Studying Structure and Functions of Nucleosomes with Atomic Force Microscopy.

Chromatin is an epigenetic platform for implementation of DNA-dependent processes. Nucleosome, as a basic level of chromatin compaction, largely determines its properties and structure. In the study of nucleosomes structure and functions physicochemical tools are actively used, such as magnetic and optical "tweezers", "DNA curtains", nuclear magnetic resonance, X-ray crystallography, and cryogenic electron microscopy, as well as optical methods based on Förster resonance energy transfer. Despite the fact that these approaches make it possible to determine a wide range of structural and functional characteristics of chromatin and nucleosomes with high spatial and time resolution, atomic force microscopy (AFM) complements the capabilities of these methods. The results of structural studies of nucleosome focusing on the AFM method development are presented in this review. The possibilities of AFM are considered in the context of application of other physicochemical approaches.

[Atomic Force Microscopy to Measure the Mechanical Property of Nanosized Lipid Vesicles and Its Applications].

Nanoparticles, including liposomes and lipid nanoparticles, have garnered global attention due to their potential applications in pharmaceuticals, vaccines, and gene therapies. These particles enable targeted delivery of new drug modalities such as highly active small molecules and nucleic acids. However, for widespread use of nanoparticle-based formulations, it is crucial to comprehensively analyze their characteristics to ensure both efficacy and safety, as well as enable consistent production. In this context, this review focuses on our research using atomic force microscopy (AFM) to study liposomes and lipid nanoparticles. Our work significantly contributes to the capability of AFM to measure various types of liposomes in an aqueous medium, providing valuable insights into the mechanical properties of these nanoparticles. We discuss the applications of this AFM technique in assessing the quality of nanoparticle-based pharmaceuticals and developing membrane-active peptides.

Assessment of sealing efficacy, radiopacity, and surface topography of a bioinspired polymer for perforation repair.

Background: Root perforation repair presents a significant challenge in dentistry due to inherent limitations of existing materials. This study explored the potential of a novel polydopamine-based composite as a root repair material by evaluating its sealing efficacy, radiopacity, and surface topography. Methods: Confocal microscopy assessed sealing ability, comparing the polydopamine-based composite to the gold standard, mineral trioxide aggregate (MTA). Radiopacity was evaluated using the aluminium step wedge technique conforming to ISO standards. Surface roughness analysis utilized atomic force microscopy (AFM), while field emission scanning electron microscopy (FESEM) visualized morphology. Results: The polydopamine-based composite exhibited significantly superior sealing efficacy compared to MTA (P < 0.001). Radiopacity reached 3 mm aluminium equivalent, exceeding minimum clinical requirements. AFM analysis revealed a smooth surface topography, and FESEM confirmed successful composite synthesis. Conclusion: This study demonstrates promising properties of the polydopamine-based composite for root perforation repair, including superior sealing efficacy, clinically relevant radiopacity, and smooth surface topography. Further investigation is warranted to assess its clinical viability and potential translation to endodontic practice.

Development of a Bio-Hybrid Mosquito Stinger-Based Atomic Force Microscopy Probe.

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The primary prevention strategy currently in use involves chemical repellents, which often prove ineffective as mosquitoes rapidly develop resistance. Consequently, the invention of new preventive methods is crucial. Such development hinges on a thorough understanding of mosquito biting behaviors, necessitating an experimental setup that accurately replicates actual biting scenarios with controllable testing parameters and quantitative measurements. To bridge this gap, a bio-hybrid atomic force microscopy (AFM) probe was engineered, featuring a biological stinger - specifically, a mosquito labrum - as its tip. This bio-hybrid probe, compatible with standard AFM systems, enables a near-authentic simulation of mosquito penetration behaviors. This method marks a step forward in the quantitative study of biting mechanisms, potentially leading to the creation of effective barriers against vector-borne diseases (VBDs) and opening new avenues in the fight against mosquito-transmitted illnesses.

Methods for making and observing model lipid droplets.

We present methods for making and testing the membrane biophysics of model lipid droplets (LDs). Methods are described for imaging LDs ranging in size from 0.1 to 40 µm in diameter with high-resolution microscopy and spectroscopy. With known LD compositions, membrane binding, sorting, diffusion, and tension were measured via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), and imaging flow cytometry. Additionally, a custom, small-volume pendant droplet tensiometer is described and used to measure the association of phospholipids to the LD surface. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes on lipid droplet monolayers.

Structural characterization of amyloid aggregates with spatially resolved infrared spectroscopy.

The self-assembly of proteins and peptides into ordered structures called amyloid fibrils is a hallmark of numerous diseases, impacting the brain, heart, and other organs. The structure of amyloid aggregates is central to their function and thus has been extensively studied. However, the structural heterogeneities between aggregates as they evolve throughout the aggregation pathway are still not well understood. Conventional biophysical spectroscopic methods are bulk techniques and only report on the average structural parameters. Understanding the structure of individual aggregate species in a heterogeneous ensemble necessitates spatial resolution on the length scale of the aggregates. Recent technological advances have led to augmentation of infrared (IR) spectroscopy with imaging modalities, wherein the photothermal response of the sample upon vibrational excitation is leveraged to provide spatial resolution beyond the diffraction limit. These combined approaches are ideally suited to map out the structural heterogeneity of amyloid ensembles. AFM-IR, which integrates IR spectroscopy with atomic force microscopy enables identification of the structural facets the oligomers and fibrils at individual aggregate level with nanoscale resolution. These capabilities can be extended to chemical mapping in diseased tissue specimens with submicron resolution using optical photothermal microscopy, which combines IR spectroscopy with optical imaging. This book chapter provides the basic premise of these novel techniques and provides the typical methodology for using these approaches for amyloid structure determination. Detailed procedures pertaining to sample preparation and data acquisition and analysis are discussed and the aggregation of the amyloid ß peptide is provided as a case study to provide the reader the experimental parameters necessary to use these techniques to complement their research efforts.

High-Speed Atomic Force Microscopy Imaging of DNA Three-Point-Star Motif Self Assembly Using Photothermal Off-Resonance Tapping.

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces. PORT has been shown to enable imaging of delicate assembly dynamics and precise monitoring of patterns formed by biomolecules. Thus far, the technique has been used for a variety of dynamic in vitro studies, including the DNA 3-point-star motif assembly patterns shown in this work. Through a series of experiments, this protocol systematically identifies the optimal imaging parameter settings and ultimate limits of the HS-PORT AFM imaging system and how they affect biomolecular assembly processes. Additionally, it investigates potential undesired thermal effects induced by the drive laser on the sample and surrounding liquid, particularly when the scanning is limited to small areas. These findings provide valuable insights that will drive the advancement of PORT mode's application in studying complex biological systems.

High-Speed Atomic Force Microscopy Reveals the Nucleosome Sliding and DNA Unwrapping/Wrapping Dynamics of Tail-less Nucleosomes.

Each nucleosome contains four types of histone proteins, each with a histone tail. These tails are essential for the epigenetic regulation of gene expression through post-translational modifications (PTMs). However, their influence on nucleosome dynamics at the single-molecule level remains undetermined. Here, we employed high-speed atomic force microscopy to visualize nucleosome dynamics in the absence of the N-terminal tail of each histone or all of the N-terminal tails. Loss of all tails stripped 6.7 base pairs of the nucleosome from the histone core, and the DNA entry-exit angle expanded by 18° from that of wild-type nucleosomes. Tail-less nucleosomes, particularly those without H2B and H3 tails, showed a 10-fold increase in dynamics, such as nucleosome sliding and DNA unwrapping/wrapping, within 0.3 s, emphasizing their role in histone-DNA interactions. Our findings illustrate that N-terminal histone tails stabilize the nucleosome structure, suggesting that histone tail PTMs modulate nucleosome dynamics.

Nanoscale colocalized thermal and chemical mapping of pharmaceutical powder aerosols.

Inhalation of pharmaceutical aerosol formulations is widely used to treat respiratory diseases. Spatially resolved thermal characterization offers promise for better understanding drug release rates from particles; however, this has been an analytical challenge due to the small particle size (from a few micrometers down to nanometers) and the complex composition of the formulations. Here, we employ nano-thermal analysis (nanoTA) to probe the nanothermal domain of a pharmaceutical aerosol formulation containing a mixture of fluticasone propionate (FP), salmeterol xinafoate (SX), and excipient lactose, which is widely used to treat asthma and chronic obstructive pulmonary disease (COPD). Furthermore, atomic force microscopy-infrared spectroscopy (AFM-IR) and AFM force measurements are performed to provide nanochemical and nanomechanical information to complement the nanothermal data. The colocalized thermal and chemical mapping clearly reveals the surface heterogeneity of the drugs in the aerosol particles and demonstrates the contribution of the surface chemical composition to the variation in the thermal properties of the particles. We present a powerful analytical approach for in-depth characterization of thermal/chemical/morphological properties of dry powder inhaler particles at micro- and nanometer scales. This approach can be used to facilitate the comparison between generics and reference inhalation products and further the development of high-performance pharmaceutical formulations.

Biophysical investigations using atomic force microscopy can elucidate the link between mouthfeel and flavour perception.

Food texture, along with taste and odour, is an important factor in determining food flavour. However, the physiological properties of oral texture perception require greater examination and definition. Here we explore recent trends and perspectives related to mouthfeel and its relevance in food flavour perception, with an emphasis on the biophysical point of view and methods. We propose that atomic force microscopy, combined with other biophysical techniques and more traditional food science approaches, offers a unique opportunity to study the mechanisms of mouthfeel at cellular and molecular levels. With this knowledge, food composition could be modified to develop healthier products by limiting salt, sugar, fat and calories while maintaining sensory qualities and consumer acceptance.

Atomic force microscopy investigation of DNA denaturation on a highly oriented pyrolytic graphite surface.

Understanding of DNA interaction with carbonaceous surfaces (including graphite, graphene and carbon nanotubes) is important for the development of DNA-based biosensors and other biotechnological devices. Though many issues related to DNA adsorption on graphitic surfaces have been studied, some important aspects of DNA interaction with graphite remain unclear. In this work, we use atomic force microscopy (AFM) equipped with super-sharp cantilevers to analyze the morphology and conformation of relatively long DNA molecule adsorbed on a highly oriented pyrolytic graphite (HOPG) surface. We have revealed the effect of DNA embedding into an organic monolayer of N,N'-(decane-1,10-diyl)-bis(tetraglycinamide) (GM), which may "freeze" DNA conformation on a HOPG surface during drying. The dependence of the mean squared point-to-point distance on the contour length suggests that DNA adsorbs on a bare HOPG by a "kinetic trapping" mechanism. For the first time, we have estimated the unfolded fraction of DNA upon contact with a HOPG surface (24 ± 5 %). The obtained results represent a novel experimental model for investigation of the conformation and morphology of DNA adsorbed on graphitic surfaces and provide with a new insight into DNA interaction with graphite.

Atomic force microscopy 3D structural reconstruction of individual particles in the study of amyloid protein assemblies.

Recent developments in atomic force microscopy (AFM) image analysis have made three-dimensional (3D) structural reconstruction of individual particles observed on 2D AFM height images a reality. Here, we review the emerging contact point reconstruction AFM (CPR-AFM) methodology and its application in 3D reconstruction of individual helical amyloid filaments in the context of the challenges presented by the structural analysis of highly polymorphous and heterogeneous amyloid protein structures. How individual particle-level structural analysis can contribute to resolving the amyloid polymorph structure-function relationships, the environmental triggers leading to protein misfolding and aggregation into amyloid species, the influences by the conditions or minor fluctuations in the initial monomeric protein structure on the speed of amyloid fibril formation, and the extent of the different types of amyloid species that can be formed, are discussed. Future perspectives in the capabilities of AFM-based 3D structural reconstruction methodology exploiting synergies with other recent AFM technology advances are also discussed to highlight the potential of AFM as an emergent general, accessible and multimodal structural biology tool for the analysis of individual biomolecules.

Localized surface plasmon resonance and atomic force microscopy study of model lipid membranes and their interactions with amyloid and melatonin.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid plaques in the brain. The toxicity of amyloid to neuronal cell surfaces arises from interactions between small intermediate aggregates, namely amyloid oligomers, and the cell membrane. The nature of these interactions changes with age and disease progression. In our previous work, we demonstrated that both membrane composition and nanoscale structure play crucial roles in amyloid toxicity, and that membrane models mimicking healthy neuron were less affected by amyloid than model membranes mimicking AD neuronal membranes. This understanding introduces the possibility of modifying membrane properties with membrane-active molecules, such as melatonin, to protect them from amyloid-induced damage. In this study, we employed atomic force microscopy and localized surface plasmon resonance to investigate the protective effects of melatonin. We utilized synthetic lipid membranes that mimic the neuronal cellular membrane at various stages of AD and explored their interactions with amyloid-ß(1-42) in the presence of melatonin. Our findings reveal that the early diseased membrane model is particularly vulnerable to amyloid binding and subsequent damage. However, melatonin exerts its most potent protective effect on this early-stage membrane. These results suggest that melatonin could act at the membrane level to alleviate amyloid toxicity, offering the most protection during the initial stages of AD.

Characterizing morphological alterations in blood related disorders through Atomic Force Microscopy.

Atomic Force Microscopy (AFM) is a very flexible method that can create topographical images from a range of materials and image surfaces. Significantly, AFM has emerged as an invaluable tool for dissecting the morphology and biochemical aspects of body cells and tissues. The high-resolution imaging capabilities of AFM enable researchers to discern alterations in cell morphology and understand the underlying mechanisms of diseases. It contributes to understanding disease etiology and progression. In the context of this review, our focus will be directed towards elucidating the pivotal role of AFM in analysis of blood related disorders. Through detailed comparisons with normal cells, we delve into the alterations in size, shape, and surface characteristics induced by conditions such as cancer, diabetes, anaemia, and infections caused by pathogens. In essence, various work described in this article highlights to bridge the gap between traditional microscopy and in-depth analysis of blood-related pathologies, which in turn offers valuable perspectives for both research and clinical applications in the field.

A method for analyzing AFM force mapping data obtained from soft tissue cryosections.

Atomic force microscopy (AFM) is a valuable tool for assessing mechanical properties of biological samples, but interpretations of measurements on whole tissues can be difficult due to the tissue's highly heterogeneous nature. To overcome such difficulties and obtain more robust estimates of tissue mechanical properties, we describe an AFM force mapping and data analysis pipeline to characterize the mechanical properties of cryosectioned soft tissues. We assessed this approach on mouse optic nerve head and rat trabecular meshwork, cornea, and sclera. Our data show that the use of repeated measurements, outlier exclusion, and log-normal data transformation increases confidence in AFM mechanical measurements, and we propose that this methodology can be broadly applied to measuring soft tissue properties from cryosections.

Atomic Force Microscopy in the Characterization of the Structure of Cell Wall Components.

Atomic force microscopy (AFM) has broken boundaries in the characterization of the supramolecular architecture of cell wall assemblies and single cell wall polysaccharides at the nanoscale level. Moreover, AFM provides an opportunity to evaluate the mechanical properties of cell wall material which is not possible with any other method. However, in the case of plant tissue, the critical step is a smart sample preparation that should not affect the polysaccharide structure or assembly and on the other hand should consider device limitations, especially scanner ranges. In this chapter, the protocols from the sample preparation, including isolation of cell wall material and extraction of cell wall polysaccharide fractions, through AFM imaging of polysaccharide assemblies and single molecules until an image analysis to obtain quantitative data characterizing the biopolymers are presented.

Atomic force microscopy correlates mechanical and electrical properties of HepG2 cells with curcumin concentration.

Hepatocellular carcinoma (HCC) is a highly prevalent cancer with a significant impact on human health. Curcumin, a natural compound, induces cytoskeletal changes in liver cancer cells and modifies the distribution of lipids, proteins, and polysaccharides on plasma membranes, affecting their mechanical and electrical properties. In this study, we used nanomechanical indentation techniques and Kelvin probe force microscopy (KPFM) based on atomic force microscopy (AFM) to investigate the changes in surface nanomechanical and electrical properties of nuclear and cytoplasmic regions of HepG2 cells in response to increasing curcumin concentrations. CCK-8 assays and flow cytometry results demonstrated time- and concentration-dependent inhibition of HepG2 cell proliferation by curcumin. Increasing curcumin concentration led to an initial increase and then decrease in the mechanical properties of nuclear and cytoplasmic regions of HepG2 cells, represented by the Young's modulus (E), as observed through nanoindentation. KPFM measurements indicated decreasing trends in both cell surface potential and height. Fluorescence microscopy results indicated a positive correlation between curcumin concentration and phosphatidylserine translocation from the inner to the outer membrane, which influenced the electrical properties of HepG2 cells. This study provides valuable insights into curcumin's mechanisms against cancer cells and aids nanoscale evaluation of therapeutic efficacy and drug screening.

Profiling native pulmonary basement membrane stiffness using atomic force microscopy.

Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) substructure measuring only ∼100-400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the precise assignment of the Young's modulus E of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young's modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young's modulus E using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of Young's moduli and generation of Young's modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1-2 d.

AFM Imaging Reveals MicroRNA-132 to be a Positive Regulator of Synaptic Functions.

The modification of synaptic and neural connections in adults, including the formation and removal of synapses, depends on activity-dependent synaptic and structural plasticity. MicroRNAs (miRNAs) play crucial roles in regulating these changes by targeting specific genes and regulating their expression. The fact that somatic and dendritic activity in neurons often occurs asynchronously highlights the need for spatial and dynamic regulation of protein synthesis in specific milieu and cellular loci. MicroRNAs, which can show distinct patterns of enrichment, help to establish the localized distribution of plasticity-related proteins. The recent study using atomic force microscopy (AFM)-based nanoscale imaging reveals that the abundance of miRNA(miR)-134 is inversely correlated with the functional activity of dendritic spine structures. However, the miRNAs that are selectively upregulated in potentiated synapses, and which can thereby support prospective changes in synaptic efficacy, remain largely unknown. Using AFM force imaging, significant increases in miR-132 in the dendritic regions abutting functionally-active spines is discovered. This study provides evidence for miR-132 as a novel positive miRNA regulator residing in dendritic shafts, and also suggests that activity-dependent miRNAs localized in distinct sub-compartments of neurons play bi-directional roles in controlling synaptic transmission and synaptic plasticity.

On machine learning analysis of atomic force microscopy images for image classification, sample surface recognition.

Atomic force microscopy (AFM or SPM) imaging is one of the best matches with machine learning (ML) analysis among microscopy techniques. The digital format of AFM images allows for direct utilization in ML algorithms without the need for additional processing. Additionally, AFM enables the simultaneous imaging of distributions of over a dozen different physicochemical properties of sample surfaces, a process known as multidimensional imaging. While this wealth of information can be challenging to analyze using traditional methods, ML provides a seamless approach to this task. However, the relatively slow speed of AFM imaging poses a challenge in applying deep learning methods broadly used in image recognition. This prospective is focused on ML recognition/classification when using a relatively small number of AFM images, aka small database. We discuss ML methods other than popular deep-learning neural networks. The described approach has already been successfully used to analyze and classify the surfaces of biological cells. It can be applied to recognize medical images, specific material processing, in forensic studies, even to identify the authenticity of arts. A general template for ML analysis specific to AFM is suggested, with a specific example of the identification of cell phenotype. Special attention is given to the analysis of the statistical significance of the obtained results, an important feature that is often overlooked in papers dealing with machine learning. A simple method for finding statistical significance is also described.