Getting a feel for AFM

Getting a feel for AFM

Since its development in 1985, atomic force microscopy has contributed to major advances in physics and engineering. Moreover, following our understanding that mechanical cues like stiffness can impact cellular behaviour, AFM has now too secured itself as a valuable tool for biologists in this field.

Despite its clever simplicity, the technique is not without hurdles. This is especially true for interdisciplinary research when biologists or chemical engineers dip a toe into AFM without any support structures to help them navigate the potential pitfalls of this approach. Many cantilevers will break, and initial force curves will look more like modern abstract art than an indentation curve. However, in developing and writing this protocol we hope to pool together our years of experience to make AFM an accessible tool for biological applications.

In developing this protocol, reproducibility was important to us.  The idea that mechanobiological mechanisms should no longer be ignored is gaining traction across all of biology, ranging from basic research into embryonic development to translational disease modelling. Biomaterials like hydrogels have been vital for in vitro approaches to mechanobiology and cell therapies, thus the ability to accurately characterise the mechanical properties of these hydrogels is crucial. Moreover, for AFM to become a reliable tool in this field, we will need standardized protocols to allow for meaningful comparisons of results between groups. Each AFM lab has its own quirky differences and not everyone agrees on the best way to do things. That said, we hope this protocol still functions as a blueprint for those new to AFM to reproducibly acquire and interpret data.

While AFM can quite easily be used as a technique to characterise the properties of simple acellular hydrogels, this protocol also extends into more complex territory. A research question in our group led us to investigate how hydrogel properties are changed when a biological component is introduced.  First, we were able to use AFM to reveal that single stem cells do not only passively respond to their mechanical environment, but also actively shape their peri-cellular environment, giving stem cells control over their own fate1. Next, we looked into how complex, multicellular structures like gut organoids behave within these biomaterials, especially when challenged by immune cells that stimulate tissue remodelling2. Whilst fascinating in the context of inflammatory disease, the application of AFM to unravel the mechanical nature of organoid-immune interactions was not without important limitations, and we found that these experiments required a fine balance between pragmatism and idealism.

We have only begun to scratch (…or indent) the surface of this issue.  AFM can be an important tool in a multi-pronged approach for characterising matrix remodelling, and hopefully, this protocol can act as a platform for other researchers to take this concept further. Indeed, paired with more complex imaging techniques like microrheology and immunocytochemistry, AFM could become a vital tool in our exploration of biomechanics in the context of development and disease. A bad worker blames their tools, but hopefully, they will no longer need to blame their protocols. 



  1. Ferreira, S.A., Motwani, M.S., Faull, P.A. et al. Bi-directional cell-pericellular matrix interactions direct stem cell fate. Nat Commun 9, 4049 (2018).
  2. Jowett, G.M., Norman, M.D.A., Yu, T.T.L. et al. ILC1 drive intestinal epithelial and matrix remodelling.  Nat Mater. 20, 250–259 (2021).


Please sign in or register for FREE

If you are a registered user on Springer Nature Protocols and Methods Community, please sign in