Proper sample preparation plays an essential role to obtain the right information in scanning electron microscopes (SEMs). To achieve this, the samples must undergo a complex preparation process to help them withstand the environment inside the microscope.
You must consider the sample’s size, shape, state, and conductive properties before starting any preparation. If you’re interested in electron microscopy and want to discover more about it, you should learn how to prepare an SEM sample in eight steps.
Sample Cleaning
A clean SEM sample is vital for image clarity. You must use suitable buffers or distilled water to clean the samples. Use a surfactant if the sample requires more vigorous cleaning. To remove oils from the surface of the sample, wash it with suitable solvents.
Primary Fixation
Use a fixative such as glutaraldehyde or formaldehyde to stabilize the structure before further processing.
Secondary Fixation
Bilipid membranes are fixed to prevent solvent extraction during dehydration. Keep in mind that If a fixative uses a phosphate-based buffer for its preparation, salt deposits can interfere with the image quality of the sample.
Solvent Dehydration
Use a graduated series of alcohol and finish the final dehydration step with 100% alcohol or acetone. Note that the solvent concentration increases gradually so that the water is removed smoothly, without shrinking the sample.
Drying
Before placing the SEM sample in a high vacuum environment, it must be completely dry. If you allow acetone or ethanol to simply evaporate, it would develop artifacts and create micro-ripping of the surface upon leaving.
In this step, the water vaporization should not obstruct the electron beam and interfere with the clarity of the image. You must use hexamethyldisilazane (HMDS) or liquid CO2.
- HMDS is frequently used in cell preparations and after 3 minutes of incubation it’s removed and the excess is allowed to evaporate.
- On the other hand, Liquid CO2 is applied to tissues in a critical point dryer where it’s brought to a temperature and pressure where it vaporizes.
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 split the sample in the right direction to reveal its internal details.
Sample Stubs, Adhesives, & Mounting
SEM sample stubs or holders are available in different diameters, making them convenient for imaging different types of samples. Make sure the stubs are clean and handle the sample with clean forceps.
Note: The material you use as an adhesive to stick the specimen to the stub must be non-toxic and must not fall to the sides of the specimen.
Mounting on a Stub
Double-Coated carbon conductive tape is the most common adhesive for this purpose. You must ensure that there is a conducting path while mounting the specimen. If the sample requires a conductive coating, be sure to mount it before coating so that both the sample and the plug receive the coating.
Sample Storage
Store the scanning electron microscope (SEM) sample and stubs in a dry, clean environment. Also, it’s highly important to use gloves during the entire sample preparation stage.
Electron Optics Instruments Is Your Answer
If you’re interested in purchasing a scanning electron microscope, Electron Optics Instruments is the leading SEM distributor in the United States. We have over 30 years of experience and offer customized solutions based on our customers’ needs.