Christmas is coming and you know what that means? Time to catch up on your latest AFM papers reading (whilst consuming your favourite festive foods, of course)!
What fascinates us about AFM is that it is a technique brimming with potential, and an instrument constantly under development as new and improved ways of using it emerge from research. Time and again we hear stories of AFM being used in new contexts, but more often than not this happens by a lucky accident, as outside traditional physics courses AFM is rarely taught.
That’s part of why we believe the AFM Community is so important. By sharing ideas and knowledge about the different ways in which AFM is and can be used, we can push the boundaries of its potential even further. With this in mind, over the past couple of months we’ve been asking YOU, the AFM Community, what papers you have found most useful in the work you’ve been doing over the last year.
Below we’ve highlighted seven of those papers we think are most interesting from an AFM community perspective.
Five of them examine different ways in which AFM has been developed, new techniques, applications, and improvements in measurements. A sixth paper applies Topostats (a programme for automated tracing of biomolecules from AFM images) to AFM images of DNA minicircle conformations. We’ve highlighted a paper discussing Topostats previously in our top papers of 2020 blog post, but it’s such a useful, freely available programme, we felt it was worth sharing again.
Finally, we couldn’t resist sharing a paper on the work undertaken using AFM to understand Alzheimer’s disease, not least because we are proud that the research used our probes.
1. Uses of Micro-Bead Force Spectroscopy (MBFS)
Pattem et al. (2021) have identified how a polymer has potential use in aiding the removal of bacteria from eye infections preventing the onset of permanent blindness (microbial keratitis). Their approach opens the possibility of assessing alternative approaches to antimicrobial resistance in eye infections.
Specifically, they used a novel micro-bead force spectroscopy approach to look at the effect of a thermo-reactive polymer on bacterial removal from a cornea. They found that the polymer reduced the work done in bacterial aggregate removal when activated to human body temperature.
To learn more about Pattem et al.’s (2021) paper published in Scientific Reports, click here: Development of a novel micro-bead force spectroscopy approach to measure the ability of a thermo-active polymer to remove bacteria from a corneal model | Scientific Reports (nature.com)
“Showing (a) AFM Topview light microscope image of S. aureus-Van-HB-PNIPAM target adsorbed to glass slide under PBS conditions, (b) 50 µm modified spherical AFM probe functionalised with poly-DOPA approaching target (c) 50 µm modified spherical AFM probe functionalised with poly-DOPA contacting target, (d) Removal of 50 µm functionalised poly-DOPA-S. aureus-Van-HB-PNIPAM spherical AFM probe, (e) Rabbit cornea in AFM Temperature cell and (f) 50 µm functionalised S. aureus-Van-HB-PNIPAM spherical AFM probe approaching rabbit cornea in PBS at 22 ֯C” (Pattem et al., 2021).
2. Protocol for Mechanical Measurements of Soft Surfaces and Hydrogels Using AFM
AFM is a brilliant tool with enormous interdisciplinary potential. However, Michael Norman explains in his blog post that despite its advantages the technique is not without hurdles, especially for biologists or chemical engineers less familiar with it.
To this end Norman et al. (2021) have developed a standardised protocol to make AFM an accessible tool for biological applications.
Specifically, the protocol enables users to reliably reproduce results characterising the mechanical properties of synthetic and naturally derived polymeric hydrogels, including more complex multicellular structures like gut organoids. This research includes a protocol for measuring the elastic modulus of soft 2D culture surfaces and three-dimensional hydrogels using AFM.
Read more of Norman et al.’s (2021) paper in Nature Protocols here: Measuring the elastic modulus of soft culture surfaces and three-dimensional hydrogels using atomic force microscopy | Nature Protocols
The AFM probe detects the stiffness of both the cluster of cells and hydrogel. Over time, the AFM probe can detect changes in E caused by the cells remodelling their surrounding environment by matrix degradation and extracellular matrix (ECM) production (Norman et al., 2021).
3. Bimodal AFM for High Quantitative Accuracy and High-Spatial Resolution Mapping
Magnetic force microscopy (MFM) is a powerful technique to investigate magnetic materials, but its biggest drawback is that we cannot obtain quantitative data from it.
Gisbert et al. (2021) have worked to overcome this problem by demonstrating that with bimodal MFM it is possible to have ‘real magnetic numbers’ in MFM images. The bimodal technique involves having the cantilever driven into two resonant modes so that double the information is obtained.
In an extra step, the team also elaborated a theory for interpreting these images and quantifying the information obtained with bimodal MFM. To explore more of Gisbert et al.’s (2021) paper published in Nanoscale, click here: Quantitative mapping of magnetic properties at the nanoscale with bimodal AFM - Nanoscale (RSC Publishing)
“(a) Tip's oscillation in bimodal AFM. The cantilever is excited at its first two eigenmodes. Upon interaction with the sample, the components of the tip's response are processed. This step generates several observables. (b) Second pass scheme applied in bimodal AFM for imaging magnetic interactions. 1st pass: the height profile of the surface is obtained over a single scan line; 2nd pass, the tip is lifted to a certain distance and displaced along the same line by following the recorded height profile. The bimodal observables are influenced by the magnetic interaction” (Gisbert et al., 2021).
4. Localization AFM (LAFM) to Overcome Resolution Limitations of AFM
Super-Resolution Microscopy (SRM) is a revolutionary technique which won the 2014 Nobel Prize in Chemistry because it enables us to see objects smaller than a wave of light, something believed to be impossible by scientists for nearly 150 years.
Heath et al. (2021) have taken the principles behind SRM and applied them to AFM, enabling the imaging of objects as small as individual amino acids, the building blocks of proteins!
Localisation AFM has huge benefits over other techniques that can also see objects as small as this (e.g. X-Ray Crystallography and Electron Microscopy) as it can actually observe small molecules moving around, and it can do this in conditions that match biological environments.
By applying localization image reconstruction algorithms to many repeat AFM images of the same sample, they were able to see smaller objects than the sharp AFM probe, something believed to be impossible by most of us until last year.
Learn more about Heath et al.’s (2021) paper published in Nature here: Localization atomic force microscopy | Nature
Average AFM maps, LAFM maps and surface representations of X-ray structures of AqpZ (Heath et al., 2021).
5. Electrostatic Force Microscopy (EFM) Used to Study Charge-Polarised Interfacial Superlattices
Van der Waals (vdW) heterostructures are combinations of 2D materials stacked layer-by-layer in a precisely chosen sequence. One of the most promising avenues for controlling their properties is to adjust the rotational angle (twist angle) between the stacked 2D crystal layers.
Woods et al. (2021) assembled hexagonal boron nitride (hBN) crystal layers at an intentionally small twist angle. This can give rise to stacking configurations with different relative positions of the interfacial Nitrogen and Boron atoms, resulting in different orientations of dipoles (either BN or NB). This leads to the formation of superlattices of charge-polarized macroscopic triangular domains at the interface of the stacked crystals.
While standard AFM topography images did not show any superlattice pattern, electrostatic force microscopy (EFM) and kelvin probe force microscopy (KPFM) revealed the triangular ferroelectric-like electrostatic domains, caused by the different stacking in hBN vdW structures.
This work provides a fascinating platform to study superlattice phenomena and engineer novel synthetic ferroelectric vdW heterostructures. It also demonstrates a simple and non-destructive AFM technique able to visualize moiré-superlattice electrostatic potentials.
Read more of Woods et al.'s (2021) paper in Nature Communications here: Charge-polarized interfacial superlattices in marginally twisted hexagonal boron nitride | Nature Communications
“(e) AFM topography image of an hBN crystal covering a bilayer step in the bottom crystal (inset: the step profile). (f) Corresponding dc-EFM image. The triangular modulation is visible on both sides of the step marked in yellow” (Woods et al., 2021).
6. DNA Minicircle AFM Images Matched to Simulation
This paper by Pyne et al. (2021) is a fantastic collaboration between experimentalists and computational scientists. It opens up the world of dancing DNA molecules, revealing how DNA moves and interacts.
Small ‘minicircles’ of DNA were imaged with AFM to show the variety of shapes these small and relatively constrained structures can take on. When these DNA molecules were unwound, the shapes became more dynamic, with strands crossing over each other and sharp bends appearing.
AFM images revealed these shapes matched up with molecular dynamics simulations of DNA minicircles. The output videos of these simulations visualise atom-by-atom what happens in the minicircles when they are unwound, showing how the DNA moves in its different configurations.
Another AFM highlight of the paper is the high-resolution imaging of the iconic major and minor grooves of the DNA double helix around the entire minicircle allowing the exact number of times the two strands of the double helix wrap around each other to be measured.
Read more of Pyne et al.’s (2021) paper in Nature Communications here: Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides | Nature Communications
The paper also uses the automated AFM image analysis software Topostats (https://www.sciencedirect.com/science/article/pii/S1046202321000207) for image processing and DNA tracing, this is worth checking out if you work with AFM images and want to get some numbers out of them.
“AFM images of DNA minicircle populations show increased writhe and compaction at increased negative superhelical density. Images are processed to obtain individual minicircles (red) for analysis. Height scale (inset): 4 nm and scale bar: 50 nm” (Pyne et al., 2021).
7. AFM and Alzheimer’s Disease
Nirmalraj et al. (2021) used AFM in the study of Alzheimer’s disease to identify blood-based biomarkers which could, alongside existing tests, provide crucial information on the disease stage.
Specifically, they quantified the physical differences of protein aggregates (physical biomarkers) implicated in Alzheimer’s disease in red blood cells (RBC). Red blood cells profiled from healthy patients and those with neurocognitive complaints showed age- and stage of neurocognitive disorder-dependent differences in size, shape, morphology, assembly, and protein aggregates on RBCs.
We are particularly pleased that our SCOUT 70 HAR Rau probes were used to image the red blood cells in this paper seen below. Read the full paper in Science Advances here: Spatial organization of protein aggregates on red blood cells as physical biomarkers of Alzheimer’s disease pathology (nih.gov)
Protein imaging in blood from individuals with Alzheimer’s disease (left) and Large-Area Imaging of red blood cells (right), using our SCOUT 70 HAR RAu probe on a Multimode 8 (with e-scanner) and Dimension Icon (Nirmalraj, Schneider and Felbecker, 2021).
A special thank you to Dr Jacob Pattem, Dr Pablo Ares, Dr Laurent Bozec, Dr Jamie Goodchild, Eddie Rollins and Dr Miriam Jaafar for their contributions to this blog post.
If you enjoyed this blog post you might also like Around the world with AFM in 5 Papers or 2020 Review: Top 5 AFM related papers in 2020.
