The goal of this project was to examine various iridescent feathers from several types of birds in order to determine how the iridescent colors are caused. It is a well-known fact that butterfly wings have a characteristic surface structure which causes light to be reflected in such a way as to cause constructive or destructive interference, affecting the color visible to the human eye. Certain iridescent bird feathers work in a similar way, although the structure is not on the surface itself, but within the barbs and/or barbules of the feather. Four types of feathers were examined for this project: pigeon, parrot, turkey, and of course, peacock.

Light has a wave nature, which is what makes interference,and in turn iridescence possible. When two light waves reflect off of something in such a way that the peaks of light waves (with the same wavelength) fall on top of each other, the result is constructive interference, meaning that the two amplitudes add. If a peak falls on a trough, however, the result is destructive interference, and no wave is seen at all. Of course, the peaks do not necessarily either fall on other peaks OR on troughs. The amount that the peak of one light wave is shifted from another is called the phase shift. Phase shifts can be caused by gratings, slits, and thin films. The small structure present in bird feathers in effect acts as a grating, which in turn causes the color of the feathers to appear different depending on angle. (See Source 5).

Light reflecting off of a grating is concentrated at very small and well-defined angles. A very small range of wavelengths will be visible to the viewer at a particular angle, but if the viewer changes his or her angle, then the wavelength will also change. This is what causes a grating to appear to change color as a viewer changes his or her angle with respect to the grating, and this phenomenon is known as iridescence (Falk, p. 320).

LIGHT MICROSCOPY

All four of the feather samples were examined using a light microscope. Although the depth of field in such a microscope is not very good (note that in the following images there are always portions which are out of focus and portions which are in focus) the light microscope does allow us to see the color that is present in the fine structure of the feathers.

(a) Barbules of a pigeon feather, 10x magnification.	(b)Barbules of a parrot feather, 5x magnification.
(c) Barbules of a turkey feather, 10x magnification.	(d)Barbules of a peacock feather, 20x magnification.

The smaller structures that take up most of the image area in each of the preceding images are called the barbules. The longer structures from which the barbules protrude are called the barbs. Note that in Fig. 1b, the image of the parrot feather, the barbs appear to contain most of the color. For this reason, the cross section of the parrot barbs, rather than the barbules, was examined. In all the other types of feathers cross sections of the feather barbules were the primary focus.

SAMPLE PREPARATION

One barb was plucked off of the main stem for each type of feather. This barb was then held down to a piece of tissue at either end with small pieces of tape. A razor blade was used to carefully cut off some of the barbules, and then the barb was fastened to a sample stub using a piece of carbon tape. (Except in the case of the parrot feather, in which the barbs were sliced across at a diagonal and then fastened to a sample stub.) The samples were then placed in the sputter coating chamber at an angle, and were sputter coated with gold for ~20 secs. at a current of ~15 mA. The peacock sample was sputter coated for an additional 40 secs. at this current. The sample stubs were placed into the SEM chamber flat, and then the sample stage was tilted to an approximately 90 degree angle in order for the cross sections of each sample to be viewed.

SEM IMAGING

All of the following images were taken using secondary electron imaging mode. Both the SE and the Mix mode were used in this project. It was decided, however, that backscattered electron imaging mode would not be worth taking extra time to use. The working distance was generally kept as short as possible, no longer than 14 mm. (The reason it had to be even this long was due to the tilt of the sample stage, which did not allow the sample itself to get very close to the final condensor lens aperture.) The accelerating voltage used was usually 5 kV, but occasionally a 10 kV voltage was used. The aperture setting was usually held to 1, while occasionally a setting of 2 was used if charging did not seem to be too much of a problem.

One of the biggest problems with the project, aside from the difficulty of getting cross sections of the tiny barbules, was charging. It was difficult to get the samples coated at the right angle, and additionally, charging could usually be seen on either the top or the bottom surface of the feather barbule. Therefore charging is present to a small extent in almost all of the images that follow.

Recently, a team of scientists from China led by Jian Zi examined the nature of the color production in peacock feathers. According to Zi, the mechanism for the coloration in peacock feathers comes from a periodic crystal lattice structure present within the barbules. In Figure 2, it is clear from looking at either surface that there are small, periodic circular shaped structures, which are actually melanin rods. The differences in color in different parts of the feather can be accounted for by the spacing of these melanin rods and the number of layers of rods. The above figure shows a section of a peacock feather in the blue portion of the eye of the feather. Pictures taken by Zi et al. of the green and brown portions can be found here. It is clear just by a visual examination of the images, which all have a scale of 500 nm, that the spacing of these melanin rods is indeed different for the different colored portions of the feathers. The periodic structure is not very clear in the above figure, both due to focusing problems and probably the amount of gold that needed to be put on the sample to prevent charging. According to Zi, the lattice constant for the blue barbules should be around 140 nm, however, due to the poor resolution of the periodic structure it was not possible to accurately measure this.

Figure 3 shows more examples of the periodic stucture visible in the blue barbule of a peacock feather. Clearly there is a lattice structure near both the top and bottom surfaces of the feather, with a different structure making up the center portion of the feather barbule. Click on the figure for a larger view.

Of course, not all microscopic, color-producing structure will look like this. An ornithologist from the University of Kansas, Richard Prum, has studied the feathers of nearly 30 different bird species, and he concluded that although these iridescent patterns are indeed produced by tiny particles arranged in a pattern, the "orderliness" of the patterns differs depending on the bird, and that the more orderly the pattern, the purer the resulting color (Pennisi).

In addition to the peacock, three other types of bird feathers were examined in this experiment. However, none produced a pattern that was quite so regular as that shown in the figures above. Probably the next most regular looking pattern belonged to the parrot, which as expected, also has the most vivid color of the three remaining birds. Images of the cross sections of parrot, pigeon, and turkey feathers are shown below.

In Figure 4, the different layers that make up the barb of a parrot feather can be seen clearly. The inner portion that has the "honeycomb" structure is refered to as the "spongy" or "cloudy" layer (Hesford). In parrot feathers, the green colors are produced both by pigments and by structural features within the barbs. A base pigment, usually yellow, is present in the barb, and the structures present create iridescent blue. The combination of these two colors is responsible for the vivid green color of parrot feathers (Falk, p. 321).

* All of the above images have been sharpened using Jasc Paint Shop Pro *

X-RAY IMAGING

According to the literature available about the structure of iridescent bird feathers, the tiny periodic structures visible in most of these images are actually particles of melanin, a protein. Most of these melanin particles are either brown or black, but their spacing and size enables light reflecting off of them to appear in a vast array of colors. As an organic particle, we would expect melanin to be composed of elements such as carbon, oxygen, and nitrogen. In addition, when looking at an x-ray spectrum we would also expect to see some gold peaks due to the sputter coating of the sample.

An x-ray spectrum was taken of a turkey feather for this experiment (see Figure 7). Since it is believed that all of the structures in all of the feathers are made of the same material, x-ray spectrums were not taken for the other types of feathers.

Figure 7. X-ray analysis of a turkey feather barbule

We can see from Figure 7 that other elements, such as sulfur, are also present in small amounts. This is not entirely unexpected, as some published material indicates that melanin often contains sulfur (Hallegot).

FALSE COLOR IMAGING

The following are images that were false colored using Jasc Paint Shop Pro. Images were colorized and the brightness and contrast were also adjusted. An attempt has been made to present a color scheme for each type of feather, with the colors that the feathers appear at different angles being the only colors used in the false color images.

(a) Pigeon cross section before coloring. Note that the image has been sharpened.	(b) Pigeon cross section after coloring. Only purples and blues were used because these are the colors that this particular feather appeared
(c) Parrot cross section. Note that the image has been sharpened using Paint Shop Pro	(d) Parrot cross section colored using greens and blues, which were close to the colors that the feather appeared
(e) Turkey cross section. Note that the image has been sharpened using Paint Shop Pro	(f) Turkey cross section colored using rich browns and yellows, which were close to the gold colors that the feather appeared
(g) Peacock cross section. Note that the image has been sharpened using Paint Shop Pro	(h) Peacock cross section colored using teals and blues

Figure 8. Original images and false color images created in Paint Shop Pro

CONCLUSIONS

Unfortunately, due to the method used in preparing the samples, it was very difficult to obtain a good cross-section of a tiny feather barbule. Many attempts were made with the razor, and the samples had to be "searched" very carefully using the SEM before suitable locations for imaging could be found. Therefore the views of the periodic structures present in the feathers used for this project are probably not optimal, and it also did not seem feasible to attempt to measure the distances between periodic structures. The analysis presented in this project is therefore only qualitative, verifying that such structures do indeed exist and also that they are strikingly different for different types of bird feathers.

SOURCES

1. Zi, Juan et al. "Coloration strategies in peacock feathers." PNAS, vol. 100, no. 22, pp. 12576-12578. October 28, 2003.

2. Pennisi, Elizabeth. "Shedding Light on Avian Iridescence." Science, Vol 299. January 24, 2003.

3. Hallegot, Philippe, et al. "In-Situ Imaging Mass Spectrometry Analysis of Melanin Granules in the Human Hair Shaft." The Journal of Invesitgative Dermatology, Vol 122, pp. 381-386, 2004.

4. Hesford, Clive. "Colour in the Parrots." Article retrieved from http://www.birdhobbyist.com/parrotcolour/elements.html on April 20, 2005.

5. Article retrieved from http://www.physics.utoledo.edu/~lsa/_color/14_int.htm on April 28, 2005.

6. Falk, David et. al. Seeing the Light: Optics in Nature, Photography, Color, Vision, and Holography. John Wiley & Sons, New York: 1986.

A special thank you to Koko for the generous donation of some of his feathers