Micrograph Analysis

Micrographs taken with optical microscope were processed with focus stacking, to obtain a larger depth of field. Those images shows what the ham looks like on micro scale, with top illumination, observed from normal direction to the surface. It is noticeable that the ham is still iridescent after water-exchanged with ethanol, or after CPD drying, as shown in Figure 2 on this page. This means that the color is not related with the water in meat, but might be related with the solid structure composed by proteins. It was also noticed that the iridescences on the same region do not change much in color, when the sample was tilted. The iridescence only faded away when the sample was tilted to a high angle (about >40°). This is quite like the behavior of film interference of light. Besides, when the samples were rotated along normal axis, the colors did not change. Usually, iridescence caused by grating will change when the sample is rotated.

Metallic colored areas on beef and ham

Fig. 1. Optical microscope micrographs of iridescent areas on (a) fresh ham; (b) raw beef.

 

Fig. 2. Optical microscope micrographs of (a) ham after water-exchanged with ethanol; (b) dehydrated ham.

 

As a powerful tool in scientific study, scanning electron microscope (SEM) can only give black and white micrographs, lacking the real color information of the sample. Researchers need to spend tons of time colorizing the micrographs with pseudo colors. In my research, colorful optical micrographs and SEM micrographs of the same areas were combined, to present the color information and microstructure information in one frame. I call this "all-natural" colorization. Figure 3 and Figure 4 show some of the all-natural colorized SEM micrographs.

Fig.3. Colorization of the SEM micrograph of a piece of ham (1.2mm×1.4mm): (a) optical micrograph; (b) SEM micrograph; (c) all-natural colorized SEM micrograph.

Fig. 4. All-natural colorized SEM micrographs of ham cross section.

Figure 4 shows how the iridescent areas look like in surface microstructure. Each large domain is the cross section of one single muscle fiber. It was noticed that the color seems to be always on the regions with multi-layered film structures. In the later study, it was found that those structures are composed of aligned protein filaments, with the axial direction perpendicular to the surface. When the meat was cut in to slices, the way those filaments being cut resulted in these terraces. Also, in some regions, the edges of neighboring terraces are parallel with each other at a relatively stable distance, showing some grating like structure.

 

Fig. 5. Pseudo colorized SEM micrographs of beef muscle fiber: (a) cross section; (b) details of muscle fiber.

Figure 5 shows the cross section of a single muscle fiber in beef and how it was composed of finer protein filaments. Those filaments composed of smaller sections with the diameter of around 300-700nm and axial length of about 1.5μm. For the biological terms and descriptions of these structures, please visit wikipedia.org.

 

Fig. 6. (a) SEM micrographs of beef muscle fiber cross section; (b) FFT image of selected area; (c) inverse FFT of a intensive spot in FFT image; (d) natural grating structure on butterfly wing scale.

Grating Structures are found on both ham (Fig.4) and beef samples (Fig.6). Figure 6 shows the grating on the cross section of a beef muscle fiber. After Fast Fourier Transform (FFT), some high intensity spots (in orange) can be found, as shown in Figure 6(b). With inverse FFT to one of the strongest spot, corresponding structure information can be rebuilt, as shown in Figure 6(d). Figure 6(c) shows the natural grating structures on butterfly wing scale, which can cause some iridescent colors.

 

Fig. 7. AFM result of a 10μm× 10μm area on CPD dried ham cross section: (a) 2D view; (b) 3D view.

Figure 7 shows the height profile of a certain area on a CPD dried ham, obtained with atomic force microscope (AFM). Each domain with the diameter of about 1μm represents one small bunch of protein filaments. Compare it with Figure 5 can give you a better understanding. Since the surface of the sample have a slope as the background, it is very hard to get the accurate height information. However, by comparing it with SEM micrographs, an estimated height of hundreds of nanometers can be made.

 

Fig. 8. (a) TEM micrograph and  (b) SEM BSD image of  finer filaments in beef muscle fiber.

Figure 8 shows the results of two alternative methods of imaging the muscle fiber, using Transmission Electron Microscopy (TEM) and Backscattered Electron Detector in SEM. The TEM results are not ideal to me. The micrographs are lack of contrast because the sample (composed of light atoms, H, C, O, N and so on) was not stained with metal ions, which is a necessary procedure in biological TEM sample preparation to give better contrast. However, fibers with 1μm diameter can still be observed. I also used Energy-dispersive X-ray spectroscopy (EDS or EDAX) analysis to obtained some chemical elements information of the beef, and the results showed that it was mainly composed of C, O, Si, K, Na, N and P (H is not detectable with EDS). However, I cannot find the result file in the SEM computer....so cannot show it here. :P

 

Discussion

It was noticed that the iridescent colors always remained the same color when the sample is tilted, or rotated. The colors are most obvious when observed from the direction almost normal to the surface with top illumination. This kind of behavior is very like the result of film interference of light, instead of grating diffraction result which will usually varied in color when tilted and showing all spectrum. Also, in all-natural colorized micrographs, it was noticed that the colorful areas were always associated with film like structure. The areas with obvious grating structures only took a small portion of the whole sample surface. :)

Summary

Both multilayer thin film structure and 2D grating structure were observed on the cutting surfaces of ham and beef, with characteristic distance comparable with the wavelength of visible light (400-700nm). The iridescent color behaves more like the result of multilayer interference of light, correlated with the thin film like structures on meat.

References:

[1] Brian McIntyre , Notes and slides for Opt 307/407 Electron Beam Methods In Microscopy 2011

[2] http://en.wikipedia.org/wiki/Thin-film_interference

[3] http://en.wikipedia.org/wiki/Diffraction_grating

[4] S. Kinoshita et al.,Structural Colors in Nature: The Role of Regularity and Irregularity in the Structure. Chem Phys Chem 2005, 6, 1442 – 1459

[5] S.-W. Chu et al.,Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy. Journal of Microscopy, Vol. 208, Pt 3 December 2002, pp. 190–200

[6] X. Sheng et al., Integration of Self-Assembled Porous Alumina and Distributed Bragg Reflector for Light Trapping in Si Photovoltaic Devices. IEEE Photonics Technology Letters, 2010,22(18)

 

Please enter any comments, criticisms, questions, etc. below.

Your name:

Email address: