Atomic Force Microscopy (AFM) - A key tool for nanotechnology

(Nanowerk Spotlight) Whenever you read an article about nano this or nano that, chances are you come across a large number of confusing three-letter acronyms – AFM, SFM, SEM, TEM, SPM, FIB, CNT and so on. It seems scientists earn extra kudos when they come up with a new three-letter combination. One of the most important acronyms in nanotechnology is AFM – Atomic Force Microscopy. This instrument has become the most widely used tool for imaging, measuring and manipulating matter at the nanoscale and in turn has inspired a variety of other scanning probe techniques.
Originally the AFM was used to image the topography of surfaces, but by modifying the tip it is possible to measure other quantities (for example, electric and magnetic properties, chemical potentials, friction and so on), and also to perform various types of spectroscopy and analysis. So far, over 20,000 AFM-related papers have been published; over 500 patents were issued related to various forms of scanning probe microscopes (SPM); several dozen companies are involved in manufacturing SPM and related instruments, with an annual worldwide turnover of $250–300 million, and approx. 10,000 commercial systems sold (not counting a significant number of home-built systems).
The term microscope in the name is actually a misnomer because it implies looking, while in fact the information is gathered by feeling the surface with a mechanical probe. Unlike other imaging techniques like X-ray diffraction, AFM does not use lenses, which means its resolution is not limited by the diffraction limit. The operation principle of an AFM is based on three key elements:
1) an atomically sharp tip (the "probe"), placed at the end of a flexible cantilever beam, that is brought into physical contact with the surface of a sample. The cantilever beam deflects in proportion to the force of interaction;
2) a piezoelectric transducer to facilitate positioning and scanning the probe in three dimensions over the sample with very precise movements; and
3) a feedback system to detect the interaction of the probe with the sample.
The AFM has inspired a variety of other scanning probe techniques
The AFM (center) has inspired a variety of other scanning probe techniques. Originally the AFM was used to image the topography of surfaces, but by modifying the tip it is possible to measure other quantities (for example, electric and magnetic properties, chemical potentials, friction and so on), and also to perform various types of spectroscopy and analysis. (Image: Christoph Gerber; copyright Nature Publishing Group)
To put the AFM in its context: The reason why nanosciences and nanotechnologies have taken off with such amazing force over the past 20 years is because our ongoing quest for miniaturization has resulted in tools such as the AFM (invented in 1986) or its precursor, the scanning tunneling microscope (STM; invented in 1982). Combined with refined processes such as electron beam lithography, this allowed scientists to deliberately manipulate and manufacture nanostructures, something that wasn't possible before.
This principle has been the basis for one of the most important nanoscience tools and allowed the visualization of nanoscale objects where conventional optics cannot resolve them due to the wave nature of light.

What is atomic force microscopy?

Atomic force microscopy (AFM) is a type of scanning probe microscopy (SPM) that is used to measure the topography, mechanical, and electrical properties of a sample at a nanometer scale. The AFM technique is based on the interaction between a sharp tip and the surface of a sample. The tip is attached to a cantilever, which is deflected by the forces between the tip and the surface. The deflection of the cantilever is measured using a laser and a position-sensitive photodetector.
The AFM tip is typically made of silicon, and it has a radius of curvature in the order of nanometers. The tip is brought into contact with the sample surface or placed in very close proximity to the surface. The interaction between the tip and the surface can be attractive or repulsive, depending on the nature of the forces involved. The tip-sample interaction can be due to van der Waals forces, electrostatic forces, or chemical interactions.
In contact mode AFM, the tip is scanned across the surface of the sample in a raster pattern, while maintaining constant contact with the surface. The deflection of the cantilever is kept constant by adjusting the height of the tip above the sample surface. The tip-sample interaction forces cause the cantilever to bend, and the bending is measured by the position-sensitive photodetector. The deflection signal is used to create an image of the surface topography of the sample.
In non-contact mode AFM, the tip is oscillated close to the surface of the sample at a frequency near the resonance frequency of the cantilever. The amplitude of the oscillation is maintained constant, while the tip is scanned across the sample surface. The tip-sample interaction causes a change in the resonance frequency of the cantilever, which is detected by the laser and photodetector system. The change in the resonance frequency is used to create an image of the surface topography of the sample.
AFM can also be used to measure other properties of the sample, such as its mechanical and electrical properties. In force spectroscopy mode, the tip is used to apply a known force to the sample surface, and the deflection of the cantilever is measured. The deflection signal is used to determine the mechanical properties of the sample, such as its elasticity and stiffness. In electric force microscopy mode, the tip is used to measure the electrical properties of the sample, such as its dielectric constant and surface potential.
Overall, AFM is a powerful technique that allows for the measurement of nanoscale features and properties of samples with high resolution and sensitivity. It has many applications in various fields, such as materials science, biology, and nanotechnology.

High-speed atomic force microscopy

Traditional atomic force microscopy works by using a very small tip to scan the surface of a sample. This tip is attached to a small cantilever that follows the sample's vertical profile, creating a force on the cantilever that can be measured. By relating the force to the position of the tip on the sample, a height map can be generated that provides structural information about the sample.
In high-speed AFM, the cantilever is made to oscillate near its resonance frequency, and variations in the amplitude or frequency of the cantilever's oscillation due to the tip's interaction with the sample's surface are recorded. This information provides a measure for the local "z" value, or the height of the sample at that location.
High-speed AFM generates a video of the sample, with the time interval between frames determined by the speed of the image generation process. Researchers at Kanazawa University have developed HS-AFM further so that it can be used to study biochemical molecules and biomolecular processes in real-time. They used the method to study the molecular dynamics of a Cas9-DNA interaction process, which is relevant for ongoing research on the CRISPR-Cas9 genome editing tool.
A recently published article in the Encyclopedia of Life Sciences, written by Martijn de Jager and John van Noort, both from the University of Leiden in The Netherlands, gives an excellent overview of Atomic Force Microscopy and its applications in life sciences. Below we are summarizing some of the key information from this article.
The AFM can be operated in a number of modes, depending on the application but four modes are most commonly used for AFM imaging: contact mode (or constant height mode), where the deflection of the cantilever is directly used as a measure for the height of the tip and the normal force applied to the sample scales directly with its height. In constant force mode, the normal force the cantilever deflection under scanning reflects repulsive forces acting upon the tip, and at sufficiently small scanning velocities the force feedback can reduce the normal force. Tapping mode (or noncontact mode), where the tip is vibrated (oscillating at its resonance frequency) perpendicular to the specimen plane to avoid gouging the specimen as the tip is scanned laterally and the lateral forces are reduced.
In a fourth mode of scanning, the force–distance mode, the tip is brought to the sample at frequencies far below the resonance frequency of the cantilever while at the same time the deflection is recorded. This allows one to measure the local interaction as a function of the tip-sample distance.
As de Jager and van Noort write in their article, large numbers of various biological samples, including cells, cell compartments and biomolecules, have been studied with AFM. "In some of these studies, AFM is used as a plain imaging tool to investigate the topography of immobilized and/or fixed samples, complementing existing methods such as electron microscopy, with the advantage that sample preparation is generally more straightforward.
For other experiments, the use of AFM is a prerequisite to look at nonfixed materials and even their dynamics in aqueous environment. Besides its imaging capabilities AFM is becoming increasingly important as a nanomanipulation tool. The single-molecule analysis of interaction forces, elasticity and tertiary protein structure in intact biological materials is uniquely possible using AFM."
Introducing this vast body of research is beyond the scope of any article. Let's just take a look at two examples illustrated in the paper:
Imaging cells
"AFM imaging of living cells provides a direct measurement of cell morphology with nanometer resolution in three dimensions. Because of its noninvasive nature and the absence of fixation and staining, even dynamic processes like exocytosis, infection by virus particles and budding of enveloped viruses have been successfully visualized in successive scans. Owing to the high elasticity of the cell membrane, the tip can deeply indent the cell without disrupting the membrane. Making use of this effect, even submembraneous structures such as cytoskeletal elements or organelles like transport vesicles can be revealed. However, due to the elasticity of the cell the contact area between the tip and the sample increases with increasing applied force. The elastic modulus of living cells varies between 10 and 100 kPa, which results in a tip sample contact area of 50–100nm in the softest region of the cell. Therefore, the (sub-) nanometer resolution that is routinely achieved on more rigid samples cannot be achieved on membranes of intact cells."
Structure, function and interaction of single DNA and protein molecules
"Besides the analysis of cells and cell membranes, AFM-based methods to study purified single molecules like proteins, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) have developed rapidly in the past decade. Unique details on the mechanism and function of DNA- and RNA metabolizing proteins can directly be obtained by quantification of the number, position, volume and shape of protein molecules on their substrate. Like other single molecule techniques all individual instances of the entire population of structures are revealed, also showing rare but important species. Further insights in the mechanism of a reaction can be obtained from image analysis by measuring parameters such as protein-induced DNA bending, wrapping and looping. Besides topography imaging, force spectroscopy has been successful in unraveling tertiary structure in proteins, RNA and other polymers."
Although it already is an essential tool for structural analysis and manipulation of complex macromolecules and living cells, it is to be expected that AFM-based applications will be further extended in the future. Technical developments will advance the AFM system itself, by improvement of resolution, image rate, sensitivity and functionality. A combination with complementary techniques will fill in some limitations of AFM.
To fully exploit the potential of AFM to study functional biomolecules and their interactions, de Jager and van Noort say that video microscopy would be needed to capture dynamic events. "Currently, the scan rate is limited by the mechanical response of the cantilever and the piezo. Smaller cantilevers will result in higher resonance frequencies, allowing faster scanning rates. By reducing the size of the cantilevers one order of magnitude, the frame rate can be reduced from typically a minute down to video rate, allowing the study of a significantly larger range of biomolecular processes."
The two researchers expect the most important developments for the tip itself. "Image resolution in all modes is dependent on tip geometry. The reduction of tip size, increase of its aspect ratio and its resistance to wear as a result of scanning will have a considerable impact on all AFM applications."
For instance, researchers at Harvard and Stanford universities have developed a specially designed AFM cantilever tip, the torsional harmonic cantilever (THC), which offers orders of magnitude improvements in temporal resolution, spatial resolution, indentation and mechanical loading compared to conventional tools. With high operating speed, increased force sensitivity and excellent lateral resolution, this tool facilitates practical mapping of nanomechanical properties.
We'll leave you with this amazing image:
golden pyramid - tip of an atomic force microscope
Developing new instruments to be able to "see" at the nanoscale is a research field in itself. Shown here is the tip of an atomic force microscope (AFM), one of the foremost tools for imaging, measuring and manipulating matter at the nanoscale. Here, a platinum electrode measuring one hundredth of a nanometer has been deposited on the tip of this pyramid shaped AFM tip via focused ion beam (FIB) deposition. (Image: C. Menozzi, G.C. Gazzadi, S3 (INFM-CNR), Modena. Artwork: Lucia Covi)
More beautiful images made with scanning probe microscopes are here.
Michael Berger By – Michael is author of three books by the Royal Society of Chemistry:
Nano-Society: Pushing the Boundaries of Technology,
Nanotechnology: The Future is Tiny and
Nanoengineering: The Skills and Tools Making Technology Invisible
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