Application Of Atomic Force Microscopy Research Articles

Automated High-Throughput Atomic Force Microscopy Single-Cell Nanomechanical Assay Enabled by Deep Learning-Based Optical Image Recognition.

Mechanical forces are essential for life activities, and the mechanical phenotypes of single cells are increasingly gaining attention. Atomic force microscopy (AFM) has been a standard method for single-cell nanomechanical assays, but its efficiency is limited due to its reliance on manual operation. Here, we present a study of deep learning image recognition-assisted AFM that enables automated high-throughput single-cell nanomechanical measurements. On the basis of the label-free identification of the cell structures and the AFM probe in optical bright-field images as well as the consequent automated movement of the sample stage and AFM probe, the AFM probe tip could be accurately and sequentially moved onto the specific parts of individual living cells to perform a single-cell indentation assay or single-cell force spectroscopy in a time-efficient manner. The study illustrates a promising method based on deep learning for achieving operator-independent high-throughput AFM single-cell nanomechanics, which will benefit the application of AFM in mechanobiology.

Applying the Atomic Force Microscopy Technique in Medical Sciences-A Narrative Review.

Penetrating deep into the cells of the human body in real time has become increasingly possible with the implementation of modern technologies in medicine. Atomic force microscopy (AFM) enables the effective live imaging of cellular and molecular structures of biological samples (such as cells surfaces, components of biological membranes, cell nuclei, actin networks, proteins, and DNA) and provides three-dimensional surface visualization (in X-, Y-, and Z-planes). Furthermore, the AFM technique enables the study of the mechanical, electrical, and magnetic properties of cells and cell organelles and the measurements of interaction forces between biomolecules. The technique has found wide application in cancer research. With the use of AFM, it is not only possible to differentiate between healthy and cancerous cells, but also to distinguish between the stages of cancerous conditions. For many years, AFM has been an important tool for the study of neurodegenerative diseases associated with the deposition of peptide amyloid plaques. In recent years, a significant amount of research has been conducted on the application of AFM in the evaluation of connective tissue cell mechanics. This review aims to provide the spectrum of the most important applications of the AFM technique in medicine to date.

Deep Learning Image Recognition-Assisted Atomic Force Microscopy for Single-Cell Efficient Mechanics in Co-culture Environments.

Atomic force microscopy (AFM)-based force spectroscopy assay has become an important method for characterizing the mechanical properties of single living cells under aqueous conditions, but a disadvantage is its reliance on manual operation and experience as well as the resulting low throughput. Particularly, providing a capacity to accurately identify the type of the cell grown in co-culture environments without the need of fluorescent labeling will further facilitate the applications of AFM in life sciences. Here, we present a study of deep learning image recognition-assisted AFM, which not only enables fluorescence-independent recognition of the identity of single co-cultured cells but also allows efficient downstream AFM force measurements of the identified cells. With the use of the deep learning-based image recognition model, the viability and type of individual cells grown in co-culture environments were identified directly from the optical bright-field images, which were confirmed by the following cell growth and fluorescent labeling results. Based on the image recognition results, the positional relationship between the AFM probe and the targeted cell was automatically determined, allowing the precise movement of the AFM probe to the target cell to perform force measurements. The experimental results show that the presented method was applicable not only to the conventional (microsphere-modified) AFM probe used in AFM indentation assay for measuring the Young's modulus of single co-cultured cells but also to the single-cell probe used in AFM-based single-cell force spectroscopy (SCFS) assay for measuring the adhesion forces of single co-cultured cells. The study illustrates deep learning imaging recognition-assisted AFM as a promising approach for label-free and high-throughput detection of single-cell mechanics under co-culture conditions, which will facilitate unraveling the mechanical cues involved in cell-cell interactions in their native states at the single-cell level and will benefit the field of mechanobiology.

The influence of substrate temperature on the structure and optical properties of NiO thin films deposited using the magnetron sputtering in the layer-by-layer growth regime

Vanadium oxide (VO x ) thin films are promising materials, exhibiting electrical, optical, and mechanical properties highly tunable by processing and structure. This work uniquely applying atomic force microscopy (AFM) nanoindentation correlated with X-ray diffractometry and Raman spectroscopy structural analysis to investigate the intricate connections between VO x post-annealing, phase composition, and resulting nanoscale mechanical functionality. Utilizing an ultra-sharp diamond tip as a nanoscale indenter, indentation is performed on VO x films with systematic variations in structure – from mixed insulating oxides to VO 2 -dominated films. Analytical modeling enables extraction of hardness and elastic modulus with nanoscale resolution. Dramatic mechanical property variations are observed between compositions, with order-of-magnitude increases in hardness and elastic modulus for the VO 2 -rich films versus insulating oxides. Ion implantation further enhances nanomechanical performance through targeted defect engineering. Correlating indentation-derived trends with detailed structural and morphological characterization elucidates explicit structure-property relationships inaccessible by other techniques. The approach provides critical mechanics-driven insights into links between VO x synthesis, structure evolution, and property development. Broader implementation will accelerate processing optimization for electronics and advanced fundamental understanding of nanoscale structure-functionality relationships

Nanomechanical properties of polycrystalline vanadium oxide thin films of different phase composition

Vanadium oxide (VO x ) thin films are promising materials, exhibiting electrical, optical, and mechanical properties highly tunable by processing and structure. This work uniquely applying atomic force microscopy (AFM) nanoindentation correlated with X-ray diffractometry and Raman spectroscopy structural analysis to investigate the intricate connections between VO x post-annealing, phase composition, and resulting nanoscale mechanical functionality. Utilizing an ultra-sharp diamond tip as a nanoscale indenter, indentation is performed on VO x films with systematic variations in structure – from mixed insulating oxides to VO 2 -dominated films. Analytical modeling enables extraction of hardness and elastic modulus with nanoscale resolution. Dramatic mechanical property variations are observed between compositions, with order-of-magnitude increases in hardness and elastic modulus for the VO 2 -rich films versus insulating oxides. Ion implantation further enhances nanomechanical performance through targeted defect engineering. Correlating indentation-derived trends with detailed structural and morphological characterization elucidates explicit structure-property relationships inaccessible by other techniques. The approach provides critical mechanics-driven insights into links between VO x synthesis, structure evolution, and property development. Broader implementation will accelerate processing optimization for electronics and advanced fundamental understanding of nanoscale structure-functionality relationships

Characterizing Morphometric and Nanomechanical Malignant Cell Features in a Rare Paediatric γδ T-acute Lymphoblastic Leukaemia: Insights from a Single Case Study Using Atomic Force Microscopy

The most common cancer in paediatric age is acute lymphoblastic leukaemia (ALL) which accounts for nearly a quarter of all cases of paediatric cancer. ALL cases are classified as B-cell or T-cell precursor ALL, based on their immunophenotypical features. Patients with T-cell ALL (T-ALL) comprise 10–15% of all newly diagnosed cases and are more prevalent in boys and in older age compared to the overall incidence peak age of ALL. In this case report, we present the morphometric and nanomechanical features of T-lymphoblasts derived from an 11-month-old infant with a rare subtype of γδ T-ALL with aggressive biological behavior. To investigate these features, we employed atomic force microscopy (AFM) and compared the blast cell deviations found in their nanostructure and elasticity to those of normal lymphocytes from a healthy child. The malignant T-lymphoblasts exhibited reduced roughness and Young’s modulus values. This single case analysis demonstrates the potential of the AFM method to provide additional information regarding the characteristics of malignant cells and suggests its potential as a complementary approach for distinguishing neoplastic cells from normal cells. The application of AFM could potentially facilitate the introduction of in vitro tests to determine the efficacy of anti-leukemic treatments.

Use of atomic force microscopy for characterization of model membranes and cells

Background: To provide a fundamental understanding of the potential and use of atomic force microscopy (AFM) in medicine and the life sciences, this work presents a thorough description of imaging and non-imaging atomic force microscopy modes for characterizing model membranes and cells at the nanoscale. Methods: The imaging and non-imaging AFM modes are described with examples in terms of the characterization of topographic, morphological, and nanomechanical sample properties. Results: AFM imaging of supported lipid bilayers (SLBs) revealed the effects of temperature and medium composition on SLB topography in the gel and fluid phases, and on the bilayer thickness. Non-imaging AFM showed the strengthening of the SLB in both phases by the ion binding process.Imaging of neuronal and neuroblastoma cells with and without treatment revealed morphological changes in shape, volume, roughness, and Feret dimension. Non-imaging AFM showed the change in cell elasticity induced by the treatment with H2O2 with and without quercetin and by the treatment with copper and myricetin. The measurements of cells elasticity revealed a reorganization of the cytoskeleton and filament structures. Conclusions: Diverse applications of imaging and non-imaging AFM can provide important information about the underlying processes in biologically relevant systems. AFM, as a complementary technique to other biomedical methods, allows screening and monitoring of physiological changes at the nanoscale.

Characteristics and Functionality of Cantilevers and Scanners in Atomic Force Microscopy.

In this paper, we provide a systematic review of atomic force microscopy (AFM), a fast-developing technique that embraces scanners, controllers, and cantilevers. The main objectives of this review are to analyze the available technical solutions of AFM, including the limitations and problems. The main questions the review addresses are the problems of working in contact, noncontact, and tapping AFM modes. We do not include applications of AFM but rather the design of different parts and operation modes. Since the main part of AFM is the cantilever, we focused on its operation and design. Information from scientific articles published over the last 5 years is provided. Many articles in this period disclose minor amendments in the mechanical system but suggest innovative AFM control and imaging algorithms. Some of them are based on artificial intelligence. During operation, control of cantilever dynamic characteristics can be achieved by magnetic field, electrostatic, or aerodynamic forces.

Soft matter analysis via atomic force microscopy (AFM): A review

Soft materials or soft condensed matter are being globally developed for various technological applications for chemical, consumer goods, pharmaceutical, agri-business, and petroleum industries. As such, there is a growing research interest in understanding the dynamics, structure, and morphology of soft materials, particularly in soft interfacial materials. While atomic force microscopy (AFM) is conventionally used for solid matter research studies, this review focuses on the emerging application of AFM as an effective analytical tool for investigating soft materials. By harnessing the capabilities of AFM, researchers can explore soft condensed matter in novel ways. Specifically, this review discusses the three AFM modes; contact, non-contact, and tapping modes, highlighting their working principles and recent applications in soft matter analysis. Moreover, derivatives of each principal AFM mode, such as Lateral Force Microscopy (LFM), nanolithography, force spectroscopy, Conductive AFM (CAFM), Scanning Polarization Force Microscopy (SPFM), and PeakForce Tapping (PFT) are included and discussed in detail. Critical analysis and comparison of these imaging modes are presented, shedding light on their advantages and disadvantages in soft matter analysis. This review aims to provide researchers with valuable insights for further advancements in the field by presenting a comprehensive overview of AFM modes in soft matter research.

Application of AFM on Identifying Mechanical Properties of Individual Minerals and Surface Properties of Crack with High Resolution in Shale

Improving the resolution and accuracy of the mechanical properties of organic-rich shale is very important. The results can reveal the mechanical properties of shale from micro scale and serve as a guide for the design of hydraulic fracture optimization parameters. This study introduced an advanced technique to obtain the mechanical properties of shale with high resolution (58.6 nm/pixel) by combining SEM, EDS, and Atomic Force Microscopy (AFM). To locate the target area in SEM and AFM accurately, a positioning technique that uses special distributions of pyrite was established. AFM PeakForce QNM mode was selected due to its advantages at capturing topography and mechanical properties in material. Results illustrated the ability of AFM to obtain the mechanical properties (modulus) of individual mineral components in shale, the detailed topography of crack, and mechanical properties of minerals in a specific area. In particular, the mechanical properties of minerals around crack explained the layered distribution of minerals around the fractures, and the cracks developed in the clay mineral layer was detected. This article demonstrates the great potential application of AFM in shale.

Fucose Binding Cancels out Mechanical Differences between Distinct Human Noroviruses.

The majority of nonbacterial gastroenteritis in humans and livestock is caused by noroviruses. Like most RNA viruses, frequent mutations result in various norovirus variants. The strain-dependent binding profiles of noroviruses to fucose are supposed to facilitate norovirus infection. It remains unclear, however, what the molecular mechanism behind strain-dependent functioning is. In this study, by applying atomic force microscopy (AFM) nanoindentation technology, we studied norovirus-like particles (noroVLPs) of three distinct human norovirus variants. We found differences in viral mechanical properties even between the norovirus variants from the same genogroup. The noroVLPs were then subjected to fucose treatment. Surprisingly, after fucose treatment, the previously found considerable differences in viral mechanical properties among these variants were diminished. We attribute a dynamic switch of the norovirus P domain upon fucose binding to the reduced differences in viral mechanical properties across the tested norovirus variants. These findings shed light on the mechanisms used by norovirus capsids to adapt to environmental changes and, possibly, increase cell infection. Hereby, a new step towards connecting viral mechanical properties to viral prevalence is taken.

Atomic Force Microscopy Methods to Measure Tumor Mechanical Properties

Atomic force microscopy (AFM) is a popular tool for evaluating the mechanical properties of biological materials (cells and tissues) at high resolution. This technique has become particularly attractive to cancer researchers seeking to bridge the gap between mechanobiology and cancer initiation, progression, and treatment resistance. The majority of AFM studies thus far have been extensively focused on the nanomechanical characterization of cells. However, these approaches fail to capture the complex and heterogeneous nature of a tumor and its host organ. Over the past decade, efforts have been made to characterize the mechanical properties of tumors and tumor-bearing tissues using AFM. This has led to novel insights regarding cancer mechanopathology at the tissue scale. In this Review, we first explain the principles of AFM nanoindentation for the general study of tissue mechanics. We next discuss key considerations when using this technique and preparing tissue samples for analysis. We then examine AFM application in characterizing the mechanical properties of cancer tissues. Finally, we provide an outlook on AFM in the field of cancer mechanobiology and its application in the clinic.

Insights into the Morphology and Surface Properties of Microalgae at the Nanoscale by Atomic Force Microscopy (AFM): A Review

Atomic force microscopy (AFM) is a method that provides the nanometer-resolution three-dimensional imaging of living cells in their native state in their natural physiological environment. In addition, AFM’s sensitivity to measure interaction forces in the piconewton range enables researchers to probe surface properties, such as elasticity, viscoelasticity, hydrophobicity and adhesion. Despite the growing number of applications of AFM as a method to study biological systems, AFM is not yet an established technique for studying microalgae. Following a brief introduction to the basic principles and operation modes of AFM, this review highlights the major contributions of AFM in the field of microalgae research. A pioneering AFM study on microalgae was performed on diatoms, revealing the fine structural details of diatom frustule, without the need for sample modification. While, to date, diatoms are the most studied class of microalgae using AFM, it has also been used to study microalgae belonging to other classes. Besides using AFM for the morphological characterization of microalgae at the single cell level, AFM has also been used to study the surface properties of microalgal cells, with cell elasticity being most frequently studied one. Here, we also present our preliminary results on the viscoelastic properties of microalgae cell (Dunaliella tertiolecta), as the first microrheological study of microalgae. Overall, the studies presented show that AFM, with its multiparametric characterization, alone or in combination with other complementary techniques, can address many outstanding questions in the field of microalgae.

Membrane Lesions and Reduced Life Span of Red Blood Cells in Preeclampsia as Evidenced by Atomic Force Microscopy.

Preeclampsia (PE) presents with maternal de novo hypertension and significant proteinuria and is one of the leading causes of maternal and perinatal morbidity and mortality with unknown etiology. The disease is associated with inflammatory vascular response and severe red blood cell (RBC) morphology changes. This study examined the nanoscopic morphological changes of RBCs from PE women versus normotensive healthy pregnant controls (PCs) and non-pregnant controls (NPCs) applying atomic force microscopy (AFM) imaging. The results revealed that the membrane of fresh PE RBCs differed significantly from healthy ones by the presence of invaginations and protrusions and an increased roughness value (Rrms) (4.7 ± 0.8 nm for PE vs. 3.8 ± 0.5 nm and 2.9 ± 0.4 nm for PCs and NPCs, respectively). PE-cells aging resulted in more pronounced protrusions and concavities, with exponentially increasing Rrms values, in contrast to the controls, where the Rrms parameter decreased linearly with time. The Rrms, evaluated on a 2 × 2 µm2 scanned area, for senescent PE cells (13 ± 2.0 nm) was significantly higher (p < 0.01) than that of PCs (1.5 ± 0.2 nm) and NPCs (1.9 ± 0.2 nm). Furthermore, the RBCs from PE patients appeared fragile, and often only ghosts were observed instead of intact cells at 20-30 days of aging. Oxidative-stress simulation on healthy cells led to RBC membrane features similar to those observed for PE cells. The results demonstrate that the most pronounced effects on RBCs in PE patients are related to impaired membrane homogeneity and strongly altered roughness values, as well as to vesiculation and ghost formation in the course of cell aging.

DNA at conductive interfaces: What can atomic force microscopy offer?

This review is devoted to the analysis of the capabilities of atomic force microscopy (AFM) in the study of the behavior of DNA at conducting solid–liquid and solid-air interfaces under electrochemical control and after the influence of the potential differences. The first part of the review presents the main aspects of AFM, the physical and chemical properties of DNA, and the behavior of DNA at conductive interfaces under the action of an electric field. The second part highlights the main categories of AFM applicability for DNA analysis at conducting interfaces from the point of view of “electrical stimulus-structure”, “process-structure”, and “process-structure-electrochemical properties” correlation. The applicability of AFM's main capabilities is shown: (i) visualization (of both monolayers and single molecules and hybridization complexes of DNA on a conductive surface), (ii) single-molecule force spectroscopy under electrochemical control, (iii) manipulation of DNA position at the nanoscale level. This review can be a starting point for further multidisciplinary DNA studies at electrochemical interfaces.

Hippo/YAP1 inhibition by verteporfin attenuates osteophyte formation in a mouse surgical osteoarthritis model

Osteophyte is an outgrowth of cartilage formed at the margins of the affected joint through endochondral ossification-like processes, and is one of the most common radiographic features of osteoarthritis (OA) that has been used to define the stage of disease. Osteophyte has been regarded to adapt the joint to the altered biomechanics of OA patients, limits joint movement and represent a source of joint pain, however, the mechanism of osteophyte formation, the morphology characteristics and biomechanical properties of osteophyte cells are remained unclear. In the present study, we isolated osteophyte cells and chondrocytes from late-stage OA patients who underwent total knee replacement surgeries, by applying Atomic Force Microscopy (AFM), we identified osteophyte cells were in irregular shape with dendrites, shrunk cell body, smooth surface and high elastic modulus (23.3 ± 5.4 kPa) when compared with chondrocytes (6.5 ± 1.8 kPa). In addition, osteophyte cells showed higher proliferation ability and colony formation capacity than chondrocytes. Mechanistically, we identified YAP1, the core transcriptional factor of Hippo signaling pathway, was highly expressed in osteophyte cell both at protein and RNA levels. Inactivation of Hippo/YAP1 signaling pathway by Verteporfin is sufficient to inhibit osteophyte cell proliferation in vitro and attenuate osteophyte formation in vivo. In conclusion, the morphology characteristic and biomechanical property of osteophyte cells at single cell level are quite different from chondrocytes, although we could not exclude other regulatory mechanisms, our findings suggested that Hippo/YAP1 is of great importance for osteophyte formation.

Investigation of Soft Matter Nanomechanics by Atomic Force Microscopy and Optical Tweezers: A Comprehensive Review

Soft matter exhibits a multitude of intrinsic physico-chemical attributes. Their mechanical properties are crucial characteristics to define their performance. In this context, the rigidity of these systems under exerted load forces is covered by the field of biomechanics. Moreover, cellular transduction processes which are involved in health and disease conditions are significantly affected by exogenous biomechanical actions. In this framework, atomic force microscopy (AFM) and optical tweezers (OT) can play an important role to determine the biomechanical parameters of the investigated systems at the single-molecule level. This review aims to fully comprehend the interplay between mechanical forces and soft matter systems. In particular, we outline the capabilities of AFM and OT compared to other classical bulk techniques to determine nanomechanical parameters such as Young's modulus. We also provide some recent examples of nanomechanical measurements performed using AFM and OT in hydrogels, biopolymers and cellular systems, among others. We expect the present manuscript will aid potential readers and stakeholders to fully understand the potential applications of AFM and OT to soft matter systems.

Correlation between Friction and Wear in Cylindrical Anchorages Simulated with Wear Machine and Analyzed with Scanning Probe and Electron Microscope.

This paper presents the possibilities of applying atomic force microscopy (AFM) techniques to the study of the wear of prosthetic biomaterials. In the conducted research, a zirconium oxide sphere was used as a test piece for mashing, which was moved over the surface of selected biomaterials: polyether ether ketone (PEEK) and dental gold alloy (Degulor M). The process was carried out with constant load force in an artificial saliva environment (Mucinox). An atomic force microscope with an active piezoresistive lever was used to measure wear at the nanoscale. The advantage of the proposed technology is the high resolution of observation (less than 0.5 nm) in the three-dimensional (3D) measurements in a working area of 50 × 50 × 10 µm. The results of nano wear measurements in two measurement setups are presented: zirconia sphere (Degulor M and zirconia sphere) and PEEK were examined. The wear analysis was carried out using appropriate software. Achieved results present a tendency coincident with the macroscopic parameters of materials.

Envision and Appraisal of Biomolecules and Their Interactions through Scanning Probe Microscopy

Scanning probe microscopy (SPM) has gifted a novel eye to envision nanotechnology and nanoscience. The SPM technique involves a sharp probe that moves over the sample surface and leads to produce signals, which facilitates a deep understanding of the structural, electronic, vibrational, optical, magnetic, (bio) chemical, and mechanical properties of the material. Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) measure various kinds of physical properties such as electric current, force, and capacitance. Moreover, AFM shows a prominent feature over STM that it accepts insulating surfaces and can work under physiological conditions, making it feasible to study biological molecules. Herein, the SPM approach toward biomolecule imaging and appraising different physiological processes with molecular resolution is highlighted. The review raises awareness regarding current obstacles in biological sample preparation and possible ways to conquer these difficulties. Subsequently, the recent applications of STM and AFM on various biomolecules with representative examples like DNA, protein, and carbohydrates are discussed. The conductance measurement studies of the biological samples emphasize applications like drug delivery, biosensors, molecular electronics, and spintronics fields. This is followed by an outlook of future perspectives making a pavement toward the basic science and applied field of biomolecules using SPM technology.

The dynamic interplay of dietary acid pH and concentration during early-stage human enamel and dentine erosion

Dental erosion continues to be a significant global health concern affecting nearly 30% of adults world-wide. With increasing soft drink consumption predominantly driving its prevalence, strategies for prevention and control are often implemented when erosion is severe, or rates are high in the populace. While factors affecting dental erosion such as pH on enamel has received much attention, the effect of dietary acid concentration when factored out to a commercially available pH has yet to be determined. Furthermore, understanding these effects on dentine, which is known to be more susceptible to erosion than enamel can unravel structure property relationships between acid characteristics and hard tissue types. This study aimed to develop structure-property relationships between dietary acid concentration, and pH, on the nano-textural and nano-mechanical properties of human enamel and dentine during short-term simulated drinking. To achieve this, a novel sample preparation methodology and analysis approach was developed by applying atomic force microscopy (AFM) in quantitative imaging mode. This enabled simultaneous measurement of enamel and dentine morphology and mechanical properties. Flow-cells were used to simulate drinking, exposing polished and smear layer free human enamel and dentine to 30 s repeated cycles of unbuffered citric acid 6% (pH = 1.88) and 1% (w/v) (pH = 2.55) and commercially available buffered pH = 3.8 states, for up to 180 s. The same 50 µm × 50 µm area of specimen morphology was analysed using in-house developed nanotextural analysis using the bearing area curve (BAC) with a focus on roughness (Ra), normalised peak (PA) and valley areas (VA). Mechanical properties were simultaneously measured for stiffness (N/m) after each 30 s. While all studies agree pH is a major factor in the erosion of enamel, here its dominance over the treatment time varied, with concentration surpassing the importance of pH after initial acid contact. Conversely, dentine erosion showed concentration-dependent changes in morpho-mechanical properties only. These results not only highlight the dynamic process of erosion, but how the interplay between acid characteristics and dental tissue type impact the progression of very early-stage erosion.