Since the advent of scanning probe microscopy (SPM) with the invention of the scanning tunneling microscope in the 1980s, scientists have had an alternative to scanning electron microscopy (SEM) to image target samples.
Image Credit: Ralf Maassen (DTEurope) / Shutterstock.com
The need for non-destructive methods when studying structures at the atomic level drove the development of SPM. Whereas SEM requires a pure vacuum (rendering a sample inert) and needs complex sample preparation, SPM does not.
One common variant of SPM is atomic force microscopy (AFM).
What is AFM?
Atomic force microscopy (AFM) builds images of nuclear structures by raster scanning the target sample along an x-y grid and reading the differences in height to build a 3D image. Like other types of SPM, it differs from SEM, the other common type of microscopy that can be used to analyze samples at the atomic level.
SEM uses a focused beam of electrons in a pure vacuum to build three-dimensional images of atomic surfaces, which can have its drawbacks.
AFM operates by using a cantilever with a fine, sharp point. Similar to a vinyl record needle, though instead of sound, readable data is produced that can be converted to a three-dimensional image of the target molecule. The interaction between the cantilever tip and the sample results in changes in the height of the cantilever.
The action of the cantilever is converted into an electrical signal, which produces peaks and troughs relevant to surface features. A laser can also be used to accurately measure changes in the height of the cantilever during the scanning process. This produces precise mapping of nuclear structures.
During the x-y scan, AFM signals (including sample height and cantilever deflection) are recorded on a computer. These are then plotted to create a pseudocolor image.
Various methods of detection can be used in AFM, including optical levers, interferometry, and the piezoelectric method. In order to control the interaction between the tip and the sample, a simple electrical feedback loop is commonly employed, which keeps the probe-sample force constant and returns the detector to a user-defined point (the setpoint.) This achieves measurements to within a small degree of error.
There are two main modes commonly used in AFM to create images. Each mode is suitable for different applications, but they do have their drawbacks. These are:
- Contact mode – the sample is scanned in a rectangular manner while checking the shift in cantilever deflection. A curve is generated from the lateral force applied to the sample that is easy to interpret. However, this constant lateral force exerted on the sample can lead to issues. As this can be quite high, sample damage or the movement of loosely attached objects can occur.
- “Tapping” mode – images are produced by gently “tapping” the cantilever rapidly on the surface of the target during scanning. This negates the lateral force on the sample, avoiding damage. By utilizing amplitude modulation detection with a lock-in amplifier, the curve is constructed by adding short-range repulsive and long-range attractive forces. However, the feedback is inherently unstable, meaning that the process is difficult to automate.
Applications of AFM
Atomic force microscopy has been used for diverse applications in a number of fields. One of the most significant discoveries made with AFM was of the porosome in 1997, which is a universal secretory body on the cell membrane of eukaryotic cells. AFM can be used in dynamic studies of live cells and their interaction at the atomic level, which gives a distinct advantage over SEM in biomedical research (though the two methods are often employed in tandem.)
Other applications include uses in the integration of nanotechnology with biological research. Pyrgiotakis et al. attached engineered CeO2 and Fe2O3 nanoparticles to the AFM tip to study their interaction with target cells. This has implications in drug delivery using nanotechnology.
There are many potential uses for this cutting-edge technology, with variants including Scanning gate microscopy (SGM), Non-contact AFM and AFM-IR (atomic force microscope infrared-spectroscopy having been developed. It is being employed more widely in scientific studies and represents a technology that is showing exciting potential for future research in a variety of fields.
- Anderson, L.L (2007) Discovery of the ‘porosome’ the universal secretory machinery in cells Journal of Cell and Molecular Medicine Vol 10. Issue 1 pgs. 126-131 https://doi.org/10.1111/j.1582-4934.2006.tb00294.x
- Van Helleputte, H.R.J.R et al. (1995) Comparative study of 3D measurement techniques (SPM, SEM, TEM) for submicron structures Microelectric Engineering Vol. 27 Issues 1-4 pgs. 547-550 https://doi.org/10.1016/0167-9317(94)00164-P
- Pyrgiotakis, G et al. (2014) Real-Time Nanoparticle–Cell Interactions in Physiological Media by Atomic Force Microscopy ACS Sustain Chem Eng. Vol. 2 Issue 7 pgs. 1681–1690 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105194/
- Meyer, G, and Nabil, M.A (1988) Novel optical approach to atomic force microscopy, Appl. Phys. Lett. Issue 53, pg. 1045 https://doi.org/10.1063/1.100061