Zooming into Disease at the Nanoscale: High-Speed Atomic Force Microscopy in Biomedical Discovery

Keesiang Lim; Richard W Wong

doi:10.1021/acsnano.5c20737

. 2026 Feb 10;20(7):5397–5422. doi: 10.1021/acsnano.5c20737

Zooming into Disease at the Nanoscale: High-Speed Atomic Force Microscopy in Biomedical Discovery

Keesiang Lim ^†,^*, Richard W Wong ^†,^‡,^§,^∥,^*

PMCID: PMC12947739 PMID: 41666273

Abstract

The dynamic properties of biomolecules are important to the biogenesis of essential building blocks, the maintenance of biological homeostasis, and the cellular responses to external stimuli critical for survival. Structural studies define biomolecular functions, and their dynamic behavior provides mechanistic insights into these processes. Real-time tracing of dynamic biomolecular structural changes and interactions is highly desirable. Current methodologies such as cryo-EM and X-ray crystallography could provide detailed structural information; however, the images are rather static. Insufficient temporal resolution precludes the observation of crucial transient intermediates. Furthermore, sample preparation and non-physiological imaging environments can compromise the native state of biomolecules. On the other hand, other tools, such as FRET and NMR, could detect dynamic changes of targets at high temporal resolution but lack real-time observation. Atomic force microscopy (AFM) enables nanoimaging and biophysical characterization of biomolecules but is limited by slow scanning speeds and strong tapping forces. Later, high-speed AFM (HS-AFM) with high spatiotemporal resolution and gentle tapping forces, emerged as an ideal nanoimaging approach for studying the functions of delicate biological samples. Building on these developments, we and others have contributed to expand HS-AFM toward biomedical applications, including the direct visualization of disease-relevant organelles and nanostructures under near-physiological conditions. In this review, we examine HS-AFM applications in biomedical science, emphasizing real-time nanoimaging of structural dynamics across biological systems relevant to infectious diseases, infertility, cancer, and neurodegeneration. We also critically discuss the technical limitations of HS-AFM and mitigation strategies.

Keywords: high-speed atomic force microscopy, biomedical research, nanoscopic imaging, structural dynamics, nuclear pore complex, infectious diseases, infertility, cancers, neurodegenerative diseases

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Introduction

Living organisms are made of various complicated biological systems that are interlinked together to maintain survival as well as to reproduce to preserve their existence in the ecosystem. These biological systems require a vast number of biomolecules to work together with organelles to orchestrate cellular functions. Under such a diverse and dynamic environment, it is difficult to understand the mechanisms of biological activities by solely looking at the structural information and ignoring their dynamic properties.

Various structural methodologies have been developed to help biologists to decipher biological activities and phenomena in terms of the structure and dynamics of biological entities. Electron microscopy (EM) and X-ray crystallography are often used to characterize their structures. These robust approaches allow rapid characterization, especially during pandemics, when viral proteins need to be urgently evaluated for therapeutic and vaccine production. Despite generating detailed structure information, their limited temporal resolution produces static images, making them unable to resolve the continuum of conformational changes in nanoscale biological architectures. Fluorescent labeling allows real-time visualization of structural dynamics of biological structures under a near-physiological environment using fluorescent or confocal microscopy. However, the diffraction limit (∼200 nm) restricts the spatial resolution needed to observe nanoscale biomolecular structures. Furthermore, the observed dynamics often reflect the behavior of the conjugated fluorophores rather than the intrinsic motions of the biological assemblies. For example, Cosentino et al. reported that fluorophores can bias the aggregation behavior of insulins and β-amyloid peptides, such that STED imaging revealed only selected aggregation pathways. In addition to spatiotemporal resolution, sample fixation and non-physiological imaging environments (e.g., vacuum) can further perturb the native structures of biological samples.

The invention of atomic force microscopy (AFM) in 1986 was a key breakthrough in nanotechnology, providing a method to investigate surfaces by regulating the interactions between a nanoscale tip and a sample. , This was followed by atomic-level imaging and nanoimaging on non-conductive materials in a vacuum by manipulating a wide range of environments and temperatures. − Later, AFM nanoimaging in liquid at near-physiological conditions was a transformative milestone, enabling direct visualization of biomolecules, membranes, organelles, and cells at (sub) nanometer resolution. − The chronology of AFM development is summarized in Figure A.

Development of HS-AFM nanoimaging for biological samples. (A) Chronology of AFM development (adapted and modified from ref ). The development of AFM in the 1980s marked a key milestone in nanotechnology, followed by the addition of an optical beam laser and buffer chamber for contact-mode nanoimaging of biomolecules (Bio-AFM). Subsequently, various AFM modalities were introduced to enhance the versatility of AFM in bioimaging. Dynamic-mode AFM (DM-AFM) uses oscillating tips to reduce friction during scanning; force–distance curve AFM (FD-AFM) records a full-force distance curve pixel-by-pixel while scanning the surface of a biological sample; multiparametric AFM (MP-AFM) enables the investigation of physical or chemical properties of samples during scanning; molecular-recognition AFM (MR-AFM) allows imaging and mapping of specific interactions in biological samples; multi-frequency AFM (MF-AFM) uses a cantilever oscillating at multiple frequencies to map various physical parameters; combined optical imaging and AFM (Opto-AFM) enables the study of intricate biological systems; and HS-AFM accelerates scanning by approximately a factor of 1000, enabling the elucidation of dynamic and transient biological processes. Several improvements of HS-AFM have been developed, including (i) ultra-fast scanning, (ii) only trace imaging/OTI (con: conventional), and (iii) ultra-wide imaging. Reproduced under the terms of a Creative Commons Attribution 4.0 International License (CC-BY-4.0) from ref . Copyright 2022 Shimizu et al. Reproduced under the terms of a Creative Commons Attribution 4.0 International License (CC-BY-4.0) from ref . Copyright 2021 Fukuda et al. Reproduced under the terms of a Creative Commons Attribution 4.0 International License (CC-BY-4.0) from ref . Copyright 2021 Marchesi et al. (B) Schematic diagrams show the basic principles of contact (left) and dynamic (right) AFM imaging modes (NPC: nuclear pore complex; adapted and modified from ref ). In contact mode, a constant cantilever deflection is maintained by continuously adjusting the vertical position between the tip and sample to ensure constant force. Any changes in surface height modify the cantilever deflection, which the feedback system compensates for by repositioning the tip. In dynamic mode, the cantilever is driven near or at its resonance frequency. Variations in surface height affect the cantilever oscillation, which the feedback system uses to modulate the tip–sample separation. (C) A schematic diagram illustrates the setup of HS-AFM nanoimaging. Bare mica substrate can be functionalized with (3-aminopropyl) triethoxysilane (APTES), poly-l-lysine (PLL), or lipid bilayer for ideal sample adsorption.

The first AFM imaging mode, contact mode, raster scans a tip while adjusting its height to maintain constant force (Figure B). The resulting height map reflects true topography, with resolution limited by tip radius, sample features, material properties, and feedback performance on soft surfaces. However, bioimaging requires strict force control, as forces above ∼100 pN can cause irreversible deformation. To reduce friction and normal loading, the dynamic scanning mode known as tapping/oscillation mode was introduced in which the cantilever is driven near its resonance during scanning (Figure B). Tip–sample forces near the surface alter the oscillation amplitude and resonance frequency, both of which can serve as feedback signals for gentle imaging. In general, amplitude modulation (AM-AFM) is more widely used than frequency modulation AFM (FM-AFM) because it requires only a single feedback loop.

AFM has been extensively applied for nanoimaging and precise measurement of biophysical properties such as stiffness, binding affinity, etc. ,− Furthermore, 3D-AFM imaging of intracellular regions yields spatial and mechanical parameters that could be linked to biological functions or phenomena. , Despite its superb atomic resolution, AFM is not ideal for investigating their dynamic properties due to the limited scanning speed. The advent of high-speed AFM (HS-AFM) , has transformed nanoimaging of delicate biological samples. − Its gentle tapping force and high spatiotemporal resolution permit the direct visualization of these samples, capturing both structural and dynamic properties. Topological features of intermediate states can be measured and analyzed to elucidate the continuum of conformational changes. Although HS-AFM is label-free, gold nanoparticle-conjugated antibodies can be applied to mark specific sites on biomolecules for targeted analysis. , Several improvements of HS-AFM including ultra-fast scanning, only trace imaging (OTI), and ultra-wide imaging have further enhanced its nanoimaging capability (Figure A). Furthermore, substrates also can be functionalized using APTES ((3-aminopropyl)triethoxysilane), poly-l-lysine, or a lipid bilayer for optimal sample adsorption and compatibility with biological samples (Figure C).

Here, we review the application of HS-AFM in biomedical science with a focus on infectious diseases, infertility, cancers, and neurodegenerative diseases. We highlight the ability of HS-AFM to resolve the molecular dynamics of biological entities that are key to the pathophysiology of these conditions, as well as its feasibility as the nanoscopic platform for therapeutics screening. Furthermore, we discuss the current limitations of HS-AFM and future opportunities. Finally, we summarize the application of HS-AFM in each section regarding the major findings and key questions answered in respective tables (Tables –).

Application of HS-AFM for Research in Infectious Diseases

Infectious diseases remain a major threat to human health at both endemic and pandemic scales. Nosocomial infections further exacerbate patient comorbidities and elevate the mortality risk. Defining the molecular mechanisms of infections is essential for developing therapeutics (drugs, antibodies, and vaccines) for management and treatment.

Pathogenic Viruses

Viruses are opportunistic pathogens that exploit hosts’ transcriptional and translational machinery to produce their progeny. − To initiate infection, they express specialized fusion proteins (FPs) whose conformational dynamics are programmed to mediate fusion of the viral envelope with host plasma or endosomal membranes, thereby enabling genome delivery into the host cell. Viral FPs are synthesized as stable precursors and typically require cleavage by host proteases to acquire a metastable and active form. These FPs engage the host surface receptors that define viral tropism. Upon receptor binding, FPs undergo conformational rearrangement, which then exposes fusion peptides critical for membrane fusion. This process often occurs within endosomes, where acidic pH or proteases facilitate the transition from the prefusion to postfusion state, a step known as the fusogenic transition.

The newly emerging β-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic since 2019. The FP of SARS-CoV-2, the spike protein (SP) consists of S1 and S2 subunits: S1 contains the receptor binding domain (RBD) that engages the host ACE2 receptor, whereas S2 subunit harbors the fusion peptide that mediates membrane fusion following cleavage by host proteases. RBD of SARS-CoV-2 SP shows a higher binding affinity for ACE2 receptor compared with its predecessor, SARS-CoV. The SP structure has been extensively resolved by cryo-EM, and computational analyses revealed that the protein remains highly dynamic on the viral surface owing to the flexibility of its stalk region. AFM imaging of SP on intact virions has been reported by Kiss et al., however, the dynamic SP and low temporal resolution of AFM hampered detailed visualization. In contrast, and working in parallel during the same year, HS-AFM imaging of recombinant SP ectodomain at the single-molecule level has captured the native structure of SP, including its closed conformation RBD and stalk (Figure A). Dynamic movement of the stalk can be directly visualized without the need for molecular simulations as reported previously. Moreover, HS-AFM directly captured SP-ACE2 interactions, revealing the RBD in the all-up conformation, while the stalk remained dynamic throughout imaging (Figure A). Real-time HS-AFM further demonstrated distinct interaction modes between small extracellular vesicles (sEVs) and SP or its S2 subunit: SP approached and contacted the sEV surface, whereas the S2 subunit docked and penetrated the vesicle membrane. This discrepancy can be attributed to the fusion peptide within S2 subunit, which embeds into the lipid bilayer during membrane fusion. Consistent HS-AFM imaging of SP was later reported by Saha and co-workers. Using AFM and magnetic tweezers, they further demonstrated that RBD dynamics impedes SP-ACE2 binding but do not affect dissociation.

Applications of HS-AFM nanoimaging in pathogenic virus research to elucidate (A–C) conformational dynamics of viral fusion proteins during synthesis and viral entry, (D) structural dynamics of viral proteins during viral replication, (E, F) viral capsid protein assembly, (G, H) intrinsic properties of viral accessory protein, and (I–K) interactions between antibodies and viral proteins or viral particles. (A) HS-AFM directly visualized the SARS-CoV-2 spike protein (SP) ectodomain with a closed receptor-binding domain (RBD) and a flexible stalk, allowing real-time observation of stalk dynamics without requiring molecular simulations. Stalk flexibility likely facilitates RBD scanning for the host receptor ACE2, and interactions with ACE2 further highlight the functional importance of this flexibility. Reproduced under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0) License from ref . Copyright 2021 Lim et al. (B) HS-AFM captured structural dynamics of hemagglutinin precursor (HA0) ectodomain of avian influenza A (H5N1). HA0 changed from an ellipsoidal to a globular shape as the scanning buffer shifted from neutral to acidic pH, mimicking the pH change from the rough endoplasmic reticulum (RER; neutral) to the Golgi apparatus (GA; acidic). Reproduced with permission from ref . Copyright 2019 Elsevier B.V. (C) Structural change of active HA (H3N2) ectodomain was successfully recorded by changing buffer pH from neutral to endosomal pH, mimicking the fusogenic transition in endosome during viral entry. Protonation of the trimeric globular head domain triggered its dissociation, inducing a conformational transition from an ellipsoidal to a Y-shaped structure, in agreement with the “uncaging” model. Reproduced from ref . Copyright 2020 American Chemical Society. (D) HS-AFM demonstrated that intact influenza A vRNP together with folded single-stranded (ss) template RNA (black) allowed repetitive viral RNA synthesis, while the process was halted in deformed vRNP with a double-stranded (ds) RNA loop composed of template RNA and nascent RNAs (red). The RNAs are indicated with white arrows. Reproduced under the terms of a Creative Commons Attribution 4.0 International License (CC-BY-4.0) from ref . Copyright 2021 Nakano et al. (E) HIV capsid protein (Cp) lattices assemble stochastically, with subunits incorporating either as monomers or as preformed oligomers, reminiscent of piecing together a jigsaw puzzle. HS-AFM nanoimaging revealed: (i) mature hexameric HIV Cp lattice on flat surface; (ii) dynamic formation of HIV Cp lattice starting from nucleation, diffusion, growth, and fusion; and (iii) close observation of dynamic self-assembly of HIV Cp lattice. Reproduced from ref . Copyright 2020 American Chemical Society. (F) Intricate hepatitis B virus (HBV) capsid protein (Cp) assembly was recorded using HS-AFM. A schematic diagram displays the Cp assembly event (adapted and modified from ref ). Below the pseudocritical concentration of Cp dimers, small oligomers existed in equilibrium with free dimers via weak dimer–dimer interactions (i). On negatively charged substrates, electrostatic stabilization of diamond pentamers by the positively charged C-terminal domain suppressed disassembly and drove assembly (ii). Diamond pentamers underwent assembly through pentamer association and/or the sequential addition of dimers and other Cp oligomers (iii) + (iv) toward a dodecamer of dimers. Reproduced under the terms of the Creative Commons Attribution–NonCommercial 4.0 International License (CC BY-NC 4.0) from ref . Copyright 2021 Buzón et al. (G) A schematic diagram illustrates the Vif–EloC–EloB–CBFβ (VCBC) complex bound to a single-stranded (ss) DNA (adapted and modified from ref ). HS-AFM imaging demonstrated the intrinsic conformational dynamics, displaying globular, triangular, and dumbbell conformations. ssDNA binding preserved flexibility, slightly reduced the triangular conformation. Random transitions highlight VCBC dynamics, potentially critical for viral replication. Reproduced under the terms of a Creative Commons Attribution 4.0 International License (CC-BY-4.0) from ref . Copyright 2020 Pan et al. (H) The intrinsic property of SARS-CoV-2 accessory protein open reading frame 6 (ORF6), self-oligomerization, has been revealed using HS-AFM nanoimaging. The fluidic lipid surface promoted ORF6 oligomerization, with protofilaments forming dynamically yet stably on the lipid surface, in contrast to their absence on mica. Oligomerization was mediated by hydrophobic interactions, consistent with the filaments’ sensitivity to 1,6-hexanediol (1,6-HD) and cyclohexanediol (CHD). Reproduced from ref . Copyright 2023 American Chemical Society. (I) Direct visualization of the interaction between SARS-CoV-2 SP ectodomain and its neutralizing antibody at the nanoscopic level. Firm and transient interactions between Fab and receptor-binding domains were recorded. Additionally, interaction between the neutralizing antibody and SP- expressing sEV was successfully recorded. Reproduced from ref . Copyright 2023 American Chemical Society. (J) HS-AFM nanoimaging of soluble IgA (S-IgA) antibodies purified from nasal wash samples of vaccinated donors revealed S-IgA multimeric states, including monomers, dimers, trimers, and tetramers (i). Remarkably, large polymeric S-IgA was observed for the first time, and its real-time interaction with the HA ectodomain was captured using HS-AFM (ii). Reproduced with permission from ref . Copyright 2015 National Academy of Sciences. (K) Antibody dynamics are dictated by the valency of antibody–antigen binding. A schematic diagram illustrates that bivalent 8F5 demonstrated “bipedal walking”, whereas monovalent 3B10 showed wagging motion on human rhinovirus-A2 (HRV2) capsids (adapted and modified from ref ). These dynamic patterns enable distinct antiviral mechanisms: 8F5 movement promotes transient clustering to trigger immune response, while 3B10 cross-links virions, inducing aggregation.

Influenza A virus is the primary cause of seasonal flu, particularly during the winter. In elderly individuals with comorbidities, seasonal influenza markedly increases the risk of pneumonia. Moreover, the threat of cross-species transmission of highly pathogenic avian influenza A (HPAIV) should not be overlooked, as reported cases exhibit high mortality rates. , The influenza A FP, hemagglutinin (HA), undergoes a pH-dependent conformational change at endosomal pH (∼pH5) to trigger membrane fusion. HA is synthesized as a stable precursor (HA0) and requires furin-mediated cleavage into HA1 and HA2 subunits to become fusion-competent. While the prefusion and postfusion structures of HA have been extensively characterized by cryo-EM, the conformational intermediates that arise during the fusogenic transition remain unknown.

Real-time HS-AFM imaging of the HA precursor (HA0) ectodomain from avian influenza A (H5N1) showed that HA0 did not undergo large-scale conformational changes but remained sensitive to acidic conditions, as reflected by increased circularity and area, and decreased height (Figure B). The results recapitulated the conformational dynamics of HA0 during trafficking from the neutral rough endoplasmic reticulum (RER) to the acidic Golgi apparatus (GA) prior to plasma membrane assembly. The subtle transition supports a fusion-incompetent state, preventing premature fusogenic activation of HA and ensuring virion infectivity. Subsequently, the active HA of human influenza A (H3N2) ectodomain was monitored during fusogenic transition at endosomal pH, revealing clear conformational changes (Figure C). Two models have been proposed previously to explain the mechanism of fusogenic transition: the “fusion peptide release” model and the “uncaging” model. Results indicated that protonation of the trimeric globular head domain (HA1) induced its dissociation, driving a conformational transition from an ellipsoidal to a Y-shaped structure, consistent with the “uncaging” model. The Y-shaped conformation likely represents a stable HA intermediate during the fusogenic transition, in which the fusion peptide remains sequestered until membrane engagement. Under acidic conditions, HA-sEV interaction triggered fusion peptide exposure and insertion into the sEV membrane, leading to membrane deformation or rupture; these effects were absent under neutral conditions. These findings demonstrate that HS-AFM can directly capture metastable fusogenic intermediates and membrane engagement events, providing mechanistic insights into viral entry that complement and extend ensemble-averaged structural models.

Viruses initiate active replication only after entry into host cells. The conformational dynamics of viral proteins involved in genome replication are therefore critical to investigate. Nakano et al. performed HS-AFM to visualize influenza A viral ribonucleoprotein complexes (vRNPs) during RNA synthesis. Following incubation for RNA synthesis, HS-AFM imaging resolved two distinct vRNP structures associated with nascent RNAs: a helical, rod-shaped vRNP linked to a folded single-stranded template RNA and a deformed vRNP attached to a looped double-stranded RNA comprising nascent RNA and template RNA (Figure D). These findings suggest that maintenance of the helical vRNP structure is critical for efficient repetitive RNA synthesis, whereas the deformed vRNP likely arises stochastically and may impair replication. Carlero et al. further demonstrated that vRNP in annular organization provided an ideal condition for HS-AFM to capture structural changes of vRNP during RNA synthesis.

Following the biogenesis of the viral genome and viral proteins, viral assembly proceeds to generate new virions. A key step in the process is the dynamic assembly of viral capsid proteins (Cp), which form a lattice that serves as a container for viral genetic materials. Valbuena et al. performed HS-AFM imaging to visualize the real-time self-assembly of HIV Cp (Figure E). Their study found that Cp lattices form stochastically, with subunits either assembling individually or first forming oligomers before being incorporated into the lattice. Notably, Cp subunits were also observed to dissociate from the lattice, underscoring that the HIV capsid assembly is a complex and chaotic process involving multiple pathways. It resembles a jigsaw puzzle in which the final structure forms through local interactions, iterative attempts, and gradual refinement. These features are difficult to capture using bulk- or ensemble-averaging methods.

Buzón and colleagues combined fluorescence optical tweezers and HS-AFM to investigate HBV Cp assembly. HS-AFM imaging identified two structural intermediates during assembly: a diamond-shaped pentamer of dimers and a dodecamer of dimers (Figure F). Their findings suggest that HBV Cp assembly proceeds through the formation of pentameric and dodecameric intermediates, rather than relying solely on canonical 5-fold-symmetric pentamers. Below the pseudocritical concentration of Cp dimers, small oligomers existed in equilibrium with free dimers and assembly was governed by weak dimer–dimer interactions. Diamond pentamers were stabilized on negatively charged substrates, such as nucleic acids or a surface, through electrostatic interaction with the positively charged C-terminal domain. The substrate limited the mobility of Cp dimers during oligomerization, thereby suppressing disassembly and providing a strong thermodynamic driving force for assembly. Once the diamond pentamer was nucleated on the substrate, further assembly proceeded through association with other diamond pentamers and/or the stepwise addition of dimers and other Cp oligomers toward the dodecamer of dimers.

Viruses often express accessory proteins that reprogram the host environment to promote replication and persistence. HIV-1 expresses the accessory protein virion infectivity factor (Vif) to promote viral replication by antagonizing host apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) restriction factors and inducing cell cycle arrest. Vif orchestrates polyubiquitination and proteasomal degradation of A3 proteins by hijacking host factors, including cellular cofactor core-binding factor β and the cellular cullin-RING ligase 5 E3 ubiquitin ligase complex comprised of cullin 5, elongin B and C complex (EloB/EloC), and RING-box subunit 2 (RBX2). Using HS-AFM, Pan et al. reported that the Vif–EloC–EloB–CBFβ (VCBC) complex showed high intrinsic conformational dynamics, adopting three primary states: globular, triangular, and dumbbell (Figure G). Single-stranded DNA bound away from Vif’s flexible linker, preserving or slightly enhancing VCBC flexibility. DNA binding modestly reduced the triangular conformation while promoting dimerization of the globular form. Transitions between three different conformations occurred randomly. The work demonstrates that VCBC’s dynamics are intrinsic and may be important for its function in viral replication and represent a potential drug target.

Pathogenic viruses frequently hijack host nucleocytoplasmic trafficking to suppress antiviral signaling pathways. ,,, SARS-CoV-2 expresses the ORF6 protein, which suppresses host interferon signaling in this manner. , Nishide et al. observed that ORF6 possesses self-assembly properties, forming protofilaments as visualized by HS-AFM (Figure H). ORF6 self-assembly was enhanced by fluidic surfaces such as lipid bilayers or by elevated temperature. Formation of ORF6 oligomers on lipid membranes was further supported by a subsequent NMR study. ORF6 oligomerization was driven by hydrophobic interactions, as the protofilaments were dissociated when treated with 1,6-hexanediol (1,6-HD) and cyclohexanediol (CHD) (Figure H). Exogenous ORF6 oligomers were internalized by host cells, where they further assembled into larger aggregates. These findings suggest that ORF6 self-assembly may contribute to long COVID-19 complications, particularly neurodegenerative complications driven by cytotoxic protein aggregates.

Antiviral antibodies neutralize viruses by blocking their interaction with host receptors. HS-AFM enables direct visualization of the dynamic interactions between viral antigens and antibodies. For example, the binding of the neutralizing anti-SARS-CoV-2 SP antibody MM43 (IgG) to SP was captured in real time, revealing dynamic engagement between the antibody Fab region and the RBD of SP (Figure I). HS-AFM was further applied to probe potential SP conformational changes upon antibody binding, which may contribute to antibody-dependent enhancement (ADE). , Besides protein–protein interaction, HS-AFM visualized binding of MM43 to SP-expressing sEV (SP-sEVs), comparing its affinity for Wuhan versus Delta variants. Similarly, HS-AFM quantified the binding affinity of banana lectin to SARS-CoV-2 SP, demonstrating viral neutralization by blocking entry. In addition to IgG, Suzuki et al. conducted HS-AFM imaging to investigate structural properties of an influenza A HA-specific secretory IgA antibody (S-IgA) purified from nasal wash samples of vaccinated donors (Figure J). S-IgA was observed in dimeric, trimeric, tetrameric, and higher-order polymeric forms, undergoing conformational changes upon HA binding. Notably, trimeric, tetrameric, and larger polymeric S-IgAs demonstrated greater neutralizing potency than monomers and dimers. A higher proportion and greater avidity (i.e., more binding sites) of polymeric S-IgA confer enhanced protection against the influenza A virus.

Repetitive antigenic structures, common in non-enveloped viruses, serve as key targets for vaccine design. Preiner et al. reported stochastic “bipedal walking” of antibodies on surface-bound antigens (Figure K). This motion is driven by steric strain, which transiently weakens bivalent antibody–epitope interactions, allowing one Fab arm to detach and rebind nearby. Steric strain is specific to bivalent binding, as monovalent antibodies showed only wagging or static behavior, exemplified by step-like motion of the bivalent 8F5 antibody versus the wagging of monovalent 3B10 antibody on human rhinovirus (HRV) capsids. These distinct dynamics reflect diverse antiviral mechanisms: bipedal walking promotes transient antibody clustering, facilitating immune responses through complement activation and phagocytosis, whereas 3B10 mediates neutralization by cross-linking virions and inducing aggregation.

Pathogenic Bacteria

HS-AFM has also been applied to study pathogenic bacteria in various aspects such as bacterial dormancy, immunoevasion, , host antimicrobial activities, , and solutions for antibiotic resistance. − Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, represents a persistent global health threat due to its ability to establish chronic pulmonary infections that are difficult to eradicate. A key feature of Mtb pathogenesis is its capacity to enter a dormant state, thereby evading host immunity and resisting antibiotic treatment. The mechanisms underlying dormancy remain elusive, but increasing attention has been focused on the role of nucleoid-associated proteins in regulating genome organization and bacterial physiology. Nishiyama and colleagues recently performed HS-AFM nanoimaging to investigate the histone-like protein known as mycobacterial DNA-binding protein 1 (MDP1), a DNA-binding protein that contains an HU-like region (HUR) at its N-terminus, and a long intrinsically disordered region (IDR) at its C-terminus. The team found that HUR mediated the initial DNA recognition and binding, allowing the polycationic IDR to mediate side-by-side alignment of DNA strands, functioning analogously to “molecular double-sided tape” to promote DNA compaction (Figure A). The disordered region also disrupted the histone-like HU protein (a chromatin-associated protein) function to strongly suppress bacterial growth. Collectively, these findings suggest that MDP1 exerts a dual function: (1) promotes DNA compaction and (2) disrupts HU protein function to trigger Mtb dormancy, providing new mechanistic insights into how nucleoid-associated proteins facilitate persistence of this formidable pathogen.

Host immune system establishes multiple strategies to eliminate invading bacteria, most prominently through phagocytosis and the complement system. Phagocytes engulf bacteria and destroy them by lysosomes, whereas complement activation forms membrane attack complex (MAC) to perforate bacterial membrane. Nevertheless, some bacteria have evolved sophisticated immunoevasion mechanisms to circumvent these defenses. Listeria monocytogenes, a facultative intracellular pathogen, relies on the cholesterol-dependent cytolysin known as Listeriolysin O (LLO) to breach the phagolysosomes, a critical step for survival and intracellular dissemination. Ruan et al. conducted HS-AFM nanoimaging to investigate the molecular dynamics of LLO during membrane attack (Figure B). Real-time imaging revealed the sequential steps of LLO action, including the kinetics of oligomer assembly, the prepore to pore transition, and subsequent membrane disruption. These analyses showed that both the efficacy and mechanism of LLO-mediated membrane perforation are highly sensitive to membrane cholesterol levels and environmental pH, consistent with the phagolysosomal milieu characterized by 20–40% membrane cholesterol and acidic conditions encountered during infection. , Notably, LLO not only formed arc-shaped pores at acidic pH but also acts as a lineactant (pH-independent but cholesterol-dependent), destabilizing lipid bilayers and promoting large-scale membrane defects. This dual activity provides a mechanistic basis for efficient phagolysosomal escape, enabling L. monocytogenes to rapidly access the host cytosol. Altogether, these findings highlight how a single bacterial toxin disrupts the membrane mechanics to subvert host immunity.

Staphylococcal protein A (SpA), a major virulence factor of Staphylococcus aureus and a leading vaccine target, evades host immunity by interfering with antibody effector functions. Cruz et al. demonstrated that SpA inhibits IgG-mediated complement activation by preventing the assembly of IgG hexamers. Using an integrative approach that combined native mass spectrometry and HS-AFM, the authors showed that SpA exerts this effect by competitively binding to the Fc–Fc interaction interface on individual IgG molecules, thereby obstructing the formation of the hexameric IgG platform required for complement engagement (Figure C).

Perforin-2 (encoded by the MPEG1 gene), a member of the MAC/perforin (MACPF) family, is critical for the intracellular killing of pathogens engulfed by phagocytes. Using HS-AFM, Ni et al. directly visualized the dynamic assembly of murine perforin-2 from prepore intermediates to fully formed pores, revealing a unique activation mechanism in which the pore-forming domain was oriented away from the membrane in the prepore state (Figure D). Cryo-EM results indicated that acidification triggered a 180° conformational rotation, rearranging the pore-forming domain into a bacterial membrane. This delayed activation strategy ensures that pore formation happens only within acidic phagosomes, preventing premature host membrane disruption and assuring pathogen-selective cytotoxicity. This study suggests that this novel regulatory mechanism may represent an ancient and conserved step in the evolution of perforin-family proteins.

Parsons et al. carried out HS-AFM to study the sequential assembly of the human complement MAC, composed of complement proteins including C5b, C6, C7, C8, and multiple C9 subunits (Figure E). Whereas the rate-limiting steps of MAC formation have remained poorly defined, their findings demonstrated that oligomerization occurred directly within the target membrane, a mechanistic divergence from structurally related bacterial pore-forming toxins. The study found that membrane engagement was initiated by C5b-C7, which facilitated the subsequent recruitment of C8. Surprisingly, the insertion of the first C9 molecule constituted the kinetic bottleneck of pore formation, after which rapid C9 polymerization completed the transmembrane channel. This kinetic framework highlights a critical regulatory point at which CD59, a membrane-bound inhibitor, intercepts MAC assembly to protect host cells from complement-mediated bystander damage.

Antimicrobial resistance poses a major challenge to clinical care, prompting interest in novel membrane-targeting agents such as AMC-109, a cationic tripeptide with potent activity against S. aureus. Using a combination of HS-AFM, molecular dynamics simulations, fluorescence assays, and lipidomic profiling, Melcrová et al. have elucidated a two-step mechanism of action of AMC-109 (Figure Fi). AMC-109 first self-assembles into stable aggregates with a hydrophobic core and cationic surface, exhibiting a high specificity for negatively charged bacterial membranes. Upon integration into the membrane, individual peptides insert into the outer leaflet, disrupting lateral membrane organization and dissolving lipid nanodomains without forming transmembrane pores. This domain dissolution is proposed to interfere with essential processes, such as protein sorting and cell wall synthesis. Mechanistically, AMC-109 mirrors the action of the disinfectant benzalkonium chloride (BAK), but with improved selectivity toward bacterial membranes over host membranes.

The same group used both conventional AFM and HS-AFM to study the mechanism of action of N-alkylamide 3d, a small cationic antimicrobial compound (Figure Fi). Using full lipid extracts from S. aureus to mimic native membrane complexity, they observed that N-alkylamide 3d was first incorporated into the membrane, slowing and dissolving laterally organized lipid microdomains. The initial perturbation was followed by the gradual attachment and growth of supramolecular aggregates (e.g., spherical structures, carpets, and rod-like assemblies) that eventually covered the membrane surface. The dual-step mechanism not only disrupts membrane organization but also forms a physical shield that could block external access and internal processes such as signaling and waste disposal. The study underscores the under-appreciated role of lipid domain disruption in the action of small-molecule antimicrobials.

Chen et al. utilized cryo-electron tomography (cryo-ET) and HS-AFM to directly visualize the membrane-disruptive action of the de novo-designed antimicrobial peptide pepD2M on Escherichia coli (Figure Fii). Using bacterial minicells to overcome cryo-ET thickness limitations, they captured high-resolution 3D images of membrane disruption in a near-native state. Unlike the pore-forming peptide melittin, which preferentially targets mammalian-like membranes, pepD2M demonstrated strong selectivity for negatively charged bacterial membranes and operated through a carpet/detergent-like mechanism (a nonpore-forming model), causing lipid removal and cluster formation. HS-AFM further revealed the real-time dynamic disruption of lipid bilayers by pepD2M at the nanoscale. Collectively, these results provide direct conformational and temporal evidence of how antimicrobial peptides (AMPs) compromise both the outer and inner membranes of Gram-negative bacteria, offering critical mechanistic insights into this class of membrane-targeting therapeutics.

Fantner and colleagues used microfabricated small cantilevers for HS-AFM live imaging of individual bacterial cells at 13 s intervals at the nanoscopic level (Figure Fiii). This method allowed real-time visualization of AMP CM15 acting on E. coli. The study reported a biphasic killing process: a variable and cell-dependent incubation phase (lasting seconds to minutes), followed by a rapid and consistent execution phase where surface corrugation developed within ∼1 min. By combining HS-AFM with fluorescence viability staining, the team confirmed that surface roughening is directly associated with cell death. Markedly, response onset times varied significantly between adjacent cells, highlighting single-cell heterogeneity in AMP susceptibility. The study illustrates how HS-AFM can bridge nanoscale dynamics with population-level kinetics, offering unprecedented insights into the mechanistic progression of antimicrobial action.

Application of HS-AFM in Infertility Research

While infectious diseases often involve aggressive pathogen–host interactions, infertility stems from subtle, intrinsic dysfunctions in highly regulated physiological systems. Both, however, share a reliance on dynamic molecular assemblies and structurally fragile intermediatesmaking them ideal targets for high-resolution, real-time visualization.

Infertility represents a growing global health challenge, which is increasingly exacerbated by aging demographics. In response, intensive research efforts have sought to uncover the cellular and molecular underpinnings of infertility in both females and males. Beyond hostpathogen interactions, HS-AFM is uniquely suited for nanoimaging biological processes that are intrinsically dynamic and physiologically delicate. In the context of infertility, HS-AFM enables the direct observation of biomolecular dynamics associated with gametogenesis. Together with AFM, these approaches provide nanoscale insights into the biophysical and structural determinants of reproductive competence that are difficult to accomplish by using conventional imaging techniques.

In the female reproductive system, follicle development is governed by a dynamic mechanical microenvironment whose disruption contributes to infertility-related conditions such as polycystic ovary syndrome (PCOS) and premature ovarian insufficiency (POI). Recent AFM-based studies have revealed distinct age- and region-specific mechanical properties within the ovary. Notably, the ovarian cortex exhibits greater stiffness and altered stress relaxation kinetics compared to the medulla, particularly in reproductively older individuals, features that correlate with hyaluronic acid distribution. Complementary AFM mapping in intact mouse ovaries identified the stiffest regions within zones enriched in large, advanced follicles, refuting prior assumptions that stromal collagen is the dominant mechanical determinant. High-resolution scans (∼5 μm) further revealed microscale heterogeneity, offering a refined biomechanical framework for folliculogenesis and informing the design of biomimetic systems for infertility treatment.

In the male reproductive system, oxidative stress is a critical factor that impairs sperm function and fertility. AFM has facilitated the development of electrochemical sensors for detecting hydrogen peroxide (H₂O₂), a key oxidative stress marker in semen. By characterizing nanoparticle-modified electrode surfaces with AFM, researchers engineered sensitive platforms capable of quantifying H₂O₂ in complex seminal matrices, enabling improved assessment of male infertility. Beyond sensing, AFM has been instrumental in evaluating sperm ultrastructure, particularly chromatin condensation, which is essential for fertilization. Condensation defects, detectable even in morphologically normal sperm, were inversely correlated with in vitro fertilization success but not intracytoplasmic sperm injection, highlighting their potential as predictive biomarkers. AFM imaging revealed increased nuclear thickness, surface roughness, and mitochondrial disorganization in defective sperm, suggesting that aberrant nuclear shaping may disrupt protein dynamics vital for fertilization.

To dissect the molecular choreography of sperm chromatin compaction, AFM has been pivotal in visualizing protamine-DNA complexes. Early in vitro reconstitution studies using conventional AFM indicated that protamine condensed DNA into toroidal structures but critically demonstrated that their geometry and aggregation state were highly sensitive to sample preparation. This work underscored a major limitation of static AFM: it could only record “snapshots” of end-point or trapped intermediate structures, making it difficult to distinguish native condensation pathways from preparative artifacts (Figure A). Later, HS-AFM directly overcomes this limitation by providing high spatiotemporal resolution. Nishide et al. performed HS-AFM nanoimaging to observe, in real time, the stepwise condensation of DNA by protamine. This dynamic imaging captured the initial DNA coiling, formation of rod-like intermediates, and final assembly of toroids, revealing a sequence of events that was previously inaccessible. These observations culminated in the proposed CARD (coil–assembly–rod–doughnut) model, which describes the temporal and hierarchical progression of chromatin compaction (Figure B).

Applications of HS-AFM nanoimaging in DNA compaction mediated by protamine. (A) Protamine promotes DNA condensation to form a compacted toroidal ring, which can be clearly observed using conventional AFM. The condensed DNA adopted a vertically stacked architecture of four to five turns, with each coil containing as little as 360–370 bp of B-form DNA. (B) With higher temporal resolution, HS-AFM nanoimaging captured the intermediates of DNA-protamine complexes in different patterns including coiled complex, rods, and doughnuts, indicating that protamine-driven DNA compaction into toroidal ring by protamine follows the CARD (coiled – assembly – rod – doughnut) model. The event was observed under the near-physiological environment.

While conventional AFM excels in static, high-resolution mapping of mechanical and topographical features, HS-AFM uniquely enables live, real-time visualization of dynamic molecular events under near-physiological conditions. Together, these tools bridge structural, mechanical, and temporal scales, offering an integrated biophysical lens into the mechanisms of infertility. Their applications are rapidly reshaping our understanding of gamete biology and guiding the development of novel diagnostics and therapeutic strategies for reproductive health.

Application of HS-AFM in Cancer Research

The same molecular precision and temporal coordination required for gametogenesis are also fundamental to maintaining cellular homeostasis and genome integrity. When these dynamic processes are corrupted, as in cancer, the consequences can be profound. High-fidelity molecular dynamics, essential for normal physiology, as exemplified in reproductive biology, becomes profoundly dysregulated in malignancies. Carcinogenesis is a complex, multistep process driven by diverse etiological factors, including chromosomal abnormalities, genetic mutations, epigenetic dysregulation, chronic inflammation, and mechanical stress. Alteration in chromosome numbers or structure and mutation in genetic sequence can profoundly affect the protein abundance and conformation. Oncoproteins are often overexpressed, whereas tumor suppressors are downregulated. , Structural changes in proteins can lead to oncogenic-gain-of-function phenotypes, as reported in mutated receptor kinases and chimeric kinases, which acquire constitutive activation to drive malignant progression. Beyond enzymatic activation, chimeric proteins such as Nup98- Homeobox A9 (HOXA9) undergo liquid–liquid phase separation (LLPS), a thermodynamically driven demixing process that creates coexisting protein-rich (dense) phase and solvent-rich (dilute) phase, to form condensates that sequester super enhancers and amplify oncogene transcription. Epigenetic modification of histones, including acetylation and methylation, further remodels chromatin architecture and gene accessibility. At the cellular level, structural alterations in organelles are associated with carcinogenesis. Cancer cells possess enlarged, pleomorphic nuclei with high nuclear-to-cytoplasmic ratios and hyperchromatic features, together with fragmented mitochondria. , During metastasis, reversible transitions between epithelial and mesenchymal phenotypes, orchestrated by epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET), drive cancer cells dissemination and colonization in accordance with the “seed and soil” model. The dynamic structural transition embodies physical manifestations of cancers. HS-AFM overcomes the diffraction limit of optical microscopy, allowing real-time nanoscale visualization of molecular and organelle dynamics that are important for understanding tumor initiation and progression.

Nuclear pore complex (NPC) is a megadalton protein complex composed of more than 30 different types proteins, collectively known as nucleoporins (Nups), which regulate nucleocytoplasmic transport and gene expression. − Nups with phenylalanine (F)-glycine (G) repeats or FG-Nups generate a selective permeable barrier to regulate nuclear transport, and they are often overexpressed in cancer cells to accelerate biomolecule trafficking to boost signal transduction. − To date, the exact conformation of the selective permeable barrier remains elusive and has been described by several models, including the polymer brush, hydrogel, two-gate, forest, and Kap-centric models. Using high-speed AFM, we directly visualized the FG-nucleoporin network within the central channel as a highly dynamic, web-like architecture resembling a spider cobweb, in which flexible FG filaments form transient cross-links that continuously rearrange in space and time. This spider cobweb model, derived from real-time HS-AFM observations, emphasizes a mechanically adaptive and topologically entangled FG meshwork that permits transport receptor threading while maintaining a robust permeability barrier against inert macromolecules. The flexible FG Nups activities in Xenopus laevis oocyte NPC, budding yeast Saccharomyces cerevisiae NPC, and DNA origami-based artificial pores were characterized using HS-AFM.

HS-AFM has been indispensable in elucidating the dynamic nanoscale behavior of intrinsically disordered FG-Nups within the NPCs of colorectal cancer (CRC) cells. , By enabling real-time observation with high spatiotemporal resolution, HS-AFM visualized distinct biophysical states of FG-Nup filaments, including extension, bending, and entanglement, which differ significantly between normal and cancer cells. These studies further demonstrated that cellular stress or chemotherapeutic treatment, such as the Aurora A inhibitor MLN8237 (Alisertib) or the phase separation inhibitor trans 1,2-cyclohexanediol, disrupted FG-Nup network integrity, leading to loss of structural resilience, pore collapse, and impaired transport function, correlating with cell death in CRC cell lines and patient-derived CRC organoids (Figure A). Moreover, nanoscopic tracking of central plug dynamics by HS-AFM, showed that the central plug is a reversible FG-Nup condensate, and highlighted the clinical relevance of specific Nups, such as Nup214, which is overexpressed in colorectal cancer and is associated with poor prognosis. Altogether, these results establish FG-Nup dynamics as a key biomechanical feature of NPC function and a potential diagnostic marker and therapeutic target in oncology.

Applications of HS-AFM in cancer research related to (A, B) cancer biology, (C, D) cancer diagnosis, and (E, F) cancer therapeutics. (A) FG-Nups in the central channel of NPCs build a selectively permeable barrier to regulate biomolecule trafficking, which resembled spider cobweb under HS-AFM imaging. Aberrant expression of FG-Nups increases protein density to accelerate signaling pathways to support hallmarks of cancer. HS-AFM imaging revealed that Alisertib (MLN8237) treatment disrupted the cytoplasmic region of NPCs in colorectal cancer cells. Furthermore, MLN8237 and the LLPS inhibitor cyclohexanediol (CHD) disrupted FG Nups interactions in the central channel, thereby abolishing the selectively permeable property. , Reproduced under the terms of the Creative Commons Attribution–Noncommercial–Noderivatives 4.0 International License (CC BY-NC-ND 4.0) from ref . Copyright 2020 Mohamed et al. Reproduced from ref . Copyright 2017 American Chemical Society. (B) Estrogen receptor alpha (ERα) plays a vital role in luminal breast cancer by enhancing the transcription activity of estrogen-response-element. The orientation of ERα was located at the side and on top of DNA strands. Dimerization of ERα occurred either directly, when another ERα approached and bound to a DNA-binding ERα, or indirectly, when two DNA-binding ERα met and merged with each other during HS-AFM scanning. The dynamic interaction patterns were found similar regardless of the presence of its ligand, estrogen (E2), but ligand enhances the binding activity. LBD: ligand-binding domain; DBD: DNA-binding domain; ERE: estrogen response element. Reproduced from ref . Copyright 2025 American Chemical Society. (C) Internal tandem duplication (ITD) of *FLT3* gene could lead to polymorphism of amplicon length after PCR. Direct visualization and measurement of amplicon length using HS-AFM allows the detection of *FLT3* amplicon with ITD in the presence of abundant wild type *FLT3* amplicons. (D) Functionalized cantilever tips, such as the macrocyclic peptide-conjugated cantilever (aMD4-C), can distinguish human (h) MET receptor from murine (m) MET receptor. When both hMET and mMET were deposited on the same substrate, difference in their phase-delay signature allowed the identification of hMET from mMET in phase-contrast images but not in topography images. The results suggest that HS-AFM offers fast and selective molecular recognition at the nanoscopic level, with potential application in nanoscopic diagnosis in future. Reproduced from ref . Copyright 2021 American Chemical Society. (E) Macrocyclic peptide inhibitor (HiP-8) binds specifically to active hepatocyte growth factor (HGF) (two-chain HGF/tcHGF) to sterically block the HGF-MET receptor interaction to inhibit MET receptor activation. HS-AFM images illustrate distinct single-chain HGF (scHGF) and tcHGF conformations. The SP domain of scHGF preferentially bends toward the N-terminal kringle 1 (NK1) domain, but the domain was highly flexible in tcHGF. In contrast, the SP domain of tcHGF became static in the presence of HiP-8, indicating an interaction between two molecules. The position of NK1 domain remained unchanged during scanning because it was adsorbed onto the substrate. Arrow: NK1 domains attached to substrate; arrowhead: serine protease-like (SP) domain. Reproduced with permission from ref . Copyright 2019 Sakai et al. (F) Nanoscopic elucidation of dynamic interaction between cancer-specific DNA aptamer (Apt-7) and cytochrome P450 family member (CYP24) using HS-AFM. HS-AFM images depict the (i) stem-loop Y-shaped structure of Apt-7; (ii) CYP24 domains (D1–3); and (iii) two Apt-7 bound to a single CYP24 molecule, as indicated by two white arrows. A schematic diagram shows that the Apt-7-CYP24 interaction inhibits CYP24 function, thereby restoring the vitamin D tumor suppressor pathway. Reproduced from ref . Copyright 2022 American Chemical Society.

Nanoscopic observation of interaction between DNA and DNA-binding proteins using HS-AFM provides important insights into epigenetic regulation and transcription factor activities. Dynamic interactions between histone protein and DNA have been observed using HS-AFM , in a real-time manner. These studies suggest that HS-AFM could be used to compare the DNA wrapping behavior on histone proteins with different types of epigenetic modifications in the near future. Interaction between DNA and transcription factors regulates the expression of oncogenes and tumor suppressor genes in carcinogenesis. Nishide et al. performed HS-AFM nanoimaging to visualize the real-time dynamics of estrogen receptor alpha (ERα) binding to estrogen response elements (EREs). The team found that ERα can bind DNA even in the absence of estrogen (E2), but ligand binding significantly enhances the dimerization stability, binding precision, and structural conformation. They proposed a ligand-induced dimerization model, where estrogen promotes optimal ERα loading onto DNA via stabilized dimer formation (Figure B). These findings provide direct mechanistic insights into the ERα role in gene regulation and have pivotal implications for hormone-dependent breast cancers, where treatment strategies aim to block ERα dimerization and nuclear import. Mutations in the ERα that change these dynamics are linked to resistance against hormonal therapies.

Besides cancer biology research, HS-AFM has the potential to be used as a nanoscopic diagnostic platform. For example, nucleic acid-based diagnostics are available for Fms-related receptor tyrosine kinase 3 (FLT3) internal tandem duplication (FLT3-ITD). FLT3-ITD is a poor-prognosis mutation in acute myeloid leukemia (AML), arises from tandem insertion of a duplicated segment within the juxtamembrane-coding region of the FLT3 gene. When HS-AFM is coupled with digital PCR, the resulting FLT3-ITD PCR amplicons, which are longer than their wild-type counterparts, can be quantitatively visualized and measured. The technique allowed direct imaging of ITD-positive amplicons at the single-molecule level, offering unparalleled sensitivity and specificity for minimal residual disease detection (Figure C). HS-AFM functionalized with macrocyclic peptide-conjugated cantilevers enabled the real-time, label-free detection of oncogenic human MET receptor tyrosine kinase (hMET) on intact cellular membranes. Results demonstrated that when murine (m) and human (h) MET receptors were simultaneously immobilized on the same substrate, they could be unambiguously distinguished based on their phase-delay signatures. Quantitative analysis revealed two well-separated molecular populations, with phase delays exceeding 15° uniquely associated with the hMET ectodomain. This approach circumvented the need for fluorescent tags and revealed the spatial heterogeneity of hMET distribution, thereby enhancing the potential for dynamic, quantitative assessment of receptor states in living cell or biopsy samples (Figure D).

In the context of drug screening, HS-AFM offers a valuable platform to analyze the effects of therapeutics on specific targets in conjunction with other approaches. Structural-functional study using HS-AFM elucidated the conformational impact of a macrocyclic peptide inhibitor, HiP-8, on HGF, a critical ligand in the MET signaling axis. HiP-8 was shown to sterically constrain HGF’s conformational mobility, thereby inhibiting MET receptor activation and downstream oncogenic signaling (Figure E). At the protein–ligand interface, HS-AFM elucidated the inhibitory mechanism of a cancer-specific DNA aptamer, Apt-7, targeting cytochrome P450 family member CYP24/CYP24A1a mitochondrial enzyme that attenuates vitamin D signaling and is aberrantly upregulated in several cancers. HS-AFM directly captured the binding kinetics of Apt-7 and its allosteric suppression of CYP24A1 activity, offering structural correlates for selective inhibition and paving the way for aptamer-guided reactivation of vitamin D tumor suppressor pathways (Figure F).

Collectively, these applications emphasize the unique capability of HS-AFM to interrogate the biophysical underpinnings of oncogenic signaling, genome instability, membrane receptor dynamics, and nuclear transport dysregulation. Beyond the conformational transition of biomolecules and organelles, HS-AFM can be used to quantify active cellular rheological properties. While AFM has been used for measuring rheology in soft matter, , its limited temporal resolution constrains dynamic analysis in living cells. Importantly, cellular rheological properties reflect the cytoskeletal state, which undergoes rapid remodeling during metastatic progression or in response to drug treatment. By bridging structural biophysics and systems oncology, HS-AFM is not only a next-generation imaging platform but also a functional assay capable of guiding precision diagnostics, therapeutic targeting, and biomarker discovery in cancer research.

Application of HS-AFM in Neurodegenerative Diseases

In stark contrast to malignancies, which are defined by uncontrolled proliferation, neurodegenerative diseases (NDDs) are characterized by progressive neuronal dysfunction, loss of synaptic integrity, and eventual cell death. While cancer arises from aberrant activation of growth pathways, NDDs result from the failure to maintain neuronal homeostasis, often linked to protein misfolding, aggregation, and impaired clearance. HS-AFM is uniquely positioned to visualize these nanoscale events in real time, providing direct insights into the conformational transitions, oligomerization pathways, and toxic interactions that underlie neurodegeneration.

NDDs include the progressive degeneration of neurons in the central nervous system or the peripheral nervous system, marked by the gradual breakdown of neural network architecture and function, driven by the irreversible loss of postmitotic neurons with limited regenerative capacity. Ultimately, patients experience impairments in fundamental processes such as memory, cognition, behavior, sensation, and motor control. Wilson et al. proposed the hallmarks of NDDs: pathological protein aggregation, synaptic and neuronal network failure, abnormal proteostasis, cytoskeletal defects, altered energy metabolism, DNA and RNA abnormalities, inflammation, and neuronal cell death. The accumulation of misfolded proteins leads to pathological aggregates that serve as key markers for the diagnosis and classification of NDD. NDDs caused by misfolded proteins include Alzheimer’s disease (AD), Parkinson’s diseases (PD), primary tauopathies, frontotemporal dementia (FTD), amyotrophic lateral sclerosis, synucleinopathies, Huntington’s disease, and prion diseases. Genetic mutation of several genes, including those encoding amyloid precursor proteins, tau protein, α-synuclein, TAR DNA-binding protein 43 (TDP-43), fused in sarcoma (FUS), and huntingtin, directly enhances protein aggregation, leading to NDDs. − In contrast, mutations in genes such as Presenilin 1 and 2 are indirectly associated with proteinopathy by promoting aggregation of key pathological proteins implicated in NDD. ,,,

In the amyloidogenic cascade, oligomers and fibrils are different entities with markedly distinct properties. Fibrils are thermodynamically stable, whereas oligomers are usually metastable intermediates. , Compared to the highly ordered structure of mature amyloid fibrils, oligomers are smaller, assemble more slowly, and manifest lower structural order. − In addition, their surfaces show greater hydrophobicity − and is generally more neurotoxic. According to their kinetic behavior, amyloid oligomers can broadly be categorized into fibrillar and nonfibrillar oligomers. Nonfibrillar oligomers possess disordered structure and then slowly convert into fibrillar oligomers with more ordered structure. Fibrillar oligomers rapidly elongate through monomer addition at both ends and exhibit potent seeding activity, thereby accelerating fibril propagation. Understanding the kinetic properties of amyloid oligomers, along with mechanistic insights into their association and dissociation, is essential for drug development to target the neurotoxic oligomers in NDDs.

HS-AFM has revolutionized the study of NDDs by providing real-time nanoscale visualization of dynamic protein interactions and conformational transitions critical to pathogenesis. In AD, HS-AFM has been instrumental in elucidating the aggregation mechanisms of amyloid-β (Aβ). Aβ1–42 possesses two distinct fibril growth modes: straight and spiral (Figure A). The interchanging between these growth modes is modulated by environmental factors such as salt composition. Structural polymorphism may contribute to disease heterogeneity, as variations in fibril architecture have been associated with clinical manifestations. Additionally, Feng et al. conducted HS-AFM scanning to quantify kinetic interactions among Aβ42 species by directly observing the real-time elongation of Aβ42 protein and its diverse conformations (Figure B). The study revealed that smaller oligomers dissociate via a single energy barrier, whereas larger aggregates showed heterogeneous binding energies, suggesting conformational diversity in toxic intermediates. These observations highlight the unique capability of HS-AFM to track transient, heterogeneous amyloid species without purification. , Nakano and colleagues reported that globular amyloid-β oligomer (gAβo) promoted fibrillation of low-molecular-weight (LMW) Aβ42, while gAβo itself remained nonfibrillar (Figure C). These results suggest that gAβo catalytically promotes LMW Aβ42 aggregation. The team also found that the molecular interactions of gAβo were modulated by both the presence and the quantity of mature fibril seeds in the reaction environment, emphasizing the complex and dynamic crosstalk among distinct Aβ species. These findings imply that gAβo would significantly accelerate Aβ aggregation in AD pathogenesis.

Applications of HS-AFM nanoimaging in elucidating the pathogenesis and potential therapeutics of neurodegenerative diseases (NDDs). (A) Nanoscopic observation of Aβ_1–42 using HS-AFM revealed its two distinct fibril growth modes, which were straight and spiral, regulated by environmental factors such as salt composition. Open and closed circles refer to the spiral and straight regions in fibrils. Reproduced with permission from ref . Copyright 2016 National Academy of Sciences. (B) Real-time observation of the interaction between Aβ_15–20nm and Aβ_agg during the elongation process. An Aβ_15–20nm (2) bound to an Aβ_agg (3), forming a stacked conformation (2 + 3). Subsequently, addition of Aβ_15–20nm (1 and 4) associated with stacked conformation, resulting in elongation. Representative topologies of different Aβ conformations captured by HS-AFM are presented here. Agg: aggregate; US: unstructured. Reproduced with permission from ref . Copyright 2019 Elsevier Ltd. (C) HS-AFM nanoimaging showed the slow fibrillation of low-molecular-weight (LMW) Aβ42. The fibrillation kinetics was significantly accelerated by the addition of globular amyloid-β (gAβ) oligomer (adapted and modified from ref ). Interestingly, the gAβ oligomers themselves did not undergo fibrillation. Reproduced under the terms of the Creative Common Attribution–Noncommercial–Noderivatives 4.0 International License (CC BY-NC-ND 4.0) from ref . Copyright 2024 Nakano et al. (D) α-synuclein monomers and dimers presented distinct patterns of conformational dynamics. Monomer predominantly appeared as compact, globular conformations, but also transiently exhibited extended tail-like protrusions and fully extended forms. In contrast, dimer showed reduced conformational flexibility, appearing as a dumbbell-shaped structure that changed slowly over time. Type-1 dimer consisted of two compact monomers, whereas type-2 dimer was formed by two extended monomers arranged in a nearly symmetrical manner. Reproduced with permission from ref . Copyright 2018 AIP Publishing. (E) Complexity of α-synuclein aggregation in Parkinson’s disease was revealed using HS-AFM. A schematic diagram illustrates that fibrillation of α-synuclein was regulated via cross-seeding of different α-synuclein species (wild type and mutants), resulting in deacceleration, acceleration, stoppage, or conformational changes (adapted and modified from ref ). Fibrils from self-seeding mirror the seed structure, whereas those from cross-seeding can differ. Reproduced from ref . Copyright 2020 American Chemical Society. (F) Real-time disruption of lipid membrane by pro-inflammatory protein S100A8 under neuroinflammation-like environment captured using HS-AFM. S100A8 did not affect BTLE (natural brain total lipid extract) or zwitterionic DC (DOPC/CHOL) lipid layers, but it rapidly destabilized anionic PS-containing DDDC (DOPC/DOPE/DOPS/CHOL) lipid membranes. DOPC: dioleoylphosphatidylcholine; CHOL: cholesterol; DOPE: dioleoylphosphatidylethanolamine; DOPS: dioleoylphosphatidylserine; PS: phosphatidylserine. Reproduced from ref . Copyright 2024 American Chemical Society. (G) Nanoscopic visualization of the synergistic effects of membrane compositions including ganglioside GM1 and cholesterol in promoting highly toxic mutant Aβ oligomers (Aβ_1–42 variant oG37C) binding at lipid bilayers, resulting in rapid membrane disruption. The oligomers bound to SPG (SM/POPC/GM1) and SPCG (SM/POPC/GM1/CHOL), but not to SP (SM/POPC) or SPC (SM/POPC/CHOL). Lipid disruption was found only on the SPCG layer. SM: sphingomyelin; POPC: 1-palmitoyl-2-oleoylphosphatidylcholine; CHOL: cholesterol. Reproduced with permission from ref . Copyright 2019 The Royal Society of Chemistry. (H–K) Nanoscopic therapeutic screening platform for NDD. Aβ-specific antibodies such as Lecanemab (H) and antibody 4396C (K) prevent the fibrillation of Aβ oligomers by binding either to Aβ42 oligomers (Lecanemab) or the end of fibril (antibody 4396C). Neurotoxic amyloid fibril destabilization by using chaperone, Hsc70 disaggregase (I), has been recorded using HS-AFM. (J) ALZ-801, a small-molecule Aβ inhibitor, selectively target the LMW Aβ42 into nontoxic globular aggregates while blocking fibril elongation. LMW: low molecular weight; HMW: high molecular weight. Reproduced from ref . Copyright 2023 American Chemical Society. Reproduced with permission from ref . Copyright 2021 National Academy of Sciences. Reproduced under the terms of the Creative Common Attribution-Noncommercial-Noderivatives 3.0 Unported (CC BY-NC-ND 3.0) License from ref . Copyright 2025 Muramatsu et al. Reproduced from ref . Copyright 2024 American Chemical Society.

In addition to Aβ, researchers have performed HS-AFM nanoimaging to study the structural dynamics and aggregation mechanisms of other key pathogenic proteins implicated in NDD. Zhang and co-workers used HS-AFM to compare the native structures and conformational dynamics of α-synuclein monomer and dimer (Figure D). Monomers predominantly adopt compact, globular conformations but can transiently extend tail-like protrusions and occasionally unfold into fully extended forms. In contrast, dimers exhibit reduced conformational flexibility, typically presenting a dumbbell-shaped morphology that remodels slowly over time. The team reported three different types of dimers that differ in monomer arrangement: type 1 dimer comprises two compact monomers; type 2 dimer consists of two extended monomers arranged in a nearly symmetrical manner; and type 3 dimer contains one compact and one extended monomer. Nakayama et al. have demonstrated the distinct kinetics and structural dynamics of α-synuclein fibrillation mediated by self-seeding or cross-seeding mechanisms (Figure E). Self-seeding refers to the α-synuclein fibrillation orchestrated by the same monomers (either wild-type or mutant), while cross-seeding refers to the α-synuclein fibrillation driven by two different types of α-synuclein (wild-type and mutant). The team found that in cross-seeding mode, a heterogeneous pool of α-synuclein seeds either produced distinct fibrils or no fibrillation occurred, demonstrating the complexity of α-synuclein aggregation in PD.

Besides protein aggregates, HS-AFM has also advanced our understanding of membrane interactions driving neurotoxicity. In AD, pro-inflammatory protein S100 calcium binding protein A8 (S100A8) was shown to selectively permeabilize anionic phosphatidylserine-rich membranes under low calcium conditions, mimicking intracellular environments during neuroinflammation (Figure F). Similarly, membrane compositions including ganglioside GM1 and cholesterol synergistically enhance toxic Aβ oligomer (Aβ_1–42 variant oG37C) binding at lipid bilayers, leading to rapid membrane destruction (Figure G). These findings highlight the feasibility of HS-AFM in deciphering lipid–protein interactions with spatiotemporal precision and clarifying neurotoxicity mechanisms.

HS-AFM not only provides a nanoscopic platform for elucidating NDD pathogenesis but also enables the study of potential therapeutics aimed at preventing the formation or dissolving of insoluble neurotoxic protein aggregates. HS-AFM also allows researchers to directly visualize and obtain mechanistic insights into fibrillation-inhibiting antibodies against Aβ. For example, the antiprotofibril antibody Lecanemab (Figure H) was found to bind laterally to Aβ protofibrils, stabilizing them and preventing toxic aggregate formation. Similarly, the antibody 4396C (Figure K) was found to selectively bind to the paused ends of growing fibrils, sterically hindering elongation and providing a novel strategy for inhibiting fibril propagation. In addition to antibodies, the effects of a small molecule Aβ inhibitor on amyloid fibrils have been studied using HS-AFM. ALZ-801 inhibits Aβ42 fibril formation by stabilizing low-molecular-weight (LMW) Aβ42 into nontoxic globular aggregates while blocking fibril elongation, as evidenced by HS-AFM imaging combined with Thioflavin T assays (Figure J). Endogenous proteins, Hsc70 disaggregase, for example, have been demonstrated by HS-AFM nanoimaging to dismantle toxic oligomers and short fibrils via tip destabilization and protofilament unzipping, revealing the mechanism underlying chaperone-mediated neuroprotection (Figure I).

Collectively, HS-AFM has become indispensable in neurodegenerative disease research, bridging molecular structure, dynamics, and function. By visualizing NDD key pathogenic protein aggregations − and their inhibition, − resolving membrane disruption mechanisms, , capturing chaperone activity, and decoding conformational dynamics, , HS-AFM provides a multidimensional view of NDD pathogenesis. Its integration with biophysical assays and computational modeling , positions it as a cornerstone for developing targeted therapies, offering hope for halting or reversing neurodegenerative cascades through precise mechanistic interventions.

Technical Limitations of HS-AFM and Mitigation Strategies

Despite its advantages, the intrinsic limitations of HS-AFM must be acknowledged to contextualize its findings. Discussion of these constraints is essential for assessing the biological relevance of the HS-AFM observations. Being label-free, HS-AFM recognizes targets primarily through surface topology. This entails a significant challenge for specificity, especially in complex biological mixtures. Generally, fibrillar structures, such as amyloids and cytoskeletal filaments, are easily distinguished, while globular proteins in a complex or mixture are difficult to identify. Therefore, the results obtained could be ambiguously interpreted for heterogeneous samples.

First, a functionalized cantilever, such as the macrocyclic peptide-conjugated tip as mentioned above, could provide specific nanoimaging through the interaction between the macrocyclic peptide and the target protein. Second, the integration of HS-AFM with fluorescence microscopy represents a transformative advance in nanoimaging, synergistically combining unparalleled structural resolution with a definitive molecular specificity. This correlative strategy overcomes the intrinsic limitations of each standalone technique: fluorescence microscopy precisely guides the AFM cantilever to dynamic regions of interest such as the leading edge of cells and specific organelles (e.g., NPC on GFP-labeled nuclear envelopes), thereby enabling targeted nanoimaging while minimizing sample damage. , This multimodal approach enables a simultaneous “touch-to-watch” modality, in which fluorescence microscopy identifies molecular identity and activity (e.g., clathrin or actin recruitment), while HS-AFM directly visualizes the associated nanoscale conformational dynamics (e.g., dynamic movement of FG-Nups in NPC and membrane invagination during endocytosis) in real time. ,, By correlating molecular function with structural transitions across diverse biological systems, from live mammalian cells to single protein complexes, this platform uniquely elucidates mechanistic principles underlying dynamic processes with exceptional spatiotemporal precision. , Collectively, this integration elevates nanoimaging beyond static structural visualization, enabling a comprehensive understanding of structure–function relationships with highly dynamic biological systems. Finally, computational approaches such as AlphaFold3, localized AFM, flexible fitting, and other molecule simulations can further strengthen HS-AFM analyses.

HS-AFM is also often criticized for its in vitro nature and perceived lack of biological relevance. Nevertheless, such critics undermine both the methodological rigor increasingly embedded in HS-AFM workflows and their unique capacity to cover mechanistic truths that are otherwise inaccessible. Like many transformative biophysical approaches, HS-AFM intentionally reduces cellular complexity to clarify elementary molecular events with remarkable spatiotemporal resolution, thereby prioritizing mechanistic fidelity over contextual completeness. Mitigation of artificiality can be done by manipulating three main components: the scanning environment, substrate surface, and sample incubation. Near physiological buffers include appropriate ionic strength, pH, nucleotide (e.g., GTP and ATP) concentration, and suitable macromolecular crowding agents (e.g., Ficoll and polyethylene glycol/PEG) can be considered for ensuring that observed dynamics closely approximate native behaviors. For example, crowding agents are important for the long-term stability of the nucleosome. Katan et al. demonstrated that careful selection of the crowding agent, PEG and poly(vinyl alcohol) (PVOH) but not BSA, yielded optimal results in nucleosome formation after 24 h incubation. In addition, modification of substrate surface is equally important to minimize surface-induced artifacts by using soft or biomimetic substrates, specific tethering strategies, and systematic comparisons across surface chemistries, showing that observed phenomena are intrinsic rather than adsorption-driven. ,

During sample incubation, samples may be either dried on the substrate or incubated in buffer prior to HS-AFM scanning. However, from a biological relevance standpoint, drying is generally not ideal for preserving intact structures in certain samples, such as membrane skeletons, even when fixation is applied. Similar concerns arise in EM sample preparation in which solvent evaporation is necessary for imaging under high-vacuum conditions, thereby perturbing the native organization of nanoparticles. A mitigation strategy involves the implementation of a buffer chamber for HS-AFM or graphene liquid cells for EM. , Biological relevance is further reinforced by integrating HS-AFM observations with complementary evidence, such as live-cell imaging, mutation analysis, and pharmacological perturbation, consequently establishing consistency between single-molecule dynamics and cellular phenotypes.

Although HS-AFM operates with exceptionally gentle tapping force (often <50 pN), these forces may still disrupt interactions mediated by weak interactions, such as individual hydrogen bonds or transient protein–protein interactions, raising the possibility of false-negative observations. Addressing this challenge requires continuous instrumental refinement. For example, the only trace imaging (OTI) method reported by Fukuda et al. enables HS-AFM to achieve faster and less-invasive imaging for fragile biological samples such as actin filaments and microtubules. Li and colleagues demonstrated that a high-frequency, high signal-to-noise ratio seesaw-shaped cantilever permits stable imaging at even lower effective forces and thus protects delicate biological samples.

Future Prospectives of HS-AFM in Biomedical Science

The main purpose of biomedical research is to integrate basic and preclinical research with clinical investigation, allowing translation from molecular mechanisms to patient outcomes. HS-AFM is uniquely suited to interrogate the structural properties, conformational dynamics, and molecular assembly of biological entities that underlie the pathogenesis of numerous diseases, including amyloidosis (deposition of abnormal protein aggregates), neurodegenerative diseases, and even nucleation and growth of salt crystals in lithiasis (e.g., gallstones and kidney stones). − Given that HS-AFM has been robustly applied to study various disease mechanisms described in the preceding sections, it is important to discuss the feasibility of extending HS-AFM beyond fundamental research toward tangible impacts in clinical assessment and personalized medicine. The ability of HS-AFM to characterize nanoparticles from body fluids at the single-particle level offers broad opportunities for clinical assessment.

We previously used HS-AFM to characterize exosome-like and ectosome-like sEVs in a heterogeneous pool of sEV samples. Liquid biopsy specimens of cancer patients contain sEVs that carry disease-specific signatures such as diagnostic and prognostic biomarkers (e.g., carcinoembryonic antigen/CEA and CA-125) , and predictive biomarkers (e.g., PD-L1). , Real-time visualization of dynamic antibody-sEV interaction is feasible using HS-AFM imaging, allowing the characterization of cancer biomarkers in clinical samples. Besides sEVs, amyloid-β-oligomer enriched cerebrospinal fluids from NDD patients can be profiled using HS-AFM nanoimaging to determine their oligomeric status and susceptibility to therapeutics. HS-AFM results can correlate with the deposition of amyloid-β in brain using other medical imaging approaches such as positron emission tomography. Kidney stones are often diagnosed when patients experience sudden sharp and severe pain. The recurrence of kidney stones is high, with a prevalence of 50% over 10 years. Calcium oxalate monohydrate and calcium oxalate dihydrate crystals with a wide range of sizes, from a few nanometers to tens of micrometers, could induce renal cell death resulting in inflammation. HS-AFM could play an important role in risk stratification and supportive diagnosis by assessing the minute crystals isolated from urine samples. As aforementioned, when coupled with digital PCR, direct single-molecule visualization and measurement of elongated FLT3-ITD amplicons relative to wild-type using HS-AFM provides highly sensitive and specific detection of minimal residual disease of AML.

The primary challenge for HS-AFM nanoimaging to be translated into clinical medicine is throughput, as the technique is low-throughput compared with other approaches, such as flow cytometry and blood chemistry assessment, which are often run in semi- or complete automation. Micro-/nanofluidic and nanopore technologies, including biological and solid-state platforms, enable high-throughput analyte assessment, − for example, nanopore sequencing. With the advancement of micro- and nanofabrication, the pore size is adjustable and further functionalized using chemical modification, genetic sequencing, or biomolecular coatings. − This technology could enhance HS-AFM throughput by rapidly capturing large amounts of specific analytes for subsequent characterization. Also, the integration with the application of machine learning for rapid, automated analysis of large HS-AFM image data sets is important to solve this bottleneck. Other minor challenges include technical complexity and dependence on complementary modalities, which should not be ignored. Standard operating procedures and proper training, together with user-friendly operating interfaces, could enhance user proficiency in HS-AFM imaging. HS-AFM should be fine-tuned and robustly streamlined for integration with clinical diagnostic data.

In therapeutic development, HS-AFM is also suitable for visualizing the assembly of functional biomaterials, such as peptoids that can be used as artificial lung surfactants to treat respiratory distress syndrome or lung injury. HS-AFM has been used for single-particle structural and dynamic characterization of engineered − and endogenous , nanoparticles, enabling detailed analysis of their controlled drug-release behavior and their stability under various physicochemical stresses. For example, HS-AFM can be performed to assess how pH modulation affects nanoparticle stability, both for long-term storage and for triggering drug release. − Besides, hydrostatic pressure has also been explored as a stimulus for controlled drug release, including therapeutic strategy for conditions such as high-altitude pulmonary edema.

Summary

In summary, HS-AFM is a powerful nanoimaging technique for elucidating disease pathogenesis at the single-molecule level. Its remarkable spatiotemporal resolution not only allows direct visualization of dynamic molecular events but also substantially boosts the scientific impact of such studies by revealing processes at the single-molecule level. For example, while techniques such as total internal reflection fluorescence microscopy provide population-level insights (e.g., FtsZ polymerization patterns), HS-AFM adds mechanistic value by resolving the real-time polymerization dynamics of individual FtsZ filaments. We have applied HS-AFM nanoimaging to address a range of biomedical questions, including the conformational dynamics of viral FPs (influenza A and SARS-CoV-2) involved in infection , and antibody neutralization; oligomerization of the SARS-CoV-2 accessory protein ORF6 associated with long COVID-19 complications; DNA condensation by protamines during spermatogenesis; recognition of estrogen response element by ERα; structural dynamics of sEVs under various physicochemical stresses; antibody-based characterization of sEVs; and the structural organization and FG-Nups dynamics of NPCs in nuclei isolated from cancer cell lines, organoid cultures, and mouse brain. ,, Altogether, HS-AFM is an indispensable tool in biomedical research. Its high spatiotemporal resolution provides not only insights into biophysical contexts but also unique mechanistic information that cannot be obtained by other methods alone. Nevertheless, there is no one-for-all methodology. Mutual complementarity with biochemical, biophysical, computational, and cellular approaches is paramount for generating comprehensive, persuasive conclusions and insights.

Acknowledgments

We are grateful to all members of the Richard Wong laboratory for their involvement, especially for those who are using HS-AFM for this biomedical discovery journey.

Assistant Professor WPI Nano Life Science Institute (WPI-NanoLSI), Institute for Frontier Science Initiative, Kanazawa University Kakuma-machi Kanazawa 920–1192, Japan, Email address: limkeesiang@staff.kanazawa-u.ac.jp. Professor WPI Nano Life Science Institute (WPI-NanoLSI), Institute for Frontier Science Initiative, Kanazawa University Kakuma-machi Kanazawa 920–1192, Japan, Email address: rwong@staff.kanazawa-u.ac.jp

Conceptualization: R.W.W. and K.L. Visualization: K.L. Writingoriginal draft: K.L. Writingreview and editing: K.L and R.W.W. Supervision: R.W.W.

This work was supported by the World Premier International Research Center Initiative (WPI). This work was funded by the following grants: MEXT/JSPS KAKENHI grant nos. 24K18449 (to K.L.); 22H05537, 22H02209, 23H04278, 24H01276, and 25H02360 (to R.W.W.); JST CREST grant no. JPMJCR22E3 (to R.W.W.); Takeda Science Foundation, Japan (to R.W.W.); Shimadzu Science Foundation, Japan (to R.W.W.); and Astellas Pathophysiology and Metabolism Research Association (K.L.).

High-speed atomic force microscopy (HS-AFM): an advanced scanning probe microscopy technique that enables real-time visualization of the structure and conformational dynamics of biological entities at the nanoscale level, with subsecond temporal resolution, under near-physiological buffer conditions. Fusogenic transition: dynamic conformational changes in viral fusion proteins that expose fusion peptides to mediate membrane fusion between viral and host endosomal membranes and facilitating viral genetic material entry into host cells. Liquid–liquid phase separation (LLPS): biomolecules, such as proteins and nucleic acids, demix from the surrounding solution to produce dense, membrane-free condensates that regulate biological processes such as nuclear transport and gene expression. Nuclear pore complex (NPC): a mega-Dalton protein complex comprised of over 30 different nucleoporins, which collectively regulate selective nucleocytoplasmic transport and gene expression. FG-Nups: a group of nucleoporins rich in phenylalanine and glycine that reside in the cytoplasmic region, central channel, and nuclear basket of NPCs, forming a dynamic selective barrier for nuclear transport. Small extracellular vesicles (sEVs): membrane-bound vesicles (30–150 nm) that carry proteins and nucleic acids, secreted by cells for local and distant communication. sEVs are involved in many diseases and are actively investigated as biomarkers and therapeutic nanoparticles. Liquid biopsy: a minimally invasive diagnostic technique that detects disease-correlated biomarkers, such as circulating tumor cells, cancer-derived nucleic acids, or extracellular vesicles, in body fluids, enabling early diagnosis, disease monitoring, and personalized medicine.

The authors declare no competing financial interest.

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PERMALINK

Zooming into Disease at the Nanoscale: High-Speed Atomic Force Microscopy in Biomedical Discovery

Keesiang Lim

Richard W Wong

Abstract

Introduction

1.

1. Summary of HS-AFM Nanoimaging in Infectious Disease Research (Virology).

4. Summary of HS-AFM Applications in Neurodegeneration Research.

2. Summary of HS-AFM Nanoimaging in Infectious Disease Research (Bacteriology).

3. Summary of HS-AFM Applications in Cancer Research.

Application of HS-AFM for Research in Infectious Diseases

Pathogenic Viruses

2.

Pathogenic Bacteria

3.

Application of HS-AFM in Infertility Research

4.

Application of HS-AFM in Cancer Research

5.

Application of HS-AFM in Neurodegenerative Diseases

6.

Technical Limitations of HS-AFM and Mitigation Strategies

Future Prospectives of HS-AFM in Biomedical Science

Summary

Acknowledgments

References

ACTIONS

PERMALINK

RESOURCES

Cite

Add to Collections

PERMALINK

Zooming into Disease at the Nanoscale: High-Speed Atomic Force Microscopy in Biomedical Discovery

Keesiang Lim

Richard W Wong

Abstract

Introduction

1.

1. Summary of HS-AFM Nanoimaging in Infectious Disease Research (Virology).

4. Summary of HS-AFM Applications in Neurodegeneration Research.

2. Summary of HS-AFM Nanoimaging in Infectious Disease Research (Bacteriology).

3. Summary of HS-AFM Applications in Cancer Research.

Application of HS-AFM for Research in Infectious Diseases

Pathogenic Viruses

2.

Pathogenic Bacteria

3.

Application of HS-AFM in Infertility Research

4.

Application of HS-AFM in Cancer Research

5.

Application of HS-AFM in Neurodegenerative Diseases

6.

Technical Limitations of HS-AFM and Mitigation Strategies

Future Prospectives of HS-AFM in Biomedical Science

Summary

Acknowledgments

References

ACTIONS

PERMALINK

RESOURCES

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