Atomic Force Microscopy: Revolutionizing the Future of Nano-scale Imaging

In 1683, Anton von Leeuwenhoek improved the primordial microscope and made several observations of bacteria on teeth, worms in the nose, and the epidermis of human skin. Though many were skeptical of the existence of living creatures that are invisible to the naked eye, Leeuwenhoek, who later became known as “the Father of Microbiology,” had brought to light a new world previously unknown to humans, and instilled in them the ardor to perceive the near-invisible (1).

A major breakthrough in the field of imaging was achieved with Gerd Binnig and Heinrich Rohrer’s invention of the scanning tunneling microscope (STM) in 1981. Though the STM could be used in vacuum, liquid, or gaseous media at a range of temperatures, it could only image metallic or semiconducting surfaces (2). These limitations prompted the invention of the atomic force microscope (AFM), also known as the Scanning Probe Microscope, in 1986 by Calvin Quate, Christoph Gerber, and Binnig. AFM, also termed the “Eye of Nanotechnology,” is a high-resolution imaging technique capable of resolving features as small as an atomic lattice. For the first time, researchers were able to observe features at the molecular and atomic scale (3).

The three-dimensional mapping of surfaces by AFM yields true topographic data with vertical resolution down to the subnanometer range. The field of view of the substance being examined can be larger than 125 μm, with a vertical range of 8-10 μm. Like its predecessor STM, AFM can be used to examine any rigid surface in air or immersed in a liquid. Furthermore, 3D AFM images are obtained relatively easily, without any expensive sample preparation, and yield far more quantitative height information in comparison to the 2D profiles generated from cross-sectioned samples. In addition to incorporating a closed chamber for environmental control to maintain the specimen at a constant temperature, the modern AFM can also be mounted on an inverted microscope for simultaneous imaging through advanced optical techniques to facilitate further resolution and magnification of the sample. Above all, since the sample need not be electrically conductive, no destructive artificial conditions such as metallic coating or dehydration of the sample are necessary. AFM is increasingly employed in biomedical applications due to its superior resolution and capacity to image samples under natural conditions of air and water (3,4).

Operation of the AFM

As the name suggests, atomic force microscopy uses force to image a surface. All force microscopes have five essential components: a sharp tip mounted on a cantilever spring, a means of detecting the cantilever’s deflection, a feedback mechanism to control the cantilever deflection, a piezoelectric mechanical scanning system that moves the sample with respect to the tip, and a display system to convert the raw data gathered into an image. The fundamental operation of the AFM rests on the concepts of magnetic and electrostatic forces as well as the atomic interactions between the tip and the sample (5). Thus, charge distributions are easily measured using an AFM because of the electrostatic forces between the charges on the tip and the sample atoms (6).

The AFM, which is made up of very sharp tips, flexible cantilevers, a high resolution tip-sample positioning, and a deflection sensor, very closely resembles a record player. The final image is formed as a result of the force interactions of the tip, mounted at the end of a small cantilever, which is brought in close contact with the surface. The tip-cantilever system is usually made of silicon or silicon nitride, and its accuracy is conveyed by parameters such as the sharpness of the apex, represented by the radius of curvature, and the aspect ratio of the tip. Cantilevers are classified on the basis of their resonance frequencies and spring constants. There are two types of cantilevers that are chosen depending on the properties of the substance to be imaged—low-resonance frequency cantilevers are suitable for imaging in liquid, while high-resonance frequency cantilevers are used for resonance mode in air (4).

The relative motion between the tip and the substance is measured by a laser beam reflected from the backside of the cantilever onto a position-sensitive photodetector. Even a small deflection of the cantilever will tilt the reflected beam, thereby changing the position of the beam on the detector. The difference in two subsequent signals indicates the position of the laser spot on the photodetector and therefore the angular deflection of the cantilever (4).

The electronics in the AFM drive a scanner across every line of the sample. The interactive forces between the tip and the sample are then recorded. The scanner and electronics, along with the sample, cantilever, and optical lever form a feedback loop. This feedback loop keeps the interactive forces constant by tracking the vertical control signals, resulting in a topographic image with x, y, and z coordinates (4).

Modes of Operation

There are two general modes of operation of the AFM based on oscillation of the cantilever close to its resonant frequency. The first case, known as the DC or static mode, records the static deflection of the cantilever. In the second case, known as the AC or resonant mode, the feedback loop sets a definite value for the amplitude of oscillation of the cantilever as it scans the sample (4).

The DC mode, which is often known as the contact mode or the constant force mode, is the most common form of AFM. In this mode, the tip is brought in direct contact with the sample surface while the cantilever deflection is kept constant by the feedback loop as it scans. Based on the applied force and the cantilever spring constant, an image contrast is formed. The adhesion forces, however, deform the tip and the sample so that contact occurs over a finite area. This finite area is greatly influenced by the tip sharpness and is increased by any additional spring force, which in turn increases the image contrast. However, since the tip is dragged across the surface, the sheer force generated could rupture living cells and specimens. As such, the contact mode is not widely used in biomedical imaging. It is commonly used to image in liquids, allowing a significant reduction of capillary forces between the tip and sample, and hence, damage to the surface (7,8).

In the AC or noncontact mode, the cantilever is set in oscillation as the probe is suspended 40 – 50 Å above the surface. Also known as the attractive mode, this form of AFM senses the long-ranged van der Waals attractive forces, whose strength depends on the tip sharpness and the amount of spring deflection as determined by the spring constant. This particular mode works by keeping a constant frequency shift during scanning and by maintaining this frequency with the help of the feedback loop. Though the tip-sample surface interactions are weak compared to those in contact mode, the attractive mode succeeds in providing a good vertical resolution, especially in dry samples (4,9,10).

Prospects of Imaging

Since its advent, AFM has become invaluable in the field of biomedical imaging. It has allowed researchers to perceive, at both spatial and time resolutions down to the atomic-scale, the development of diseases, and the functioning of a healthy body. For instance, AFM can help explain how proteins pathologically misfold in the human body, producing diseases such as Alzheimer’s and Parkinson’s diseases (10). In comparison to other imaging devices, AFM has a much broader application because it can image any conducting or non-conducting surface. AFM is now widely used in several fields of nanoscience and nanotechnology. Not only has AFM provided the ability to view events in real-time at the molecular level and thereby widened our understanding of systems in the fields of biotechnology, electrochemistry, and polymer science, it has also led to several new discoveries of proteins, enzymes, polymers, and drugs (3).

Despite the significant contributions made by the AFM, it still remains a part of a family of scanning probe microscopes that has great potential to grow. Even today, AFM still promises much room for improvement. Ease of use, sample accessibility, and scan speed limitations are concerns now being addressed and improved upon. Furthermore, scanners and tips are constantly being enhanced. The non-contact mode introduced in 1987 enabled even the softest samples to be imaged without causing much physical damage. In 1996, smaller cantilevers were developed to allow for higher resolution and smaller scanning times. Such cantilevers have a higher resonant frequency and a low spring constant. In the late twentieth century, a prototype known as AFM Version 5 was released, combining an optical microscope and an atomic force microscope and using a much smaller laser spot to allow for smaller cantilevers to be used (11). As advancements continue to make the AFM a superior imaging device, engineers, biomedical researchers, and material scientists can now see substances that were invisible to us just years ago (4). With every improvement in its instrumentation, the AFM has increased our understanding of the microscopic world and has blurred the distinction between the visible and invisible.

References

1. L. Anthony, Philosophical Transactions (Vol. 14, 1684), pp. 568-574.
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3. Agilent Technologies, Instruments for Nanotechnology Research: What is an AFM? (2009). Available at http://nano.tm.agilent.com/index.cgi?CONTENT_ID=809&User:LANGUAGE=en-US (28 August 2009).
4. B. Pier Carlo, D. Ricci, Atomic Force Microscopy: Biomedical Methods and Applications. (Humana Press: New York, 2003).
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8. H. L. Jeffrey,  J. Bechhoefer, Calibration of atomic-force microscope tips (1993). Available at http://scitation.aip.org/getpdf/servlet/GetPDFServlet?filetype=pdf&id=RSINAK000064000007001868000001&idtype=cvips&prog=normal (28 August 2009).
9. S. Morita, R. Wiesendanger, E. Meyer, Noncontact Atomic Force Microscopy (Springer: New York, 2002).
10. H. Paul, An Introduction to AFM. Available at http://hansmalab.physics.ucsb.edu/afmhistory.html#history (28 August 2009).
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