What is the Purpose of Sample Preparation?
Samples to be investigated by electron microscopy need preparation through various techniques to obtain the most accurate results. The sample preparation procedure may include steps such as washing, drying, fixation, mounting, and coating to achieve the best result. Not carrying out preparation steps before imaging may deteriorate the device’s performance, image quality, and sample structure.
What is SEM?
Scanning Electron Microscopy (SEM) is a powerful tool to achieve information on the structure and other nanoscale characteristics of a specimen. This type of electron microscopy provides nanoscale images which can be used to study various samples, including ceramics, metals, alloys, semiconductors, biological samples, etc.
Why Should an SEM Sample be Prepared?
SEM samples should be dry and conductive. Samples containing water cause problems when vacuuming the SEM chamber, also energetic electrons incidence on the sample may evaporate the residual water and reduce image quality. The conductivity of the sample surface is vital to prevent charge build-up on its surface (Figure 1). Accordingly, some specimens require a preparation step before SEM imaging.
SEM Sample Preparation Process
The SEM sample preparation mainly depends on the sample characteristics, it may need some or all of the following steps (Figure 2):
- Cleaning and cutting
- Fixation and stabilization
- Mounting on a stub
- Coating with a conductive material
SEM Sample Preparation Techniques
Sample Cleaning and Drying
Cleaning the samples is a crucial step in the sample prepapration process to obtain clear images. Degreasing and drying the sample reduces outgassing from possible organic contaminations and residual water in the sample. To clean and dry a sample one can follows the below steps:
- Wash the sample with volatile solvents like acetone, methanol, or isopropanol
- Use ultrasonic bath (High power baths are not recommended to prevent any physical damage to the sample)
- Drying using compressed gas
- If cleaned by water, dry the samples using hot plate or oven
- Use clean gloves during sample preparation and handling (Hand grease is a major outgassing source in a vacuum system)
Biological samples should undergoe fixation and dehydration processes, and then dried to remove the water inside them. This will allow higher vacuum SEM imaging and clearer images.
Powder samples may fly off under vacuum condition inside the SEM chamber, so they must be processed. Large amount of powder can be compressed to a pill and in small amount they should be dispersed in a volatile solvent and dropped on a suitable substrate.
Non-conducative samples (including biological ones) can trap incident beam electrons on the surface and result in shinning spots in SEM images. Also this built-up charge can cause overheat and deterioration of the sample. Coating a thin film of a conducting material on the sample surface can prevent this phenomena (Figure 3).
The coating thickness must be enough to prevent charging (You can read more about thickness uniformity here), while preserving specimen surface details (around 10 nm). Deposition of a layer of gold, silver, platinum, or chromium for SEM and carbon for EDX are ideal, since carbon spectral lines in EDX normally has less interference with the sample’s compared with gold. For more details on the coating material characteristics visit our blog article Selection of suitable material for preparing an electron microscope sample.
Vac Coat Products & SEM Sample Preparation
Vac Coat produces high-precision coating systems for a variety of purposes. Vac Coat sputter coaters equipped with vacuum rotary and turbomolecular pumps, working at low and high vacuum conditions (DSR1 and DST1), are a suitable choice for SEM sample preparation.
High-precision QCM thickness meter, electronic shutters, sample rotation, 7” colored touch-screen panel, user-friendly updatable software, and many other special features are presented in these benchtop coating systems. DST1 is presented in two chamber size, with a diameter of 170 or 300 mm. Optional features, such as RF sputtering, plasma cleaning, and substrate bias voltage and heating can be offered due to customer’s requirements.
- Polilov, A.A., Makarova, A.A., Pang, S. et al. Protocol for preparation of heterogeneous biological samples for 3D electron microscopy: a case study for insects. Sci Rep 11, 4717 (2021). https://doi.org/10.1038/s41598-021-83936-0
- Echlin, Patrick. Handbook of sample preparation for scanning electron microscopy and X-ray microanalysis. Springer Science & Business Media, 2011.
- Murtey, Mogana, Ramasamy, Patchamuthu. “Sample Preparations for Scanning Electron Microscopy – Life Sciences”. Modern Electron Microscopy in Physical and Life Sciences, edited by Milos Janecek, Robert Kral, IntechOpen, 2016. 10.5772/61720.
- Nguyen, Jenny Ngoc Tran, and Amanda M. Harbison. “Scanning electron microscopy sample preparation and imaging.” Molecular Profiling. Humana Press, New York, NY, 2017. 71-84.