Source: Peiman Shahbeigi-Roodposhti and Sina Shahbazmohamadi, Biomedical Engineering Department, University of Connecticut, Storrs, Connecticut

A scanning electron microscope (SEM) is an instrument that uses an electron beam to nondestructively image and characterize conductive materials in a vacuum. As an analogy, an electron beam is to the SEM as light is to the optical microscope. The difference is that the electron microscope yields images of much higher resolution and magnification. The best optical microscopes typically have a resolution down to 200 nm, whereas SEMs usually claim a resolution of 0.5 nm. This is due to the fact that optical microscopes are limited by the diffraction of waves, a function of the wavelength, which is around 500 nm for visible light. Conversely, the SEM uses an energized electron beam, which as a wavelength of 1 nm. This characteristic makes them very dependable tools for the study of nano and microstructures. Electron microscopes also enable the study of biological samples with feature sizes too small for optical microscopy.

This demonstration provides an introduction to sample preparation and initial image acquisition of biological samples using a scanning electron microscope. In this case, a collagen-hydroxyapatite (HA) cellular scaffold will be studied. The vacuum environment of the SEM and the induced charging by the electron beam on non-conductive samples (such as organic matter) creates challenges that will be addressed in the preparation. The advantages and disadvantages of different imaging methods as they relate to resolution, depth of focus and sample type will also be discussed. The purpose of this demonstration is to give the participant more information on SEM to determine if this microscopy module is the best fit for a type of biological sample.

1. Sample Preparation

1. Wear gloves and take precautions to avoid contamination when handling the sample.
2. Make sure that the sample on the slide is dried and there is no contamination on the sample. This is because SEM measures surface characterization, and these defects can severely hinder the signal.
3. If the sample is loaded on a standard glass slide, decrease the size of the sample by scoring the slide with a diamond tipped glass cutter in a straight line and gently push on the scored line

The SEM images in Figures 3 and 4 show that the imaged structure is highly three dimensional with microscale features. Image quality is affected by the focus and the thickness of the sputter coating.

Figure 3: The following images demonstrate how the sample focus can affect image quality.  In the image on the right, the

Here we demonstrated the depth of focus, field of view and maximum resolution and magnification of an electron microscope and how these properties can be used to view biological samples. This demonstration was designed to help viewers decide which microscopy module is the best for a certain application. As demonstrated, SEM has a very high depth of focus, much higher resolution and greater magnifications. However, it is not appropriate for all sample types.

This demonstration introduced the pr

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2. Goldstein, Joseph, et al. Scanning electron microscopy and X-ray microanalysis: a text for biologists, materials scientists, and geologists. Springer Science & Business Media, 2012.
3. Carol Heckman, et al. Preparation of cultural cells for scanning electron microscope. Nature Protocols Network, 2007, doi:10.1038/nprot.2007.504