Daniel Fletcher studies actin networks using atomic force microscopy. Credit: D. FLETCHER

“I did my doctoral work building force and optical microscopy systems,” says Daniel Fletcher, a bioengineer at the University of California, Berkeley. “I was an instrument developer and did not really get to use them, as the main goal was always to develop a new contrast mechanism or achieve the best resolution.” But all that changed when Fletcher started his postdoc training and saw cells crawling.

“I realized that although we could build these fancy instruments that control forces down to the piconewton level or below, we had no clue how to assemble complex molecular systems that do what the crawling cells could do.” This led Fletcher to study the dynamic actin networks of crawling cells using atomic force microscopy (AFM) and optical traps.

The biochemistry of the actin system has been studied for more than 20 years and actin networks can be grown in vitro, which provides the opportunity to directly probe how network assembly is involved in force generation and shape-changes in cells. “What we have been trying to do with both AFM and optical traps is to create a controlled resistance to the growth of these networks and study their behaviour,” he says. One approach that Fletcher's group uses is to grow actin networks on the ends of its AFM cantilever and then monitor how the networks adapt to different loads and how those forces influence the architecture of the structures generated.

Using AFM in this manner provided a challenge, requiring Fletcher to use his instrument-development skills. “One of the things that AFM is not good at doing is measuring sustained displacement over long time periods, because the surface can drift relative to the cantilever,” says Fletcher.

Cantilevers are not directly connected to the surface being scanned — they are held by a fluid cell, held by a mount, which is connected to a moving stage that is connected through an even longer path to the surface. The distance between cantilever and surface helps to make crucial calculations of the extent of deflection of the cantilever by the surface. Fletcher explains that because of this large mechanical path from the surface to the cantilever, which can alter the reference position of the cantilever relative to the surface due to thermal drift, the forces measured over time can change even if the sample does not. At short time scales this might not be an issue, but for his team, trying to measure actin growth continuously on the microscope tip over hours, drift of this sort can potentially influence the data collected.

Side-view AFM (left) and the two-cantilever approach (centre) for studying actin-network growth. Credit: D. FLETCHER, S. PAREKH & O. CHAUDHURI, UNIV. CALIFORNIA, BERKELEY

Fletcher decided that one approach to correcting for this drift would be a dual cantilever AFM system. Having two adjacent cantilevers allows one cantilever to always be in contact with the surface while the second measures the growth of the actin networks. Fletcher says that by always having one cantilever on the surface, they can now detect movement or drift directly and use feedback to correct the cantilever's position.

Fletcher continues to develop new approaches to making force measurements. His team recently developed an epi-fluorescence 'side-view' AFM device (most commercial systems come with the AFM system mounted on top of a microscope) to visualize growing actin networks between the cantilever and the surface. “Where people interested in single-, or even multimolecule, biophysics can continue to make contributions is in developing better tools that help to overcome the limitations of existing technologies and reveal new behaviour of biological systems.”