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Atomic force microscopy (AFM) is a very popular analysis tool for 3D surface topology visualization and other measurements on a wide range of materials at nanoscales.

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Applications of atomic force microscopes in nanotechnology are numerous. However, to obtain the best results with AFM, as well as produce accurate AFM images, it is critical to apply an effective and well-optimized sample preparation procedure.

In this article, we discuss AFM sample preparation, the differences in sample preparation for various types of nanomaterials, and some factors to consider during AFM sample preparation for best results.

Atomic force microscopy (AFM) is a robust technology that is used to visualize the surfaces of several kinds of materials like ceramics, polymers, glass, or even biological samples. The AFM probe obtains the 3D topography of the surface, identifies and measures magnetic forces, measures adhesion strength, and investigates other mechanical properties of the sample surface at the nanoscale.

For example, one study demonstrates the application of AFM to study lithium-ion batteries' electrochemical and mechanical properties, which is useful in solving the most challenging issues in efficiency and safety associated with lithium-ion battery technologies.

AFM sample preparation is a process necessary to improve the quality of results during analysis without compromising the sample’s integrity. It is the key to obtaining high-quality AFM images, but it can be labor intensive, time-consuming, and error-prone. Ideally, AFM sample preparation should be efficient, fast, simple, and compatible with many applications.

An AFM experimental setup consists of: an AFM, contact mode cantilevers, an AFM tip, a disc of the substrate material, an adhesive like poly-l-lysine for mica substrates or 3-aminopropyldimethylethoxysilane for silicon substrates, analyte nanoparticle solution, deionized water and ethanol, an optical microscope, and an AFM image analysis software.

For AFM particle imaging, the sample must be adhered rigidly and properly dispersed on the substrate; the roughness of the substrate should not be bigger than the size of the nanoparticle sample surface.

Some aspects of AFM sample preparation are discussed below:

Substrate preparation requires the selection of a substrate such as mica, silicon, glass, or metal discs, depending on the size of the nanomaterial. Then, the substrate can be processed. For example, mica discs must be cleaved for a clean surface to be produced before use as a substrate.

Activation facilitates the adhesion of the nanomaterial to the substrate. Activation generally involves imparting a charge on the substrate and the nanomaterial such that there can be a chemical or electrostatic bond between them. This is done by using adhesives like PLL solution, which can go with mica, and the nanomaterial can be imparted a charge by stabilizing with citrate ions.

The substrate and nanomaterial are bound and incubated with incubation times depending on the nanomaterial particle size. They are then rinsed with deionized water and dried with nitrogen before visualization.

Before observation with AFM, it is recommended that the sample be inspected with an optical microscope to observe the dispersion of particles and note areas of suitable dispersion that will produce the best results.

A protocol chapter published in Springer Journal outlines a detailed step-by-step procedure for AFM sample preparation for size determination, considering different substrates like mica and silicon as well as different modes of analysis.

As previously mentioned, AFM can be used on many types of nanomaterial samples. These will naturally require different methods for AFM sample preparation.

The different nanomaterial samples used can be classified as engineered or non-engineered particles. Those that are engineered can be further classified into organic and inorganic groups. These groups can further be classified as suspensions, powders, and embedded particles.

Furthermore, factors such as biocompatibility, hydrophobicity, particle size, and native environments need to be considered. There are hundreds of AFM sample preparation techniques, which can be customized according to the research. Fortunately, several combinations of nanomaterials, substrates, and adhesives work well with atomic force microscopy.

Different substrates can be used depending on the size of the nanomaterial. The smaller the size of the nanomaterial, the smoother the substrate must be. Flat substrates like mica, silicon, or glass work best for fine-size nanomaterials, while metal discs can be used for larger particles.

Various types of chemical adhesives can be used to functionalize surfaces or make them hydrophobic or hydrophilic. The adhesive should be chosen such that the affinity between the substrate and the sample is greater than between the sample and the tip.

Optimal dispersion methodologies also vary within samples and knowing the optimal method requires a lot of experimentation. For example, additives and surfactants can affect particle dispersion during washing and evaporation in particle suspensions.

Optimization for adhesives and timing are some challenges during AFM sample preparation.

When the sample is not properly adhered to the substrate, it can lead to the formation of streaks on the images due to interaction with or adherence to the AFM tip. This requires the use of a more effective adhesive.

When conducting AFM sample preparations on a large scale, it is also important to consider exposure times and dilutions of the sample solutions. Meanwhile, for smaller scales, nanoparticles can clump together or be far apart due to electrostatic and interfacial free energy.

AFM sample preparation is a critical process in AFM analysis because it’s a strong determinant of the quality of results that will be obtained.

Sample preparation generally involves selecting a suitable substrate, activating and binding the sample to the substrate, and finally visualizing. While sample preparation is relatively simple and versatile compared to other imaging techniques like scanning electron microscopy, it is important to optimize for suitable substrates and adhesives to avoid poor results.

Other factors related to the AFM device like the probe used can affect the quality of the results but are beyond the scope of this article. 

Grobelny, J., DelRio, F.W., Pradeep, N., Kim, D.-I., Hackley, V.A., Cook, R.F. (2011). Size Measurement of Nanoparticles Using Atomic Force Microscopy, in: McNeil, S.E. (Ed.), Characterization of Nanoparticles Intended for Drug Delivery, Methods in Molecular Biology. Humana Press, Totowa, NJ, pp.71–82. https://doi.org/10.1007/978-1-60327-198-1_7.

Starostina, N., West, P. (2006). Part II: sample preparation for AFM particle characterization. Probe Microscopy, pp.1–9.

Zhao, W., Song, W., Cheong, L. Z., Wang, D., Li, H., Besenbacher, F., Huang, F., Shen, C. (2019). Beyond imaging: Applications of atomic force microscopy for the study of Lithium-ion batteries. Ultramicroscopy, 204, pp.34–48. https://doi.org/10.1016/j.ultramic.2019.05.004.

Disclaimer: The views expressed here are those of the author expressed in their private capacity and do not necessarily represent the views of AZoM.com Limited T/A AZoNetwork the owner and operator of this website. This disclaimer forms part of the Terms and conditions of use of this website.

Blaise Manga Enuh has primary interests in biotechnology and bio-safety, science communication, and bioinformatics. Being a part of a multidisciplinary team, he has been able to collaborate with people of different cultures, identify important project needs, and work with the team to provide solutions towards the accomplishment of desired targets. Over the years he has been able to develop skills that are transferrable to different positions which have helped his accomplish his work.

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