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This Review Article examines the principles, advantages and limitations of emerging bioimaging modes of atomic force microscopy, including multiparametric, molecular recognition, multifrequency and high-speed imaging. Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM) 1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules 2 . Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms 3 to peak positions in high-speed AFM and conventional AFM data, we 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. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset. A localization algorithm is applied to datasets obtained with conventional and high-speed atomic force microscopy to increase image resolution beyond the limits set by the radius of the tip used.

This Review of force-distance curve-based atomic force microscopy highlights the unique capabilities of the technique to simultaneously image the architecture of complex biological systems and map their physical, chemical and biological properties at nanometer resolution. A current challenge in the life sciences is to understand how biological systems change their structural, biophysical and chemical properties to adjust functionality. Addressing this issue has been severely hampered by the lack of methods capable of imaging biosystems at high resolution while simultaneously mapping their multiple properties. Recent developments in force-distance (FD) curve–based atomic force microscopy (AFM) now enable researchers to combine (sub)molecular imaging with quantitative mapping of physical, chemical and biological interactions. Here we discuss the principles and applications of advanced FD-based AFM tools for the quantitative multiparametric characterization of complex cellular and biomolecular systems under physiological conditions.

Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.High-speed AFM imaging enables a semiquantitative, realistic description of the dynamic structure of intrinsically disordered proteins.

High-speed atomic force microscopy (HS-AFM) allows direct visualization of dynamic structural changes and processes of functioning biological molecules in physiological solutions, at subsecond to sub-100-ms temporal and submolecular spatial resolution. Unlike fluorescence microscopy, wherein the subset of molecular events that you see is dependent on the site where the probe is placed, dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of how biological molecules function. Here we present protocols for HS-AFM imaging of proteins in action, including preparation of cantilever tips, step-by-step procedures for HS-AFM imaging, and recycling of cantilevers and sample stages, together with precautions and troubleshooting advice for successful imaging. The protocols are adaptable in general for imaging many proteins and protein–nucleic acid complexes, and examples are described for looking at walking myosin, ATP-hydrolyzing rotorless F 1 -ATPase and cellulose-hydrolyzing cellulase. The entire protocol takes 10–15 h, depending mainly on the substrate surface to be used.

Confocal and surface-enhanced Raman spectroscopy (SERS) are powerful techniques for molecular characterization; however, they suffer from the drawback of diffraction-limited spatial resolution. Tip-enhanced Raman spectroscopy (TERS) overcomes this limitation and provides chemical information at length scales in the tens of nanometers. In contrast to alternative approaches to nanoscale chemical analysis, TERS is label free, is non-destructive, and can be performed in both air and liquid environments, allowing its use in a diverse range of applications. Atomic force microscopy (AFM)-based TERS is especially versatile, as it can be applied to a broad range of samples on various substrates. Despite its advantages, widespread uptake of this technique for nanoscale chemical imaging has been inhibited by various experimental challenges, such as limited lifetime, and the low stability and yield of TERS probes. This protocol details procedures that will enable researchers to reliably perform TERS imaging using a transmission-mode AFM-TERS configuration on both biological and non-biological samples. The procedure consists of four stages: (i) preparation of plasmonically active TERS probes; (ii) alignment of the TERS system; (iii) experimental procedures for nanoscale imaging using TERS; and (iv) TERS data processing. We provide procedures and example data for a range of different sample types, including polymer thin films, self-assembled monolayers (SAMs) of organic molecules, photocatalyst surfaces, small molecules within biological cells, single-layer graphene and single-walled carbon nanotubes in both air and water. With this protocol, TERS probes can be prepared within ~23 h, and each subsequent TERS experimental procedure requires 3–5 h.This protocol describes how to perform nanoscale chemical imaging using tip-enhanced Raman spectroscopy (TERS). The procedure details the preparation of plasmonically active TERS probes, alignment of a TERS system, and various example procedures.

The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical microscopy. The processive hand-over-hand movement coupled with hydrolysis of adenosine triphosphate was thereby demonstrated. However, the protein molecules themselves are invisible in the observations and have therefore been visualized by electron microscopy in the stationary states. The concomitant assessment of structure and dynamics has been unfeasible, a situation prevailing throughout biological research. Here we directly visualize myosin V molecules walking along actin tracks, using high-speed atomic force microscopy. The high-resolution movies not only provide corroborative ‘visual evidence’ for previously speculated or demonstrated molecular behaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to a comprehensive understanding of the motor mechanism. Our direct and dynamic high-resolution visualization is a powerful new approach to studying the structure and dynamics of biomolecules in action. Walking myosin V caught on video Motor proteins of the myosin family serve many important functions in eukaryotic cells and are central to actin-based motility. Our understanding of the mechanism of myosin action has been hampered by the lack of technology for simultaneously observing the structure and dynamics of biomolecules, but now Kodera et al . have used high-speed atomic force microscopy (HS-AFM) to directly observe myosin V moving along actin filaments with unprecedented time resolution. They obtain high-resolution movies that provide visual evidence supporting the 'swinging lever-arm' model for myosin motility. The high-resolution imaging possible using HS-AFM should make the technique broadly applicable in the fields of structural biology and single-molecule biology. High-speed atomic force microscopy can be used to record the structure and dynamics of biomolecules simultaneously. These authors use this method to directly observe the dynamics of the motor protein myosin V moving along actin filaments, with unprecedented time resolution. The high-resolution movies provide evidence supporting the 'swinging lever-arm' model of myosin motility, and provide important insights into the mechanism of motor movement.

Cancer progression is closely related to changes in the structure and mechanical properties of the tumor microenvironment (TME). In many solid tumors, including pancreatic cancer, the interplay among the different components of the TME leads to a desmoplastic reaction mainly due to collagen overproduction. Desmoplasia is responsible for the stiffening of the tumor, poses a major barrier to effective drug delivery and has been associated with poor prognosis. The understanding of the involved mechanisms in desmoplasia and the identification of nanomechanical and collagen-based properties that characterize the state of a particular tumor can lead to the development of novel diagnostic and prognostic biomarkers. In this study, in vitro experiments were conducted using two human pancreatic cell lines. Morphological and cytoskeleton characteristics, cells’ stiffness and invasive properties were assessed using optical and atomic force microscopy techniques and cell spheroid invasion assay. Subsequently, the two cell lines were used to develop orthotopic pancreatic tumor models. Tissue biopsies were collected at different times of tumor growth for the study of the nanomechanical and collagen-based optical properties of the tissue using Atomic Force Microscopy (AFM) and picrosirius red polarization microscopy, respectively. The results from the in vitro experiments demonstrated that the more invasive cells are softer and present a more elongated shape with more oriented F-actin stress fibers. Furthermore, ex vivo studies of orthotopic tumor biopsies on MIAPaCa-2 and BxPC-3 murine tumor models highlighted that pancreatic cancer presents distinct nanomechanical and collagen-based optical properties relevant to cancer progression. The stiffness spectrums (in terms of Young’s modulus values) showed that the higher elasticity distributions were increasing during cancer progression mainly due desmoplasia (collagen overproduction), while a lower elasticity peak was evident - due to cancer cells softening - on both tumor models. Optical microscopy studies highlighted that collagen content increases while collagen fibers tend to form align patterns. Consequently, during cancer progression nanomechanical and collagen-based optical properties alter in relation to changes in collagen content. Therefore, they have the potential to be used as novel biomarkers for assessing and monitoring tumor progression and treatment outcomes.