How can AFM be used to obtain biological information across a wide range of scales from cell structure and function to single molecule detection?
Atomic Force Microscopy (AFM) is a scanning probe microscopy technique which is commonly used not only for the observation but also for the manipulation of biological macromolecules. This technique is simple and extremely precise (Kensal E. van Holde, 1998). An AFM microscope comprised of a very small and thin tip mounted on a flexible cantilever. The forces between the tip and the sample are very small (<
) and they can be detected through the deflection of the cantilever. There are three main modes of operation in AFM: contact mode (repulsive), non-contact mode (attractive) and tapping or semi-contact mode (Li, Introduction, 1997).
CONTACT MODE (Repulsive)
In contact mode, the small tip comes in contact with the surface of the sample. “The force on the tip is a repulsive force which is set by pressing the cantilever against the sample with a piezoelectric positioning element”(Li, The Common AFM Modes, 1997) . In this mode the deflection of the cantilever is compared to a desired value of deflection. If the deflection is different from this value, the sample is raised or lowered from the cantilever, in order to get the desired value of deflection (Li, The Common AFM Modes, 1997). Some of the advantages of contact mode are:
- “Simplicity (minimum operational skills and basic hardware)”(Sokolov, 2007)
- “High scanning speeds”(Sokolov, 2007)
- “The load force can be controlled”(Sokolov, 2007)
- “Good signal to noise ratio”(Sokolov, 2007)
- “Cheaper mode of operation”(Sokolov, 2007)
Some of the disadvantages of contact mode are:
- “Possible damage of the surface”(Sokolov, 2007)
- “Provides limited amount of information compared to other modes of operation”(Sokolov, 2007)
Figure 1: AFM in Contact Mode (Lecture 7: AFM (Handout))
NON-CONTACT MODE (Attractive)
This mode was developed for instances that the contact of the tip with the sample could lead to a level of alteration of the sample. In non-contact mode, attractive Van der Waals forces are detected between the tip and the sample resulting in topographic images of the surface of the sample (Li, The Common AFM Modes, 1997). In this case, the attractive forces are very weak and the tip is slightly oscillated so “that AC detection methods can be used to measure changes in amplitude, phase or frequency of the oscillating cantilever”(Li, The Common AFM Modes, 1997). Some advantages of the non-contact mode are:
- “The ability to detect long-range forces”(Sokolov, 2007)
- “Information about surface elastic and viscoelastic properties are provided”(Sokolov, 2007)
However there are also some disadvantages such as:
- “No topographical information”(Sokolov, 2007)
- “Not suitable for biological samples as it requires a really clean and homogenous surface”(Sokolov, 2007)
Figure 2: AFM in Non-Contact Mode (Lecture 7: AFM (Handout))
TAPPING or SEMI-CONTACT MODE
The most commonly used mode of operation for biological samples is the tapping or semi-contact mode (Kensal E. van Holde, 1998). In this particular mode, the cantilever is oscillated so that the tip can come periodically in contact with the surface of the sample (Kensal E. van Holde, 1998). The surface is scanned with a steady rate with the help of a piezoelectric guide (Kensal E. van Holde, 1998). During the aforementioned process, a laser beam is reflected from the cantilever to a sensor (Kensal E. van Holde, 1998). As the tip is scanning the surface and comes across a molecule, there is an alteration of the height (Kensal E. van Holde, 1998). This alteration is detected and amplified (Kensal E. van Holde, 1998). Due to this amplification, distances in the order of 1 nm or less can be measured (Kensal E. van Holde, 1998). This mode of operation presents a lot of advantages such as:
- “No artifacts due to surface scratching”(Sokolov, 2007)
- “No damage of the surface of the sample”(Sokolov, 2007)
- “Collection of a wide range of information”(Sokolov, 2007)
There are also some disadvantages such as:
- “Demanding mode which requires operational skills and hardware”(Sokolov, 2007)
- “The load force cannot be controlled to the desired point of precision”(Sokolov, 2007)
- “Slow scanning speeds”(Sokolov, 2007)
It should also be toted that in this mode the phase signal is also acquired. “The phase signal is the difference between the applied and the real oscillation” (Lecture 7: AFM (Handout)). This signal can provide more information about the surface of the sample under examination.
Figure 3: AFM in Tapping or Semi-Contact Mode (Lecture 7: AFM (Handout))
Figure 4: Basic schematic of AFM (Meyer, 1992)
Figure 5: Forces in all of the Modes of Operation of AFM (Lecture 7: AFM (Handout))
APPLICATIONS OF ATOMIC FORCE MICROSCOPY
Atomic Force Microscopy can be found in many areas in research. Some examples are “inorganics, polymers, coatings, the topography and nanomechanical properties of coatings, the removal of nanopartical from a specific surface” and of course biological samples (Introduction to AFM-Atomic Force Microscopy, 2012).
As far as the cells are concerned, Atomic Force Microscopy can provide information about the “mechanical and viscoelastic properties of the cell as well as cell adhesion and rheological properties” (Surena Vahabi, 2013). This technique can also provide information about DNA. In particular, it can provide information for the way single RNA polymerase molecules are behaving on DNA after the process of transcription (Kensal E. van Holde, 1998). It should be noted that for the examination of living cells it is common practice that the AFM tip is activated biologically by the attachment of biological macromolecules or chemical compounds (Ch. Lioutas, 2009). This activation enables the information acquisition for several biological and chemical reactions in cellular level (Ch. Lioutas, 2009).
Figure 6: Studying living cells using AFM (Ch. Lioutas, 2009)
There is also application of this technique in the pharmacology industry and the medical industry. One of the main examples of the application in these industries is the characterization of the microbial surfaces and the quantitative study of the molecular interactions (Surena Vahabi, 2013). The high resolution provided by AFM can provide information about the “molecular forces and the physical properties on the microbial surface” (Surena Vahabi, 2013). Cardiology is another domain where AFM can be of great assistance as a detection and measurement tool. The “measurement of the stiffness of cardiac myocytes and the scanning of the renal epithelium” in order to measure property changes can help for an early detection of various diseases (Surena Vahabi, 2013).
ADVANTAGES OF ATOMIC FORCE MICROSCOPY IN BIOLOGICAL AND BIOMEDICAL SCIENCES
It is common knowledge among the scientific community that AFM is an invaluable tool in research. The biggest advantage of AFM is the fact that it provides the ability to study biological structures and samples in general, in their natural environment (Kensal E. van Holde, 1998). This means, studying a biological sample in an aqueous solution or a buffer solution (Kensal E. van Holde, 1998). Furthermore there is no need for the staining of the sample, a process which can be harmful for a biological sample (Kensal E. van Holde, 1998). The samples also do not need further stabilization. For example a specimen can be covered by a solution and placed on a surface in order to be studied (Kensal E. van Holde, 1998). This specific configuration of the biological sample allows the examination of the effect various solvents can have on biomolecules and provided that the sensor remains over a specific macromolecule, shifts in the macromolecule’s structure can be observed (Kensal E. van Holde, 1998). Alternatively, images from successive AFM scans can be used for the observation of dynamic processes such as the trajectory of RNA polymerase on single DNA molecules, a process mentioned above (Kensal E. van Holde, 1998).
To conclude, it is obvious that AFM is a valuable microscopy technique that can provide a plethora of information. It can be used to examine a wide range of materials and the fact that AFM can scan biological samples with great precision in their natural environment gives it an edge compared to other microscopy techniques.
- Ch. Lioutas, N. F. (2009). Structure Errors and Microscopy Techniques. Thessaloniki.
- Introduction to AFM-Atomic Force Microscopy. (2012). (National Physical Laboratory)
- Kensal E. van Holde, W. C. (1998). Principles of Physical Biochemistry. Upper Saddle River: Pearson Prentice Hall, Pearson Education Inc-For the translated greek version EMBPYO Publications (Athens, 2010).
- Lecture 7: AFM (Handout). (n.d.).
- Li, H.-Q. (1997). Introduction.
- Li, H.-Q. (1997). The Common AFM Modes.
- Meyer, E. (1992). Atomic Force Microscopy. Progress in Surface Science, Vol.41.
- Sokolov, I. (2007). Atomic Force Microscopy in Cancer Cell Research. In Cancer Nanotechnology. American Scientific Publishers’ Inc.
- Surena Vahabi, B. N. (2013). Atomic Force Microscopy application in biological research: A review study. Iranian journal of medical sciences Vol.38(2).