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Posted: Mar 10, 2008
A key tool for nanotechnology: Atomic Force Microscopy
(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. Today we take a look at one of the instruments that has it all made possible. 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 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.
These engineered nanomaterials, either by way of a top-down or bottom-up approach (in the former, a bulk material is reduced in size to nanoscale particles; in the latter larger structures are built or grown atom by atom or molecule by molecule), go beyond just a further step in miniaturization. They have broken a physical barrier beyond, or rather: below, which the standard laws of physics are replaced by what is called "quantum effects". Any material reduced to the nanoscale can suddenly show very different properties than to what it shows on a macro- and larger scale. For instance, opaque substances become transparent (copper); inert materials become catalysts (platinum); stable materials turn combustible (aluminum); solids turn into liquids at room temperature (gold); insulators become conductors (silicon).
A second important aspect of the nanoscale is that the smaller nanoparticles get the larger their relative surface area becomes. The larger the relative surface area, the more reactive a particle becomes with regard to other substances. The fascination with nanotechnology stems from these unique quantum and surface phenomena that matter exhibits at the nanoscale, enabling novel applications and interesting materials.
But without the AFM, all this wouldn't be happening.
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. 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.
Scanning across the surface, the sharp tip follows the bumps and grooves formed by the atoms on the surface. By monitoring the deflections of the flexible cantilever beam one can generate a topography of the surface.
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.
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:
"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:
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)