Recent advances in the application of atomic force microscopy to structural biology

Biological molecules, such as carbohydrates, lipids, nucleic acids, and proteins are involved in crucial tasks of cellular life, determining life-sustaining functions. In order to understand these functions within complex biological systems, a quantitative structural and functional characterization of their properties, which are heterogeneous at the nanoscale, is necessary. Moreover, the dynamic nature of biological processes, such as the assembly of lipids and proteins into functional domains in cellular membranes, the remodeling of the actomyosin cortex, or cell reshaping during mitosis, requires the use of highly sensitive techniques that work at high spatial and temporal resolution under physiological conditions. In this context, we will review how recent developments in AFM enabled its use to structurally characterize complex biological systems in their native-like state at (sub-)nanometer resolution and to observe dynamic biological processes in real time. Ideally, it is desirable to structurally and functionally probe proteins within the cell without the need to isolate them. In this regard, AFM offers the possibility to work in liquid conditions similar to physiological environments. We also identify current limitations of AFM operation for a diverse range of structural biology applications and the challenges lying ahead in terms of speed, lateral resolution, interplay between cantilever size and lateral resolution on soft biological samples, as well as integration with correlative techniques to achieve a multimethodological characterization of complex living biological systems.

The AFM was introduced more than three decades ago (Binnig et al., 1986) and key improvements over the past years in terms of force sensitivity (Bull et al., 2014), thermal stability (Churnside and Perkins, 2014), lateral and temporal resolution (Heath et al., 2021, Heath and Scheuring, 2019), as well as imaging modes (Dufrene et al., 2017) have expanded its capabilities from a multifunctional tool into a nanoscopic analytical laboratory (Müller et al., 2020). Figure 1 shows an overview of significant developments that led to the establishment of AFM as a powerful technique in structural, molecular and cell biology. The operating principle of the first commercially available AFM relies on raster-scanning with a sharp tip at the end of a flexible cantilever spring over a sample, and monitoring the cantilever deflection changes as a result of tip-sample mechanical interactions, in what is widely known as contact mode operation of the instrument. During an experiment, variations in surface topography or material composition are recorded as bidimensional maps. Early on, the development of a fluid chambers for imaging in buffer allowed maintaining the native state of the studied biological system, leading to applications of AFM on biomolecules, such as for reproducible imaging and dissection of plasmid DNA (Hansma et al., 1992). Although contact mode AFM is widely used to characterize solid substrates, its application to soft biological systems should be carefully evaluated, since imaging forces above 100 pN can cause irreversible deformations. Dynamic mode [DM) AFM in liquids was introduced in the early 1990ies to minimize the high imaging and lateral forces applied to biological samples (Hansma et al., 1994). Here the AFM cantilever is oscillated at its resonance frequency, causing the probe to tap on the surface only at the extreme of each modulation cycle, which minimizes frictional forces present when the probe is constantly in contact with the surface. The oscillation amplitude is maintained as feedback, and the tip-sample distance changes to maintain the oscillation constant, while scanning over the surface. The capability of AFM to measure interaction forces was soon exploited in what was initially termed chemical force microscopy and is nowadays is commonly called force spectroscopy mode, by using the cantilever as a force sensor. In this mode, the stylus is cyclically approached and retracted from the surface while monitoring the variation of the force with respect to the tip-sample distance. Recording force-distance curves with pN sensitivity and sub-nanometer vertical resolution makes it possible to extract mechanical and physio-chemical properties of a wide variety of samples. In more advanced approaches, the AFM stylus can also be turned into a nanoscopy laboratory through [targeted (Wildling et al., 2011)] functionalization with chemical groups (Frisbie et al., 1994), ligands (Florin et al., 1994), lipids, proteins, or viruses in force spectroscopy experiments (Müller et al., 2020). Combining single molecule force spectroscopy (SMFS) and imaging through the use of force-distance curve-based AFM (FD-AFM) has opened avenues for quantifying surface and structural properties of biological surfaces from reconstituted membranes to living cells, studying ligand-receptor dissociation dynamics, or localizing specific lipids, drug-binding sites, and bacterial surface-layer proteins (Müller et al., 2020, Lo Giudice et al., 2019). Precedent to FD-AFM, other dynamic (recognition) modes have been developed and applied to biological samples, such as harmonic (Dulebo et al., 2009) and torsional mode (Dong and Sahin, 2011, Husale et al., 2009), with the most prominent example being topography and recognition imaging (TREC) in which topography and molecular recognition signals are extrapolated from different regions of a magnetically driven cantilever oscillation cycle (Stroh et al., 2004). In order to increase the number of observables and decrease the data acquisition time, multifrequency AFM (M-AFM) methods have emerged [reviewed in (Garcia and Herruzo, 2012), offering the possibility to use the amplitude, phase or frequency of the different cantilever oscillation modes to quantify and map various physical parameters. In the late 2000s, AFM investigations of biological molecules have turned their focus to mechanistic studies that specifically address structural dynamics and short-lived transition states. High-speed AFM is a particular technique that has been invented to directly acquire video-like images of biological processes in 3D at high spatiotemporal resolution and with low scanning forces (Heath and Scheuring, 2019, Kodera et al., 2010). This is the result of numerous instrumental developments such as faster scanners and feedback operation, small ultrafast cantilevers, improved optical beam deflection and environmental control systems. Shortly after, microfluidic devices coupled to hollow AFM cantilevers, also named FluidFM, has been first introduced to manipulate cells or to inject or extract a small amount of solutions into cells (Meister et al., 2009). Around 2010, the advantages of combining microscopy approaches with AFM were acknowledged and implemented, making it possible to retrieve in-depth physical, chemical, and biological information of complex cellular systems (Dufrene et al., 2017, Müller et al., 2020). Combined with optical microscopy, AFM topography images and chemical maps can be correlated with larger cellular structures. However, for visualizing details and composition of smaller cellular compartments/ structures in a minimally invasive manner with high specificity, fluorescence and confocal microscopy are commonly combined with AFM. Just very recently, Heath et al. (Heath et al., 2021) presented a technique, called localization atomic force microscopy (LAFM), which overcomes current resolution limitations, facilitating single-molecule structural analysis with aminoacid resolution. By applying localization image reconstruction algorithms to peak positions in high-speed AFM and conventional AFM data, the authors were able increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions.

Overall, AFM has shown an extraordinary potential to directly visualize molecular structures at work (Figure 2), but also to biochemically image, sense and manipulate living biological systems (Müller et al., 2020). Probing out-of-equilibrium and close-to-equilibrium thermodynamic and kinetic parameters of biological bonds is also possible due to the unique ability of AFM to apply directional forces (Figure 3). When further combined with advanced data analysis and theoretical biophysical models (Merkel et al., 1999, Dudko et al., 2008, Friddle et al., 2012), as well as optical microscopy and spectroscopy techniques (Dehullu et al., 2019, Dumitru et al., 2021, Koehler et al., 2021, Miranda et al., 2021, Odermatt et al., 2015, Rygula et al., 2018, Wood et al., 2011) (Figure 4), AFM can provide a unique wealth of opportunities to simultaneously quantify structural, functional, and chemical parameters of biomolecular and cellular systems close to their native state.

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