Scanning electron microscopy (SEM) is a powerful tool that may be used to examine the nature of bulk specimens. When the primary beam of the SEM interacts with the specimen in question, electrons from the beam may be elastically scattered by the sample. Elements of higher atomic number will elastically scatter more primary beam electrons than elements of lower atomic number, yielding a direct correlation between signal intensity and atomic number. Therefore, these elastically scattered electrons, termed backscattered electrons (BSE), provide information about variation in atomic composition of the sample.
When the primary beam of the SEM interacts with the specimen in question inelastically, secondary electrons (SE) are produced. However, due to their low energy, only secondary electrons generated closely enough to the surface of the sample are able to escape from within the sample and produce a signal. Therefore, SE imaging provides information about the surface structure of the specimen.
Due to its superior ability to elucidate information about the fine topographical structure of specimens, SEM is excellently suited for examining the surface detail of insects. For this study, secondary electron, backscattered electron, and topographic backscattered electron imaging were performed on insect samples. In order to prepare the samples for imaging, samples were coated in gold using a sputter coater. Post-processing of the SEM micrographs includes colorization and anaglyphic image creation using Adobe Photoshop CS6 and analysis using ImageJ.
Insect samples were obtained from Brian McIntyre1. Samples include a beetle and two butterfly wings, each collected from a different butterfly. In order to fit on the sample stub, appropriately small pieces of the butterfly wings were cut. Samples were then mounted on SEM sample stubs using adhesive tape. In order to make the samples conductive, the samples, mounted on the sample stubs, were then coated with gold for five minutes using a gold sputter coater. Following coating, the samples were grounded to the sample stub using graphite paint.
Butterfly Wing Samples
Individual scale on a butterfly wing.
Zoom view of butterfly wing scale.
Damaged butterfly wing scale.
Head meets body of beetle, zoom view.
Scales on leg of beetle.
Leg of beetle.
Damaged portion of beetle shell.
Eye of beetle.
Backscattered Electron (BSE) images were collected using the Zeiss Auriga Crossbeam SEM belonging to the Institute of Optics at the University of Rochester. BSE and topographic BSE electron images were collected of a butterfly wing sample. In traditional BSE imaging, the four quadrents of the detector are turned on and are positively biased. In topographic BSE imaging, two of the four quadrents that are side-by-side with one another are turned off. Comparing BSE and topographic BSE images of the same sample shows variation in topography. This may be seen in the images below, where the shadow produced by the wing scale changes when the imaging mode changes.
BSE micrograph of butterfly wing scales.
Topographic BSE micrograph of butterfly wing scales.
In order to generate anaglyphs, two SE-2 micrographs were collected of the samples, differing only by a sample stage tilting of approximately two degrees. Superposition and colorization of the micrographs in Adobe Photoshop CS 6 produced the final anaglyphic image. SE-2 images were collected using the Zeiss Auriga Crossbeam SEM belonging to the Institute of Optics at the University of Rochester.
Beetle shell, covered in debris.
Butterfly wing scales.
Image colorization was performed on two butterfly wing scale images collected in SE-2 mode. Image colorization was performed using Adobe Photoshop CS6.
Butterfly wing scale connecting to the butterfly.
Butterfly wing scale with broken tip, zoom view.
Analysis of size distribution of holes on a butterfly wing’s scale was performed using ImageJ. The average size of hole was found to be 0.186 µm2. It was found that holes comprised 38.6%, or 132.125 µm2,of the image.