Author: Izaz Ul Islam
Introduction to SEM Sample Preparation
Scanning Electron Microscopes (SEMs) are indispensable tools for analyzing surface morphology, structure, and elemental composition across fields like material science, forensics, and additive manufacturing. Modern desktop SEMs enable fast, on-site analysis, but achieving high-quality results hinges on meticulous sample preparation. This guide outlines practical techniques for handling diverse samples, from conductive materials to delicate biological specimens.
Basic Sample Preparation
Every SEM is equipped with a sample holder or a loading chamber where the sample can be inserted. To load a sample in a SEM, the use of aluminum stubs is recommended. These come in different, standard sizes and are readily available on a commercial basis.
Sample adhesion to the surface of the stub is crucial before placing it in the sample holder or stage. This will prevent pieces of sample being dislodged under vacuum and contaminating the SEM column which can affect the final image quality. It may also damage the SEM imaging system which can be expensive to repair.
TIP 1: Stick the sample securely to the pin stub, by using:
- Double-sided carbon sticker
- Conductive paint
- Conductive tape
- Special clamps
- A combination of the above.
TIP 2: Remove all loose particles from your sample after adhering the sample to the pin stub by:
- Holding the aluminium stub with tweezers, tilt it by 90° and gently tapping it on its side.
- Spraying dry air on the sample.
TIP 3: Use tweezers when handing the pin stub
- This should be done in order to prevent contamination.
TIP 4: Make sure that the mounting procedure is solid
- This is so that you do not introduce mechanical vibrations due to incorrect mounting.
TIP 5: DO NOT spray dry air in the direction of any electronics
- Or a scanning electron microscope, because it might be flammable.
TIP 6: Make sure there is no condensed liquid in your spray air straw
- You can do this by first spraying away from your sample.
These precautions will help to reduce the risk of contamination of your system and sample holder and guarantee better performance over time. Below we discuss best practice sample preparation techniques for 5 common sample types which include: Non-conductive samples; Magnetic samples; Beam sensitive samples; Powders and particles and Samples containing moist or outgassing samples.
Handling Specific Sample Types
1. Non-Conductive Samples
When a non-conductive material like a biological sample is imaged, the electrons fired onto the sample surface don’t have a path to the ground potential, causing them to accumulate on the surface. The image will become increasingly bright or entirely white until details are no longer visible. Mild movement can also be detected, caused by the mutual interaction of the electrons. This will cause blurriness in the collected image.
Several Solutions Are Widely Used
- Conductive tapes or paints
By covering part of the sample with a piece of conductive tape (e.g. copper tape) or some conductive paint, a bridge to the surface of the aluminum stub is created. SEM image of sugar cube charging. SEM image of sugar cane in low vacuum. This will allow the sample to partially discharge and is enough to image mildly non-conductive samples when imaging areas close to the tape edge.
- Low vacuum
Introducing an atmosphere in the sample chamber allows beam interaction with air molecules. Positive ions are generated and attracted by the large number of electrons on the sample surface. The ions will further interact with the electrons, discharging the sample. While this technique adds some noise to the final image, you can analyse the sample faster and at lower cost without further processing.
- Sputter coating
By using a sputter coater such as the LUXOR series, it is possible to create a thin layer of a conductive material on the sample surface. This creates a connection between the surface of the aluminum pin and the ground potential. The choice of coating material is strongly dependent on the kind of analysis to be performed on the sample. Gold and platinum are ideal materials for high-resolution images because both have extremely high conductivity. Lighter elements, like carbon, can be used when Energy Dispersive Spectroscopy (EDS) analysis on non-organic samples is required. An alloy of indium oxide and titanium oxide (ITO) can create transparent, conductive layers, to be used on optical glasses to make them suitable for SEM.
However, there are disadvantages to using a sputter coater: Additional instrumentation is required, the analysis becomes more time consuming, and the samples undergo more pumping cycles. Also, any advantage of using a backscatter electron detector (BSD) to image the sample is lost, as the contrast becomes very homogeneous and there is no difference in gray intensity for different elements. The option for EDS analysis for elemental analysis is also lost.
2. Magnetic Samples
Challenge: Magnetic fields distort the electron beam, elongating images (stigmation). Solution:
Stigmation Correction: Adjust the SEM’s lens magnetic fields to restore a circular beam shape.
Re-focus after correction for optimal clarity.
Save settings for frequently analyzed samples to streamline workflows.
3. Beam-Sensitive Samples (e.g., Polymers, Biological Specimens)
Challenge: Beam heat or chemical interactions damage delicate structures.
Mitigation Strategies:
- Low Beam Energy: Use reduced voltage (1–5 kV) and beam current.
- Sputter Coating: Apply a thin carbon layer to dissipate heat.
- Cooling Stages: Use temperature-controlled holders to minimize thermal damage.
- Limit Exposure Time: Rapid imaging reduces localized heating.
- Low Magnification: Higher magnifications concentrate beam energy, increasing damage risk.
4. Powders & Particles
Challenge: Overlapping particles or loss during preparation skews analysis.
Optimized Methods:
- Manual Dispersion: Sprinkle a small amount onto carbon tape and blow off excess with dry air.
- Particle Dispersers (e.g., Nebula): Ensure even distribution for accurate size/shape analysis.
- Adjust vacuum levels: Higher for hydrophilic powders, lower for fragile particles.
5. Moist or Outgassing Samples (e.g., Plant Tissues, Gels)
Challenge: Vacuum-induced dehydration or structural collapse.
Preparation Techniques:
- Critical Point Drying (CPD): Replace water with supercritical CO₂ to preserve microstructure.
- Freezing: Flash-freeze in liquid nitrogen to avoid ice crystals (use cryo-SEM stages).
- Low-Vacuum Mode: Reduces evaporation for mildly hydrated samples (e.g., leaves).
- Minimal Sample Quantity: Deposit thin layers using a toothpick (ideal for gels/emulsions).
Key Points
Tailor preparation methods to sample type to avoid charging, contamination, or damage.
Prioritize safety: Avoid flammable hazards and column contamination.
For complex cases, consult SEM specialists to refine protocols.
By following these steps, users can achieve high-resolution SEM images while preserving sample integrity and extending instrument lifespan.
Read More: Limitations and Advantages of Computational Method
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