Abstract

The permanent effects of heat on the structure and colour of the iridescent Chrysochroa wallacei beetle were investigated by heating its wing cases (elytra) to temperatures up to 500°C. Scanning electron microscopy (SEM) was used to detect changes in the structure of the elytra as a result of heat treatment. Spectral data indicates that heat treatment causes an exponential decrease in the wavelengths of colour peaks of C. wallacei elytra, but also a change in relative intensity of colour peaks. The changes in colour are thought to be due to protein denaturation and a resultant change in structure.
Keywords: iridescence; multilayer reflector; heat; beetles; colour

Introduction

Colour is how certain sections of the electromagnetic spectrum are perceived, and the waves called visible light have a wavelength between roughly 400 and 700 nanometres. Usually, a surface appears to have a colour due to pigments in the material, which are molecules that absorb certain wavelengths of light and reflect all others. Chlorophyll for example is the molecule in plants that absorbs all wavelengths of visible light except the green wavelengths, which are reflected and observed by us.
Iridescence, however, is not due to pigmentation but structure. Iridescence is the property of surfaces that change colour when observed from different angles, meaning that iridescent surfaces reflect different wavelengths of light at different angles. Iridescence occurs when a layer of oil on water is viewed from different angles, and iridescence also occurs in animals such as the Chrysochroa wallacei beetle (Figure 1). There are plenty of theoretical evolutionary advantages of iridescence for animals, such as recognising members of the same species or a potential mate. Maybe even to scare predators, or to lure prey.^{^[1]} In addition, there are manmade applications of artificially produced iridescent structures, such as the theoretical concept of surgical threads that change colour when experiencing varying degrees of tension.
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Figure 1: The Chrysochroa wallacei beetle has iridescent multilayer reflectors, giving it green-blue colours depending on the angle of view. Notice the two large wing-like elytra on the back of the beetle (Photograph by Duncan Armour).
The commonest iridescent structure in Coleoptera (beetles) is the multilayer reflector,^{^[2]} which consists of two alternating layers (Figure 2), and the C. wallacei has multilayer reflectors. These two layers vary in chemical composition, and therefore have different refractive indices. When light travels through a multilayer reflector, some light is reflected and some light is refracted at each layer boundary.

Figure 2: A simplified multilayer reflector. The repeating layers have refractive indices n₁ and n₂, and thicknesses d₁ and d₂.
As shown in Figure 2, rays a and b originated from the same ray, but they have travelled different distances. Ray a reflected as soon as the original ray reached the multilayer, whilst ray b refracted and then reflected before it left the multilayer. Although these rays were in phase (i.e. in sync) with each other before the original ray reached the multilayer, they may be out of phase once they have both left the multilayer due to the difference in distance that they have travelled. This phase difference will result in light interference, as shown in Figure 3.

Figure 3: Constructive and destructive light interference is determined by the phase difference between two waves, i.e. how much in line they are.
When a multilayer reflector appears to have a colour, it is because certain wavelengths of visible light interfere constructively (which are seen), and other wavelengths of light interfere destructively (which are not seen). Usually, interference causes either an increase or decrease in intensity of a wavelength, because complete constructive or destructive interference is unlikely since the peaks and troughs are not likely to exactly line up. This is the reason why a multilayer reflects not only one wavelength of light, but a spectrum of wavelengths with peaks in intensity. If the relative intensity of each wavelength of light reflected by a multilayer reflector of a blue beetle were measured, a graph similar to Figure 4 would be produced.

Figure 4: The spectrum of light reflected off a mainly blue multilayer reflector. The wavelength of the most intense peak of a spectrum is called wavelength_max (or λ_max), which in this case is about 500 nm, but there are other smaller peaks too. The corresponding spectrum of colour is in the background.
Various factors will determine how wavelengths of light interfere, such as: refractive indices of the layers, thicknesses of the layers, and the angle of incidence of light. All three variables determine the distances that rays of light travel in the multilayer, which affect the phase differences of the rays, and therefore the interference of the rays, and thereby the colour. The angle of incidence also affects the ratio of light reflected and refracted at each layer boundary. Due to this change in light interference as the angle of incidence changes, the multilayer reflector will reflect different wavelengths of light at different angles, which is iridescence. As the angle of incidence increases, the wavelength at which there is a peak in constructive interference (this wavelength is called wavelength_max or λ_max) decreases. Therefore, there is a blueshift in wavelength as the angle of incidence relative to the multilayer reflector increases.
Here are spectra of light reflected off the multilayer reflector of a C. wallacei at different angles:

Figure 5: Spectra of light collected from the C. wallacei multilayer reflector from 10°-20°. The most intense spectrum is collected at the smallest angle of incidence (10°), and as the angle of incidence increases, the intensity of the spectrum decreases. This just means that the multilayer looks darker from greater angles because less light is being reflected.
Although this is not clear in Figure 5, the wavelength peak (λ_max) is changing as the angle of incidence is increasing. Therefore there is a change in colour at different angles, hence iridescence. This relationship is clearer when plotting λ_max versus angle of incidence (Figure 6).

Figure 6: The wavelength peak of a C. wallacei at 10°-20°. This graph shows a change in colour at different angles and therefore iridescence. The gradient of the line of best fit is spectral richness.
Figure 6 displays an apparent linear decrease in the wavelength of the main colour of the C. wallacei elytron as the angle of incidence increases, and this blueshift confirms that the beetle is iridescent. This relationship is not actually linear but normally distributed, though this is not noticeable within such a narrow range of angles.^{^[3]} As the relationship is so close to linear within this range (usually R²>0.90), a line of best fit can be produced using linear regression. The gradient of this line (with units nm/°) will be an estimate of how much the colour changes as the angle of view changes, and is therefore a measure of iridescence. This measure is called the spectral richness.^{^[4]}
For interference with visible light to occur, the layers of a multilayer reflector need to have the thickness of roughly a quarter of the wavelength of light,^[2] otherwise interference will occur for non-visible electromagnetic radiation. The number of layers in the multilayer also has an effect on colour, as it determines the intensity of interference. Multilayer reflectors with more layers will have less broad colour peaks, giving them purer colours. In beetles, the multilayer reflector may be at different depths of the insect exoskeleton,^[2] with brighter iridescent beetles usually having the multilayer reflector on the outside of their exoskeleton. Different sections of a beetle may have different colours due to variations in the multilayer reflector.
The bright green Chrysochroa wallacei beetle (Figure 1), native to Malaysia, is the species that was studied in this investigation. The beetle has multilayer reflectors covering its whole body, including its elytra (wing cases). The two alternating layers of the C. wallacei’s multilayer reflectors are thought to be air and chitin.^[4] Chitin, which plays important structural roles in insects, is a strong polymer similar in structure to cellulose, because its monomers are derivatives of glucose. The multilayer reflector of the C. wallacei is thought to have approximately 20 alternating layers of air and chitin, and is located on the outside of its body, which is why this beetle looks so metallic.^[4]
Although the short term effects of heat treatment on the colours of iridescent beetles have been studied,^{^[5]} the permanent effects of heat on the colour, and iridescence, have not been studied. Given that there are possible applications for thermosensitive surfaces, and for scientific interest, the long term effects of heat on the multilayer reflector of the C. wallacei beetle were investigated.

Materials and Methods

Because the elytra of beetles are the easiest part with which to experiment, only the elytra of the C. wallacei were used in this investigation, and colour was recorded using the set up shown in Figure 7. Elytra of C. wallacei specimens were removed and cut in half, with their spectra recorded at different angles. To collect that spectral data, a light source (Ocean Optics HL-2000) was shone on a piece of elytron and the reflected light was recorded using a spectrophotometer (Ocean Optics USB-650). A spectrophotometer is a device that goes through different wavelengths of light and measures the relative intensity of each wavelength, and the spectrophotometer in this investigation had a range of 350-1000nm. This spectrophotometer was connected to a computer and produced graphs such as in Figure 4 and Figure 5 using Spectrasuite software. A goniometer, a device which measures angles accurately, was used to measure the angle between the light source and the spectrophotometer, hereby allowing the angle of incidence to be measured. The angles at which spectra were collected were from 10° to 20°, in increments of half a degree.

Spectral data was collected from each piece of elytron before and after heat treatment. Each piece of elytron was heated only once (Figure 8).

Figure 7: The set-up of the equipment used to collect spectral data of the elytra of the C. wallacei. The spectrophotometer measured spectra of colour and the goniometer measured the angle between the light source and the spectrophotometer.

Using a glass kiln (Kilncare Hobbyfuser 3), elytron pieces were heated at a rate of 1000°C/hour, held at the desired temperature for 30 minutes, then left to cool to room temperature within the kiln. The elytron pieces were then removed and left for at least two hours before the collection of spectral data in order to minimise the recording of possible short term effects of heat treatment.

Figure 8: The elytra of three C. wallacei were removed and cut in half, so that each beetle provided four samples to heat. The temperatures to which each elytron piece was heated are displayed above. Each elytron piece was heated only once.
Using an SEM (Hitachi, Tabletop Microscope TM3030) from St Paul’s School in London, changes in structure of elytron pieces due to heat were attempted to be detected. Four elytron pieces were heated to 300°C, and four pieces were untreated. All elytron pieces were freeze-fractured with liquid nitrogen in order to leave the multilayer reflector intact, then gold coated by sputter deposition (Emitech K575X) in order to improve the quality of the electron micrographs.

Results

SEM Imaging:
Below are several of the SEM images, and more are available on the Beetles website (www.beetlesproject.weebly.com/images).
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Figure 9: Cross section of heat treated elytron (multilayer reflector is highlighted with arrow).
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Figure 10: Cross section of a normal C. wallacei elytron.

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Figure 11: Close up of multilayer reflector of normal elytron.
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Figure 12: Cross section of heat treated elytron, with the right side showing the multilayer reflector.
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Figure 13: Surface of normal elytron, including a blocked pore on the top left. Notice how the surface consists of hexagons.
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Figure 14: Surface of an elytron heated to 300°C.
Heat Treatment Data:
The highest temperature to which elytron pieces were heated and remained viable for analysis was 400°C; pieces heated to 500°C were reduced to dust. As the temperature of heat treatment increased, the elytra increased in curvature and brittleness. Due to those limitations, only elytron pieces heated to eight different temperatures up to 325°C had spectral data collected (Figure 15).
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Figure 15: Heat treated elytron pieces (left to right: 100°C, 200°C, 230°C, 260°C, 275°C, 285°C, 300°C, and 325°C).
Below are the spectra of normal and some of the heat treated pieces of elytra. In each graph, the most intense spectrum is at 10° angle of incidence, with each successive spectrum having a half a degree increase in the angle of incidence until 20°.

Figure 16: Colour of a normal C. wallacei.

Figure 17: Colour of normal C. wallacei heated to 100°C.

Figure 18: Colour of normal C. wallacei heated to 230°C.

Figure 19: Colour of normal C. wallacei heated to 275°C.

Figure 20: Colour of normal C. wallacei heated to 285°C.

Figure 21: Colour of normal C. wallacei heated to 300°C.

Figure 22: Colour of normal C. wallacei heated to 325°C.

Figure 23: The movement of the colour peaks of the C. wallacei after heat treatment in terms of light intensity. Also notice how all the peaks are decreasing in wavelength (moving to the left) after heat treatment.

Figure 24: The change in wavelength at which the original colour peak occurs after heat treatment.

Figure 25: The spectral richness (measure of iridescence) before and after heat treatment for the elytron pieces.

Discussion and Conclusions

As shown in Figure 10, normal elytron pieces were generally not freeze-fractured in a way that would leave the cross-section of the elytra intact, whilst heat treated elytra usually had clean cuts (Figure 9, Figure 12). This may be down to heat treated elytra being more brittle due to the denaturation of structural proteins. Therefore, changes in elytral structure due to heat were difficult to establish. A possible solution is to submerge the elytra in liquid nitrogen for longer before cleaving them.
There appeared to be a decrease in the thickness of the multilayer reflectors after heat treatment, though more data would need to be collected to confirm this. Multilayer reflectors are shown in Figure 11 and Figure 12, though the layers are so thin that they are not distinguishable.
Figure 13 and Figure 14 suggest that there is no significant change in the surface morphology of elytra after heat treatment, meaning that changes in structure due to heat are probably internal.
As shown in Figure 16 to Figure 22, the original colour peak (at about 550 nm for a normal elytron) decreases in relative intensity after heat treatment, whilst other peaks, with longer wavelengths, are increasing in relative intensity (Figure 23). After heat treatment at 285°C, the original colour peak is no longer the most intense, and the original peak is no longer present after heat treatment at 325°C. The latter may be a result of the chitinous layers in the multilayer reflector fusing together, as the melting point of chitin is considered to be approximately 300°C.^{^[6]}
The wavelengths at which colour peaks occur decrease as the temperature of heat treatment increases. This relationship is exemplified for the original colour peak in Figure 24, which shows the decrease in wavelength of the original peak versus the temperature of heat treatment, and this relationship appears to be exponential.
In summary, the heat treatment of the elytra of the C. wallacei simultaneously causes a redshift and a blueshift in colour, though the blueshift is more prominent (Figure 15). There is a redshift because colour peaks with a greater wavelengths increase in relative intensity compared to the original colour peak, but there is a blueshift because the wavelengths at which colour peaks occur decrease as the temperature of heat treatment increases.
Spectral richness, a measure of iridescence, is negative for a normal multilayer reflector because λ_max decreases as the angle of incidence increases. There is, however, generally a decrease in the magnitude of the spectral richness of elytra after heat treatment (Figure 25), meaning that the spectral richness is becoming less negative. This could be because the multilayer reflectors in the C. wallacei have evolved to be the optimally tuned, and any changes in structure, which could be caused by heat treatment, will have a detrimental impact on the spectral properties such as spectral richness. The data point at 100°C could be an anomaly or an indicator of an actual increase in magnitude of spectral richness, though more data would need to be collected to confirm this. After heat treatment at 325°C, the spectral richness has become positive, meaning that there is a redshift as opposed to a blueshift as the angle of view increases. Again, this may be because of the layers of chitin fusing together and completely distorting the structure.
One explanation for the blueshift of colour peaks after heat treatment is that the thickness of the air layers in the multilayer reflectors of the elytra could be decreasing. After heat treatment, structural proteins in the elytra could denature, which explains the observed increase in curvature and brittleness of the elytra after heat treatment. That increase in curvature could be putting pressure on the multilayer reflector on the surface of the elytra, and as half of the layers in the multilayer are air, these layers could decrease in thickness due to the pressure. That decrease in thickness of some of the layers in the multilayer will affect the interference of different wavelengths of light and therefore the colour of the structure. The wavelength of the most intense colour reflected off a multilayer reflector (at 0° angle of incidence) is given by the equation
where a and b are the alternating layers in the reflector, n is the refractive index, and d is the layer thickness.^{^[7]} According to this equation, a decrease in thickness in one of the types of layers, which for the heat treated C. wallacei elytra could be the air layers, will result in a decrease in the wavelength of the colour peak (i.e. there is a blueshift). As the blueshift appears to be exponential (Figure 24), the denaturation of structural proteins in elytra could be exponentially related to temperature.
That theory does not account for the changes in relative intensity of colour peaks after heat treatment, and the Beetles Project is currently mathematically modelling spectra of light reflected off a multilayer in order to try to explain all the changes in colour after heat treatment.
Although the data in this investigation does allude to certain relationships, more data would need to be collected for greater certainty. In addition, the age and gender of the beetles used in this investigation were not known, which could have affected results.
This investigation has shown that multilayer reflectors can change colour due to external variables, in this case heat, and there are therefore potential applications for this structure. For example, it could be artificially produced and used as an alternative to the conventional thermometer, with colour being the indicator. Industrially, heat could be used to determine the colour of surfaces that are covered with multilayer reflectors.
For a more in-depth write-up and more data of this investigation, please visit www.beetlesproject.weebly.com/papers

Acknowledgements

Dr Barbara Kirby, previously Simon Langton Grammar School for Boys, Canterbury, currently Department of Physiology, Anatomy and Genetics, University of Oxford; extensive support and guidance.
Mr McMachan, Design Department, Simon Langton Grammar School for Boys; heat treatment.
St Paul’s School, Barnes, London; use of SEM.
Dr James Perkins, previously St Paul’s School, currently Queen Elizabeth’s Grammar School, Faversham; sputter deposition and SEM guidance.
Dr David Pickup, School of Physical Sciences, University of Kent; freeze-fracturing of elytron pieces.

References

Stéphanie M. Doucet and Melissa G. Meadows, “Iridescence: a functional perspective”, J. R. Soc. Interface, 6(2), (April 2009), https://doi.org/10.1098/rsif.2008.0395.focus.
Ainsley E. Seago, Parrish Brady, Jean-Pol Vigneron and Tom D. Schultz, “Gold bugs and beyond: a review of iridescence and structural colour mechanisms in beetles (Coleoptera)”, The Royal Society, 6(2) (2009), https://doi.org/10.1098/rsif.2008.0354.focus.
Doekele G. Stavenga, Bodo D. Wilts, Hein L. Leertouwer and Takihiko Hariyama, “Polarized iridescence of the multilayered elytra of the Japanese jewel beetle, Chrysochroa fulgidissima”, The Philosophical Transactions of The Royal Society B, 366(1565) (2011): 709-723, https://doi.org/10.1098/rstb.2010.0197.
Olivier Deparis, Marie Rassart, Cédric Vanderbem, Victoria Welch, Jean-Pol Vigneron and Stéphanie Lucas, “Structurally tuned iridescent surfaces inspired by nature”, New Journal of Physics, Volume 10 (2008), https://doi.org/10.1088/1367-2630/10/1/013032.
Eiki Adachi, “Unexpected Variability of Millennium Green: Structural Color of Japanese Jewel Beetle Resulted From Thermosensitive Porous Organic Multilayer”, Journal of Morphology, Volume 268 (2007): 826-829, https://doi.org/10.1002/jmor.10557.
Chemical Book, “1398-61-4(Chitin) Product Description”. [Online], available at: http://www.chemicalbook.com/ChemicalProductProperty_US_CB9853096.aspx [Accessed 9 July 2017].
Michael F. Land, “The physics and biology of animal reflectors”, Progress in Biophysics and Molecular Biology, Volume 24 (1972): 75-106, https://doi.org/10.1016/0079-6107(72)90004-1.

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Effects of heat on colour and iridescence of Chrysochroa wallacei beetle