results were summarized in Table 1. The mean value of d₁ is found to be 4.3±0.9 μm, in good agreement with the mean value of about 4.6 μm determined from SEM observations. The value of d₁ from SEM measurements corresponds to a structure with a groove density of 220 grooves/mm. Strong iridescent colors can be observed at this density. At similar groove density values, strong iridescent colors on the shell of Pinctada Margaritifera were also observed by Liu et al [10].

 

Fig. 4. Typical diffraction patterns produced by the shell showing: (a) bright spots with higher orders overlapped (b) a set of fine fringes at the zero order position. The average spacing of these fine fringes in the central diffraction band is about an order of magnitude less than the spacing between the main diffraction bands.

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Table 1. Diffraction measurements of narrow grooves on shell surface using He-Ne laser

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Figure 4(b) shows a set of diffraction fringes with small separations, in addition to the larger diffraction spots. This pattern is about perpendicular to the lager diffraction spots. This can be explained by the observation that the wide groove microstructures are in the direction which is perpendicular to that of the fine groove microstructures. The groove spacing d₂ of this grating structure can be calculated using the relation d₂ = λD/x which x is the fringe separation as measured from the diffraction pattern. The values of d₂ are tabulated in Table 2. A mean value of d₂ = 52 ± 10 μm is obtained. This agrees fairly well to the mean value of 43 μm obtained from SEM measurements. The groove density from SEM measurements is found to be about 23 grooves/mm, which gives a weaker iridescent effect according to Liu et al [10].

Table 2. Diffraction measurements of large grooves on shell surface using He-Ne laser

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3.3 Numerical modeling of the effect of the nacreous layers

To investigate how the nacreous layers might play a part in producing the iridescence effects, the spectral reflectivity was determined by the using the matrix method as outlined by Hecht [19] for alternating aragonite-conchiolin layers. The matrix method basically makes use of boundary conditions to obtain a characteristic matrix which relates the field at two adjacent boundaries. The coefficient of reflection is then solved from the elements of the derived characteristic matrix. The numerical calculations were based on the angle of incidence and the alternating pattern of the layers with their respective thicknesses and refractive indices (n) and with the electric field vector taken as perpendicular to the plane of incidence. The aragonite platelet layer was taken to be (500 ± 50) nm with n₁ = 1.6 and the conchiolin layer to be (25 ± 5) nm with n₂ = 1.3. The values of the thicknesses are in agreement with TEM calculations made on a similar abalone shell, Haliotis iris [20], from New Zealand.

The results of the numerical modeling for 1024 layers of composite nacreous layers at various angles of incidence are shown in Fig. 5. It can be seen that there is a clear peak at about 550 nm (green) at normal incidence. This peak is found at roughly the same position for computations involving 64 or more nacreous layers. This corresponds to the greenish color found dominantly on the surface of the shell. At 60°, the peak shifts to 700 nm (red) on a broad background of evenly distributed colors. This results in a pinkish tint seen on some parts of the shell. At 80°, there is a mixture of three peaks of violet, green and red, which combines to give an overall white color. This explains the subdued nature of the greenish-blue and pinkish tints on the surface of the shell as the white color reduces the contrast of these colors. The results give some evidence that the multiple nacreous layers do play a role in producing the iridescent colors on the shell.

 

Fig. 5. Spectral reflectivity of 1024 composite nacreous layers at various angles of incidence.

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It must be noted that the above treatment does not take into account the absorption and scattering of light as it penetrates through the layers. It is also assumed that the layers are planar and with constant thickness. A total of 1024 layers are used in the modeling as this corresponds to the order of magnitude of the total thickness of the shell. The variations in color observed are more complicated than what is actually explained due to the curved surface of the shell which affects the actual incident angle.

3.4 Fourier transform infrared (FTIR) spectroscopy

A typical FTIR spectrum of the nacreous layers of the shell is shown in Fig. 6. Typical infrared absorption peaks for calcium carbonate, CaCO₃ structure as given in [21] are shown in the figure. The absorption peaks at 700, 713, 862 and 1083 cm^-1 show that CaCO₃ in the nacreous layers of the shell is basically aragonite crystalline structure, similar to that of a turtle eggshell in a study by Baird and Soloman [22]. The double peaks for the C-O in-plane bend are at 700 and 713 cm^-1 and the peak for the C-O out-of-plane bend is at 862 cm^-1, typical of aragonite structure. The infrared spectroscopy results agree and confirm the present SEM observations that the crystalline structure of the calcium carbonate layers is aragonite. The regular stacks of thin aragonite layers in the shell indicate that interference of light in these layers adds to the effect of iridescent colors in the shell. Such iridescent effect has been specifically observed for the shell of Helcion pruinosus [12] and for pearl [16].

 

Fig. 6. The FTIR spectrum of the nacreous layer of shell with double peaks for the C-O in-plane bends (700 and 713 cm^-1), giving evidence for the presence of aragonite.

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4. Conclusions

The polished shell of the abalone Haliotis glabra shows very strong iridescent colors of mostly pink and blue-green. The causes of the strong iridescent colors were studied by examining the microstructure of the surface and cross-section of the nacreous layers of the shell using SEM. A laser diffraction experiment shows that diffraction plays a major role in generating iridescent colors. Through numerical modeling, we have demonstrated that the presence of stacks of uniform nacreous layers just below the surface of the shell leads to interference effects that contribute to the iridescent colors. The SEM observations of the crystalline structure and infrared absorption peaks using infrared spectroscopy confirm the aragonite structure of the nacreous layers of the shell.