Sample Preparation for Scanning Electron Microscopy

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 Images of Non-Conductinve (Left) and Conductive (Coated) (Right) Samples
Figure 1. SEM Images of Non-Conductinve (Left) and Conductive (Coated) (Right) Samples

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):

  1. Cleaning and cutting
  2. Fixation and stabilization
  3. Dehydration
  4. Drying
  5. Mounting on a stub
  6. Coating with a conductive material
SEM Sample Preparation Process Schematic
Figure 2. SEM Sample Preparation Process Schematic

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:

    1. Wash the sample with volatile solvents like acetone, methanol, or isopropanol
    2. Use ultrasonic bath (High power baths are not recommended to prevent any physical damage to the sample)
    3. Drying using compressed gas
    4. If cleaned by water, dry the samples using hot plate or oven
    5. Use clean gloves during sample preparation and handling (Hand grease is a major outgassing source in a vacuum system)
  • Biological Samples

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

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-Conductive Samples

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.

Three Insect Heads Coated with Gold on a SEM Stub (Left) and SEM Image of One of the Heads (Right)
Figure 3. Three Insect Heads Coated with Gold on a SEM Stub (Left) and SEM Image of One of the Heads (Right)

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 coaters.

Vac Coat also provides low and high vacuum carbon coaters (DCR and DCT) for carbon deposition. Sputter and carbon coater (DSCR and DSCT) systems offer switching between these two techniques in one desktop deposition system.

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.

References

  1. 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
  2. Echlin, Patrick. Handbook of sample preparation for scanning electron microscopy and X-ray microanalysis. Springer Science & Business Media, 2011.
  3. http://www9.open.ac.uk/emsuite/services/sem-and-tem-sample-preparation-materials
  4. https://ywcmatsci.yale.edu/facilities/sem/sample-preparation
  5. 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.
  6. https://www.gu.se/en/core-facilities/sem-sample-preparation-techniques
  7. 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.
  8. https://bitesizebio.com/34150/sem-sample-prep/#fl-main-content

6 thoughts on “Sample Preparation for Scanning Electron Microscopy

  • Carlos Bay

    After completion of fixation and dehydration steps for preparing a sample for SEM analysis, is it possible to store it in drying stage for a while, before mounting on a stub?

    • Vac Coat

      To store a dry sample for a long time and preserve its structural features, it should be stored in a low humidity environment like a dessicator.

  • MohiTech

    Hello all, How to prepare a powder of a non-conducting material, like liposom, for scanning electron microscopy?

    • Vac Coat

      Hello. The main method is to spread a small amount of powder on one side of a conductive dual-sided adhesive tape, such as carbon or copper tape. Also, sputter coating of a thin gold layer (a few angstroms) on the powders results in a better electrical conductivity and clearer SEM images.

    • Vac Coat

      “SEM analysis requires a high vacuum in the sample chamber, so it is necessary to extract the oil from the lubricants and grease to image their morphology in SEM characterization. The conventional sample preparation steps include:

      1- Oil extraction by immersing the sample for one week in an organic solvent.
      2- Replacing the solvent 3 – 4 times per day until oil extraction is completed.
      3- Drying the samples at room temperature for several days.
      4- Coating the samples with a thin layer of gold by means of a sputter coater to prevent charging of the surface, reduce thermal damage, and improve the secondary electron signal required for topographic examination in the SEM.”

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