Troubleshooting: edge effect, charging, sample damage
Getting the perfect image takes knowledge and practice. Obtaining the perfect image is a trade-off between many factors. There are a number of problems that can be encountered.
1. Lack of detail of surface structures
At high kV the beam penetration and diffusion become larger and result in signal (electrons coming out of the sample) being generated from deeper within the specimen. This can obscure fine surface structures. It will also increase BSE and so the image will start to show changes in contrast based on composition. The solution therefore, for obtaining fine surface structure is to exclude these backscattered electrons by using lower kVs such as 3-10kV. Hence lower energy provides better detail of surface structure.
2. Edge effects
Edge effects are due to the enhanced emission of electrons from edges and peaks within the specimen. They are caused by the effects of topography on the generation of secondary electrons and are what gives form and outline to the images produced by the Secondary Electron detector. Electrons preferentially flow to and are emitted from edges and peaks. Poor signal intensity occurs in those regions shielded from the detector, such as depressions. Topographic contrast is also enhanced by Back Scattered electrons emitted from regions of the sample facing towards the detector. Lowering the beam kV can reduce edge effect.
Charging is produced by build-up of electrons in the sample and their uncontrolled discharge, and can produce unwanted artefacts, particularly in secondary electron images. When the number of incident electrons is greater than the number of electrons escaping from the specimen, then a negative charge builds up at the point where the beam hits the sample. This phenomenon is called charging and it causes a range of unusual effects such as abnormal contrast and image deformation and shift. Sometimes a sudden discharge of electrons from a charged area may cause a bright flash on the screen. These make it impossible to capture a uniform image of the specimen and may even be violent enough to cause small specimens to be dislodged from the mounting stub. The level of charge will relate to (1) the energy of the electrons and (2) the number of electrons. The energy of the electrons is related to the kV (i.e. high kV = high energy) so reducing kV can reduce charging. The number of electrons relates to a number of parameters including, beam current, the emission level of the gun, the spot size, and the apertures between the gun and the specimen. So reducing the number of electrons by adjusting these parameters can also reduce charging.
In this image of a small hive beetle, the horizontal bright and dark bands are a result of charging. A sample preparation solution to such a problem can be to recoat the sample with a thicker layer of platinum. Alternatively such samples can be pre-treated with osmium tetroxide vapour to enhance conductivity in the joint regions that are difficult to coat effectively with metal.
The streaking and enhanced contrast in this image of organic secretion from an adult jewel beetle are due to charging. Loose materials such as particles often suffer from charging. A sample preparation solution is to reduce the amount of sample on the mount so that all the material is in contact with the base adhesive, and coat it with a metal such as platinum.
Charging is not as apparent in backscattered electron (BSE) imaging mode so that signal can be used or signals can be mixed (BSE and SE). A fast scan rate with frame averaging can be used to advantage in some circumstances too.
If available, an SEM with low vacuum capability or an Environment Scanning Electron Microscope (ESEM) can be used to control charging.
4. Specimen damage
Irradiating a specimen with an electron beam results in a loss of the beam energy to the sample in the form of heat. A higher kV results in a higher temperature at the irradiated point and this can damage (e.g. melt) fragile specimens, such as polymers or proteins, and volatilise waxes or other sample components. This can ruin a sample (as well as contaminate the SEM chamber). The solution is to lower the beam energy, sometimes down to a few kV. Increasing the working distance can also help since it produces a larger spot size on the sample for the same beam energy but this has the disadvantage of reducing resolution.
5. Beam-related contamination
Beam-related contamination refers to the deposition of material (e.g. carbon) in a region on the sample where the beam has been scanning. One way to work around this artefact is to take micrographs at low magnification, before moving to higher magnification.
Brass surface with a rectangle of carbon contamination from scanning the beam at a higher magnification.