Atomic+Force+Microscopy+(AFM)

 In the scientific world, “seeing is believing.” Scientists urge to see things from large scales to very small scale such as telescope (invented by Galileo) or light optical microscope (invented by Hooke). However, it has been a challenge to study surfaces in atomic or molecular scales, of which behavior was found to be like “reactive” liquid phase. Not until Gerd Binnig and Heinrich Rohrer, in 1982, invented scanning tunneling microscopic (STM) instrument which earned them a Nobel Prize in 1986. Later, two scientists invented a better instrument, atomic force microscopy (AFM). Both instruments are the powerful tools for obtaining information on the packing order of molecular adsorption on a surface, and molecule-molecule and molecule-substrate interactions, as well as the types of forces responsible for the packing order at the surface. Atomic force microscopy can work on different surfaces even including fluid or conductive surfaces. The resolution of displacement and force sensing can be up to 0.1 Å and 0.1 pN, respectively (**1)**. It is a “promising tool for studying the mechanical properties of surfaces (hardness, adhesion, friction, and wear at the atomic scale)” (**2**). Figure 1 is a simple schematic of AFM, which includes a cantilever, a sample-loading xyz piezo, and feed-back system. The cantilever-tips have radius between 10 to 100 nm (**1**), and are commonly made from monolithic silicon (**3**).  One of the advantages of using AFM is to be able to collect samples in most media without having to damage the structures of the molecules. Hence, it is a great tool to study biological material such as DNA, chromatin, protein-enzyme interactions, membrane viruses, and so on (**3**). These applications will be elaborated in few examples below. The AFM technique was used in study of Alzheimer’s disease (**5**). β-Amyloid (Aβ) is a primary factor that causes the disease. A β is a short unfolded protein that causes the accumulation of different neurotoxic morphologies. This causes neural loss in Alzheimer patience (5). In this study, Liu et al. used AFM to determine the binding affinity of a therapeutical antibody, single chain variable fragment (scFv’s) to a specific region of Aβ. During the study, a scFv’s H1v2 was selected as its strong binding affinity to Aβ40 (a derivative of Aβ), and AFM was used to study this interaction of these two molecules. The AFM confirmed the Aβ/H1v2 mixture inhibited aggregation of Aβ 40 (**5**). Dague et al. (2010) studied in vitro interactions between Lactococcus lactis and Mucins using AFM, and the study was the first in using to study adhesion force to pig gastric mucin (PCM) (**6**). Mucins were found to be “overexpressed” in cancer cells and to be the target for cancer diagnosis. In addition, pathogens with high adhesion ability affected greatly on the host, and the effect was lessened with low adhesion. Moreover, adhesion was needed for bacteria such as lactic acid bacteria (LAB) to survive and growth. A model of LAB (Lactococcus lactis) was used in this study. Dague claimed that the direct adhesion forces of bacteria to mucins was still poorly understood (**6**). Since AFM works under the Hooke’s law, F = Kx, (**1**), Dague et al. were able to investigate the adhesion force to PCM by complimentarily attached mucus cells and LAB colony. By setting the AFM instrument at a constant parameters such as force variant (F), spring constant of the calivier (K), and distance (x) (Hooke’s law), the researchers were able to observe and collect quantitative data for the force between LAB and PCM(**6**). This is a cutting edge investigation that provides a “powerful framework” (**6**) for interactions between two microbial mechanism.  As in **Fig. 1**, the xyz piezo enables AFM to obtain topographic maps of the sample in 3-dimension (**1**). Fuentes- Perez et al. used AFM volumetric methods to characterize proteins and nucleic acid (**7**). The method involved the use of fiducial markers co-adsorbed with the protein and single-stranded DNA to minimize any artifacts produced during the measurement. The instrumentation was the AFM from Nanotec, and two types of tips were Pointprobeplus PPP-NCH tips and ACT-SS ultrasharp dynamic mode AFM probes. Researchers were able to obtain topographic maps of single nucleic acids and ssDNA as well. Technique can help to provide better images of DNA’s intermediate forms during DNA recombination, replication, and even DNA repair (**7)**.  Similarly, Chammas et al. used AFM to observe PCR products of DNA (**8**). In this study, homo-polynucleotide ssDNA loops of 20 base-pairs of each of the four bases (A, T, G,C) were used to serve as labelling template. The labelling templates were added to linear double-stranded DNA, and the complex was amplified with PCR. The products were mixed with imaging buffer on the 1x1 µm2 mica plate. The topographic maps in good resolution in 2-dimension and 3-D were obtained by the AFM. Researchers were able to identify individual strand by looking at the nucleic acid loops, which were clearly shown on the topographic maps. This study suggests the use of nucleic acid loops as a marker to investigate various DNA-protein interactions using AFM and other spectroscopic techniques (**8**).
 * [[image:AFM schematic diagram.JPG width="372" height="375"]] ||
 * **Figure 1 **: Schematic of atomic force microscopy (**4**). ||

3) Skoog, D.A., Holler, F.J., Crouch, S.R. (2007).//Instrumental Analysis//.pp 443-466. Stamford, CT: Cengage Learning.
====4) Noy, A., & Friddle, R. W. (2013). Practical single molecule force spectroscopy: how to determine fundamental thermodynamic parameters of intermolecular bonds with an atomic force microscope. //Methods (San Diego, Calif.)//, //60//(2), 142–50. doi:10.1016/j.ymeth.2013.03.014==== ====5) Liu, R., Yuan, B., Emadi, S., Zameer, A., Schulz, P., McAllister, C., … Sierks, M. R. (2004). Single chain variable fragments against beta-amyloid (Abeta) can inhibit Abeta aggregation and prevent abeta-induced neurotoxicity. //Biochemistry//, //43//(22), 6959–67. doi:10.1021/bi049933o==== ====6) Dague, E., Le, D. T. L., Zanna, S., Marcus, P., Loubière, P., & Mercier-Bonin, M. (2010). Probing in vitro interactions between Lactococcus lactis and mucins using AFM. //Langmuir : the ACS journal of surfaces and colloids//, //26//(13), 11010–7. doi:10.1021/la101862n ==== ====7) Fuentes-Perez, M. E., Dillingham, M. S., & Moreno-Herrero, F. (2013). AFM volumetric methods for the characterization of proteins and nucleic acids. //Methods (San Diego, Calif.)//, //60//(2), 113–21. doi:10.1016/j.ymeth.2013.02.005==== ====8) Chammas, O., Billingsley, D. J., Bonass, W. a, & Thomson, N. H. (2013). Single-stranded DNA loops as fiducial markers for exploring DNA-protein interactions in single molecule imaging. //Methods (San Diego, Calif.)//, //60//(2), 122–30. doi:10.1016/j.ymeth.2013.03.002====