Updated by Ibtisam Abbasi on 04/05/22
Atomic Force Microscopy (AFM) is a type of high-resolution scanning probe microscopy that allows for imaging, manipulation, and force measurement. Atomic Force Microscopy was first developed in 1986 as a way of overcoming the drawbacks of scanning tunneling microscopy (STM). It is used in a variety of different industries, including solid-state physics, semiconductor science, molecular engineering, polymer and surface chemistry, molecular biology, cell biology, and medicine.
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How it Works
Scanning probe microscopy scans the surface of samples with a probe to measure fine surface shapes and properties and generate an image. Scanning probe microscopy measures properties such as height, friction, and magnetism. The main types of scanning probe microscopy include atomic force microscopy, scanning tunneling microscopy (STM), and near-field scanning optical microscopes (MSOM).
AFM has a fine silicon or silicon nitride probe which is attached to a cantilever. When the probe moves across the sample surface, it measures the surface morphology on the atomic scale. The force between the tip and the sample is measured during scanning by monitoring the deflection of the cantilever. The tip can be modified in various ways to investigate different surface properties
AFM applications include biochemical imaging of molecules, cells, and tissues, chemistry, materials science, nanotechnology, and physical and biophysical applications such as measuring forces between the AFM tip and the sample surface.
AFM has progressed over the years with many novel techniques and routes performed to optimize it for specific functions.
Friction Determination
Frictional determination by AFM in the past decade has mainly been focused on biochemical materials at the nano scale. AFM has developed from the determination of frictional morphology to several mechanical parameters of friction value and process. Two aspects that determine dynamic and static properties can be achieved by multi-AFM modes. Frictional morphology, friction curve, and friction motion processes can all be measured using AFM.
AFM-based Nanoindentation
AFM nanoindentation allows for the control of microscopic penetration depth and stress, as well as an increased contact region. An article published by Ilya A. Morozov in the journal Microscopy Research and Technique indicates that this particular AFM technique is viable for obtaining tensile properties.
It is carried out by pressing a rigid tip of a specified size and shape into the specimen for a brief period of time. As the tip penetrates the specimen, the pressure mounts until it reaches the input value initially specified by the user. Once this figure is attained, the stress is either withdrawn or maintained at a steady level. Nanoindentation generates a set of indentation load-displacement (P-h) graphs from which toughness and Young's modulus are calculated. This technique is vastly employed in interface elevation topological modeling and 3D high-resolution photography, most notably in enabling in-situ assessment of the form of residual markings.
Localization Atomic Force Microscopy
Heath et al. have suggested a novel localization AFM (LAFM) methodology in their research published in the journal Nature, to circumvent the traditional design and operational limitations of AFM by employing a post-probing optimization and image-resolution enhancement technique.
The LAFM method finds characteristics that are concealed in conceptual and ordinary topographies, according to simulations. Within 10–100 pictures, the LAFM methodology surpassed typical approaches, with the highest gain in sharpness (approximately 1/5) for tip radii higher than the divergence of structural components. These investigations confirmed that the accuracy of the LAFM mapping improves as the number of data points grew until it reaches a plateau.
High-Speed Atomic Force Microscopy
High-speed AFM (HS-AFM) is a microscopy tool that was developed to overcome the current limitations of structural biology and light-based single-molecule biophysics. HS-AFM allows for the simultaneous assessment of the structure and dynamics of single protein molecules whilst in action at high spatiotemporal resolution.
HS-AFM has been successfully applied to a variety of proteins, including motor proteins, membrane proteins, antibodies, enzymes, and intrinsically disordered proteins.
Pulse-Atomic Force Microscopy
A team has recently explained a novel pulse-atomic force lithography technique in their article published in the journal Nanomaterials aimed at the synthesis of nanostructures that have varying depths. Tip-Based Nanofabrication (TBN) is a fast-growing top-down microfabrication option due to its versatility.
Mechanical TBN (m-TBN) is one of the most successful options, with Atomic Force Microscopy (AFM)-based m-TBN being the most intriguing owing to its great adaptability. It is feasible to generate 2.5D nano grooves with specified thickness levels (constant or variable), heights, and inclinations in a single process and without the use of supplementary power sources using the Pulse Atomic Force Lithography (P-AFL) process.
The AFM tip is placed in direct contact with the specimen surface in CP-AFL, and a constant-amplitude voltage pulse is delivered to the piezo-scanner. The tip distorts the substrate, producing a groove of the required depth on the nanometer scale.
Automated 3D AFM for Sidewall Roughness Analysis
Sang-Jhoo Cho et. al. has suggested sophisticated 3D metrology equipment, the 3-Dimensional Atomic Force Microscope (3D-AFM), which is used to assess the sidewall roughness (SWR) of lateral and undercutting components. Unlike traditional AFMs, the researchers' 3D-AFMs made use of a turnable Z detector.
To correctly capture sidewall characteristics, the Z scanner may be angled left or right. Measurements using 3D-AFM may be done at a deeper angle than in prior investigations, with high repeatability of less than 2%. Because this 3D-AFM is entirely automated, the study revealed a new option for industrial-level 3D-AFM use.
Nanoendoscopy-AFM
A needle-like probe is placed into a live cell, which allows rapid and direct contact with the internal anatomy of the cell enabling the comprehension of nanostructural orientation within the cell. This innovation in AFM techniques transcends the constraints of cell imaging by providing benefits such as improved resolution, microstructural modeling, and molecular identification, which broadens the spectrum of intracellular structures visible within live cells.
In short, much progress is being made in the field of AFM which paves the way for rapid imaging and analysis.
Biological Applications
AFM has become a standard technique for surface imaging, but it is now also increasingly used in biological research to look at the properties of living cells. AFM is used to look at biological issues such as the characterization of organelles, DNA-protein interactions, cell adhesion forces, and electromechanical properties of living cells can all be measured by AFM.
Changes in the mechanical properties of the cell membrane such as cell stiffness and viscoelasticity can be measured by AFM, as well as AFM having the ability to assess cell adhesion and the rheological properties of cells. Samples can be directly analyzed in their natural environment without the need for any sample preparation, saving a lot of research time.
Cancer Research
Recent advances in AFM have enabled it to be used in cancer research and diagnosis.
The physicochemical properties of live cells change when their physiological conditions are altered. When cells undergo the process of carcinogenesis from external stimuli, their morphology, elasticity, and adhesion properties change. AFM surface imaging and ultrastructural observation of live cells can be done with atomic resolution under near-physiological conditions, collecting force spectroscopy information which allows for the study of the mechanical properties of cells.
AFM can detect the changes and differences between single cancerous and non-cancerous cells, which allows for early diagnosis and treatment of cancer. AFM can also study the structure and function of cancerous cells by looking at the mechanisms involved in their spreading, the functions of anti-cancer drugs, and the interaction processes between cells.
Pharmacological Applications
The ability of AFM to scan the interaction between lipid bi-layers and drugs is a major advantage of AFM that can be used in the pharmaceutical industry.
AFM can test the interactions of drugs with receptors as well as test the contact of the drug candidate with target cell membranes. Due to the non-destructive nature of AFM, it can be used to look at soft systems under controlled environmental conditions, which then serves as a unique foundation for the in vitro development of novel drug delivery systems. Enzyme hydrolysis visualization can also be achieved by the phase imaging mode of the AFM.
AFM is used for the thorough qualitative and quantitative evaluation of pharmaceutical formulations. Surface properties can affect the final formulation characteristics and so require analysis. AFM is currently the only available technique that is capable of measuring interactions in the pN range.
Oscillatory AFM techniques
Oscillatory AFM techniques such as amplitude and frequency modulation modes have allowed for a further expansion of the applications of AFM. The destructive lateral forces in the contact mode are practically eliminated, and AFM applications have been expanded to a broad range of soft biological samples and polymers.
Atomic Force Microscopy Global Market
According to the Statistics Marketing Research Council, the Global Atomic Force Microscopes Market is expected to grow at a CAGR of 6.8% annually until 2026. The increased need for high-resolution microscopy and the use of AFM in biology and medicine is thought to be a driving factor for market growth.
References and Further Reading
Pellegrino P. et. al. 2022. Pulse-Atomic Force Lithography: A Powerful Nanofabrication Technique to Fabricate Constant and Varying-Depth Nanostructures. Nanomaterials. 12(6). 991. Available at: https://www.mdpi.com/2079-4991/12/6/991
Yoo, SB., Yun, SH., Jo, AJ. et al. 2022. Automated measurement and analysis of sidewall roughness using three-dimensional atomic force microscopy. Appl. Microsc. 52(1). Available at: https://appmicro.springeropen.com/articles/10.1186/s42649-022-00070-5
Heath, G.R., Kots, E., Robertson, J.L. et al. 2021. Localization atomic force microscopy. Nature 594, 385–390. Available at: https://www.nature.com/articles/s41586-021-03551-x
Morozov, I. A. 2021. Atomic force microscopy nanoindentation kinetics and subsurface visualization of soft inhomogeneous polymer. Microscopy Research and Technique. 84(9). 1959-1966. Available at: https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/jemt.23751
Khan, Marzia. 2022. Nanoendoscopy-AFM: An Overview. AZoNano, viewed 27 March 2022, Available at: https://www.azonano.com/article.aspx?ArticleID=600
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