Applications of Atomic Force Microscopy in the Biosciences

PEG is commonly used in tip functionalization as a linker between the tip and molecule of interest and not a sample to image by itself. It might be challenging to see differences in chain orientation if these differences are subtle or small. There has
been one publication (that I know of) out of UC Davis that used our AFM to look at PEG dedrimers. I usually do not discourage people from trying a sample just because it has not already been done. This is one of those cases and I would recommend trying to image it to see what resolution is capable.

That has been the dream application from the very beginning of AFM. We have not been able to image DNA molecules at high enough resolution to identify individual base pairs. Some research groups have had reasonable success using a nanopore (see below)
but it is still not a straight-forward experiment.

Imaging cells in general is challenging since they are soft, tall and may be loosely bound to the substrate. Although it may seem counter intuitive I always recommend using contact mode to image cells. First it is important to firmly attach cells to the substrate (glass coverslip, petri dish, etc.) which may mean pre-treating the substrate with a bioadhesive (collagen, fibronectin, etc.). You also want to use a soft cantilever with a k ~ 0.02 N/m for live cells or k ~ 0.1 N/m for fixed cells. Ideally the cantilevers should have a duller tip with a minimum radius of 20nm to prevent tearing of the
membrane surface. If the cells are tall (>10µm in height), which is the case for cancer cells, then you should use a cantilever with a taller tip and an AFM that has a larger Z range. It is also important to image at a slower scan rate. For cell imaging I prefer to look at the scan speed value instead of the scan rate since speed also takes into consideration the scan size. For live cells I recommend a scan speed of 20-30 µm/s. For fixed cells you can go faster using scan speeds of 30-50 µm/s. Finally, you should optimize the setpoint and integral gain to get the best tracking of the surface.

I have not done a lot of work in the area of nanodevices. But I can see AFM playing two roles in this field. One as a method to qualify the devices themselves by providing information such as roughness, distribution or density. Another role could be measuring the results. For example, if a particular nanodevice looks at the binding of a molecule (i.e. antibody) to the patterned surface, AFM can be used to image the device before and after the introduction of the antibody to resolve morphological differences. Although AFM may not provide specificity the way fluorescence would to a labeled molecule you will see an increase in size as the antibody binds to its target on the device.

I always begin by saying that AFM can resolve any features, large or small, on the sample surface including contaminants. Therefore, it is very important that samples have been purified and buffers are filtered. Make sure your substrate and cantilever holder are clean. Just keep in mind that when you add your buffer it will pick up all of
the dirt and salt crystals present on these surfaces. Next, make sure that you are using a fresh cantilever. In air we have the luxury of re-using the probes until they break or become dull and contaminated. In liquid, however, it is difficult to re-use the probe since they are immediately coated with the buffer and become contaminated with sample over time. There aren’t guaranteed methods to clean the probes so it is best to start with a fresh probe with every experiment. Finally, once you start scanning use gentle forces to image. For example in tapping mode decrease the setpoint enough to track the surface well but not too much to damage the surface.

There are several existing publications looking at virus structure, viral infection of cells and effects of anti-virals on viruses. I showed one example of the binding of catalytic enzymes to the H1N1 influenza virus.

I would recommend reading the recently released application note titled “Advancing
Virology Research with High-Resolution AFM Imaging”, available on Asylum Research’s
webpage:
https://www.oxinst.com/news/new-application-note-advancing-virology-researchwith-high-resolution-afm-imaging/.

We have micrometers on the XY stage which allows us to move the sample/ROI to the area beneath the cantilever but the actual scans are acquired with the XYZ piezos. The maximum XY range varies depending on the AFM but for the Infinity BIO the maximum
range is 120 µm.