Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), and optical microscopy are all techniques that analyze and visualize surfaces and structures at various scales. All three techniques are used to observe the morphology and structure of samples to better understand physical properties. Each can provide information about surface morphology at different scales. While there are many similarities in each technique, there are significant differences with resolution, depth of field, the type of information gathered, and physical principles used to collect data. Applications include material science, biology, and nanotechnology to characterize properties of materials such as roughness, defects, and morphology. Looking at the same sample under an AFM, SEM, and Optical Microscope will provide different types of information as there are different resolutions and imaging techniques from each instrument.

Butterflies are famous for their iridescent colors, but it is "unusual in how those colors are produced" (Stone 2015). The wings are "not only colored by pigments, but also by the scattering of light within micro- and nanoscale lattice structures" (Stone 2015). This case study uses SEM, AFM, and optical microscopy to analyze the micro- and nanoscale surface structures of monarch butterfly scales, highlighting the similarities and differences among these research tools.

Optical Microscope: Keyence VHX-7000

The Keyence VHX-7000 is capable of 4K ultra-high-resolution imaging (20X to 2500X), achieves a high depth of field, has flexible observation modes, able to stitch 3D and measure, automated functions, and overall, a user-friendly instrument. The ultra-high-resolution imaging and high depth-of-field allow for precise analysis and clear images, even on uneven surfaces. Furthermore, it can support multiple observation methods such as brightfield, darkfield, polarized light, and differential interference contrast, allowing for a diverse set of needs. This instrument can obtain surface features, overall morphology, and color variations.

Figure one displays a high-resolution image of a Monarch butterfly wing at ---X. At this magnification, it is possible to view the surface morphology of the scales, but not the ridges and cross ribs within an individual scale (Giraldo 2008).

Scanning electron Microscope: JOEL 6500

The JOEL 6500 SEM uses a focused beam of electrons to scan the sample. Based on the interaction of electrons (backscattered or secondary) with the sample produces signals to form an image. The SEM is capable of high-resolution imaging (10X to 500,000X) that can be used to analyze surface morphology as well as conduct elemental analysis. Even though the images are in grayscale, the magnification is significantly higher than the optical microscope and can observe nanoscale details.

Figure 2 and 3 display nanoscale details of the Monarch butterfly scale at 800X and 2,000X. While both figures display A, the cross ribs of the scale, figure three also displays the ridges, B.

Applications to SEM: Cryo-SEM

Cryo-scanning electron microscopy (cryo-SEM) rapidly freezes samples and keeps them at extremely low temperatures for high-resolution surface imaging, preserving their natural structure. The operating temperature of cryo-EM is usually around -180°C to -196°C. This technique has numerous applications including tissue and cell development. Other additional techniques during this process include cryofracture and milling revealing details about air spaces, water distribution, and subcellular structures.

Figure four shows a scanning electron micrograph (SEM) of fractured cross-sections, revealing the ridges on the upper surface of a butterfly wing scale, which are supported by underlying structures. These ridges have a layered structure that creates interference, resulting in the metallic iridescence and sheen characteristic of a butterfly's wing (ACS 2020).

Atomic Force Microscope: Dimension Icon AFM with SanAsyst

Atomic Force Microscopy (AFM) can achieve atomic-level resolution, often less than a nanometer. In this technique, a sharp probe scans the surface of a sample to measure the forces between the tip and the sample, allowing for the creation of detailed 3D surface topography. Applications of AFM include assessing surface roughness, mechanical properties (such as modulus, adhesion, deformation, and force-distance (F-z) curves), electrical properties (including Conductive AFM (CAFM), Kelvin Probe Force Microscopy (KPFM), Electrostatic Force Microscopy (EFM), Tunneling AFM (TUNA), and I-V curves), as well as magnetic properties (Magnetic Force Microscopy (MFM)). AFM is also capable of revealing molecular and atomic-level features, making it a versatile tool in nanotechnology and materials science. Figures 5 and 6 show the 10um morphologies of the Monarch butterfly scale. The height of the cross rib (A) was measured at about 511.25nm and the ridge (B) was measured at about 1.43um.

Conclusions

In conclusion, Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), and optical microscopy each offer distinct advantages in visualizing and analyzing surfaces and structures at various scales. These techniques complement one another, providing a comprehensive understanding of sample morphology and surface characteristics from micro- to nanoscale.

The application of these methods to study the micro- and nanoscale structures of monarch butterfly wings highlights their unique capabilities and the differences in resolution and imaging techniques. The butterfly wing is an excellent sample for this comparison due to its intricate and varied surface features, making it ideal for demonstrating the strengths of each microscopy technique. Utilizing all three allows for a more complete and detailed characterization of materials, contributing to advancements in fields like material science, biology, and nanotechnology.

References

Stone, M. (2015, January 22). Understanding butterfly wing color. Penn Today. University of Pennsylvania. https://penntoday.upenn.edu/2015-01-22/features/understanding-butterfly-wing-color

Giraldo, M. (2008). Butterfly wing scales: Pigmentation and structural properties. Advances in Insect Physiology, 38

American Chemical Society. (2020, July 8). Chemistry in Pictures: Butterfly effect. Chemical & Engineering News. https://cen.acs.org/materials/photonics/Chemistry-Pictures-Butterfly-effect/98/web/2020/07