Laura Greeley

Traditional light microscopes are not able to resolve images small enough to explore the details of cells. One of the techniques used to investigate nanoscale samples is atomic force microscopy (AFM). AFM uses a very fine tip (atoms in diameter) attached to a cantilever to “feel” the surface of a sample. This method is ideal for measuring microfabrication, like silicon chips, but traditionally was too harsh on living organisms. Yet, advancements in the technology and new techniques have opened new possibilities for exploring the fine details of microbes.

Atomic Force Microscopy

One advancement in this technology is high-speed atomic force microscope. This technique employs a series of small cantilevers (shown in figure) and new advancement in instrument sensivity and timing to take faster-than-ever-before AFM images. Although some of the resolution is lost, the high-speed aspect enables images to be produced every thirteen seconds while still being able to clearly process resolution of fractions of a micrometer.

The figure below shows a series of images of E. coli bacterium as its surface transitions from smooth to corrugated after exposure to an antimicrobial peptide. This corrugation is correlated to cell death. Using high-speed atomic force microscope, the timing of each bacterium’s death can be determined, showing a pattern that indicates two stages in the bacterial cell death. This knowledge increases understanding of how the antimicrobial peptides work and alludes to possible mechanism of resistance, helping to design better treatments. This technique is not limited to bacteria, but can also be used on yeast, mammalian cells, and eukaryotic cell organelles.

Measuring pore formation using AFM

Another technique of AFM being used in the life sciences utilizes a functionalized tip. A functionalized tip has one of two binding species (such as the protein in a protein-substrate interaction) covalently bound to it. As shown in the image below, this protein can then bind to its partner (in this case other proteins), which is in turn bound to a surface. The laser is used to determine changes in the angle of the cantilever and thereby the change in distance of the tip. The forces on the tips indicate an attraction to the surface caused by the binding of the two proteins. Once the distance is too great, the protein-protein interaction is severed, and a rapid change in the forces observed occurs. This technique can measure the distance of a single protein-protein interaction.

AFM measuring forces

It was this technique that was used to explore a key component of viral assembly in Dengue virus. Dengue virus is a Flaviviridae, like West Nile, that puts 2.5 billion people at risk of infection each year. Because of a lack of knowledge on the basic aspects of the viral life cycle, an effective treatment has yet to be discovered. It is known that Dengue capsid protein must interact with hepatic lipid droplets before assembly can occur.  Understanding how these droplets interact with this protein illustrates one possible pathway to target for drug studies. Previously, it was thought that the protein-lipid droplet interaction was caused by a direct association between the lipids and protein. However, using AFM with Dengue capsid protein functionalized tips in tangent with another technique known as zeta potential analysis, a research team was recently able to determine that this interaction is caused by protein-protein interactions between Dengue capsid protein and primarily perilipin 3, a native protein of these hepatic lipid droplets.

References:

Fantner, G., Barbero, R., Gray, D., & Belcher, A. (2010). Kinetics of antimicrobial peptide activity measured on individual bacterial cells using high-speed atomic force microscopy Nature Nanotechnology, 5 (4), 280-285 DOI: 10.1038/nnano.2010.29

Carvalho, F., Carneiro, F., Martins, I., Assuncao-Miranda, I., Faustino, A., Pereira, R., Bozza, P., Castanho, M., Mohana-Borges, R., Da Poian, A., & Santos, N. (2011). Dengue Virus Capsid Protein Binding to Hepatic Lipid Droplets (LD) Is Potassium Ion Dependent and Is Mediated by LD Surface Proteins Journal of Virology, 86 (4), 2096-2108 DOI: 10.1128/JVI.06796-11