Proper sample preparation plays an important role in obtaining the required information when using scanning electron microscopy (SEM). You need to consider the sample’s size, shape, state, and conductive properties prior to sample preparation. Ideally, the smallest representative sample size is the one to use. The microscope’s detection capacity is as much as 1µm from the sample surface.
Working Principles of Scanning Electron Microscopy
An electron gun at the top of the microscope directs a stream of electrons vertically down a set of electromagnetic lenses inside a vacuum chamber. There is usually one of the following electron guns that are present in a SEM:
1. Thermionic guns: these use filament heating to stimulate electrons to stream out.
2. Field emission guns: these produce a strong electric field in order to stimulate electrons out of their atoms.
The lenses are placed in a vacuum chamber in order to avoid obstruction and contamination by other particles. The lenses help direct the electrons towards the sample. The electron imprint is converted to a three-dimensional image which is visualized digitally.
Sample Preparation Starters
Some samples need to be coated to make them conductive. Metals require no preparation due to their inherent ability to conduct electricity. However, non-metals need to be coated with a conductive material. Most often, a thin layer of gold works. This requires the use of a sputter-coater.
Important parameters in preparing a sample for SEM imaging are as follows:
A clean sample is essential for image clarity. For biological samples, use appropriate buffers or distilled water for cleaning the samples. Use a surfactant if the sample requires more vigorous cleaning. If the biological property of the sample is known, then you might be able to use proteolytic enzyme cleaning. To remove oils on the sample surface, wash with appropriate solvents. Additionally, you can use ultrasonic baths for cleaning the sample. However, ultrasonic baths require caution in order to avoid damaging the sample.
Sample Fixation and Dehydration
Use a fixative like glutaldehyde or osmium vapor to maintain the structural details of the sample. Note that if a fixative uses a phosphate based buffer for its preparation, salt deposits may interfere with the sample’s image quality. For dehydration, use a graded series of alcohol and finish off the final dehydration step with 100% alcohol or acetone.
Prior to placing the sample in a high vacuum environment, it must be totally dry. Otherwise, water vaporization will obstruct the electron beam and interfere with image clarity. When using biological samples, be careful when doing critical point drying (or CPD), so as to not compromise the structural integrity of the sample. A suitable CPD instrument can help achieve this. Alternatively, you can try using freeze drying. In this regard, freeze drying causes the least amount of sample shrinkage in comparison to air drying or critical point drying. However, freeze drying carries the risk of ice crystal formation on the sample.
Sample Preparation of Tissue Sections
To observe details from tissue sections, remove the epoxy resin using organic solvents, ion beam etching, or plasma etching. You can also break the sample in the appropriate direction to reveal its internal details.
Sample Stubs, Adhesives, and Mounting Approach
Sample stubs or supports are available in different diameters making them convenient for imaging different kinds of samples. Be sure the stubs are clean and handle the sample with clean forceps. Also, use gloves during the entire sample preparation stage. The material you use as an adhesive to glue the sample to the stub should be non-toxic and should not tumble into the sides of the sample. Conductive double coated carbon tape is the most common adhesive for this purpose. You need to ensure that a conducting path exists as you mount the sample. If the sample requires a conductive coating, be sure to mount it before coating so that both the sample and plug receive the coating.
Store the sample and stubs in a dry, clean environment. Use clean forceps and gloves while handling the stubs.
In conclusion, I hope you now have a feel for what you need to start doing SEM. Happy imaging!