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.

High-resolution assessment of multidimensional cellular mechanics using label-free refractive-index traction force microscopy.

A critical requirement for studying cell mechanics is three-dimensional assessment of cellular shapes and forces with high spatiotemporal resolution. Traction force microscopy with fluorescence imaging enables the measurement of cellular forces, but it is limited by photobleaching and a slow acquisition speed. Here, we present refractive-index traction force microscopy (RI-TFM), which simultaneously quantifies the volumetric morphology and traction force of cells using a high-speed illumination scheme with 0.5-Hz temporal resolution. Without labelling, our method enables quantitative analyses of dry-mass distributions and shear (in-plane) and normal (out-of-plane) tractions of single cells on the extracellular matrix. When combined with a constrained total variation-based deconvolution algorithm, it provides 0.55-Pa shear and 1.59-Pa normal traction sensitivity for a 1-kPa hydrogel substrate. We demonstrate its utility by assessing the effects of compromised intracellular stress and capturing the rapid dynamics of cellular junction formation in the spatiotemporal changes in non-planar traction components.

Application of single cell force spectroscopy (SCFS) to the assessment of cell adhesion to peptide-decorated surfaces.

The adhesion forces of cells to peptide-coated functionalized materials were assessed through the Single Cell Force Spectroscopy (SCFS) technique in order to develop a methodology that allows the fast selection of peptide motifs that favor the interaction between cells and the biomaterial. Borosilicate glasses were functionalized using the activated vapor silanization process (AVS) and subsequently decorated with an RGD- containing peptide using the EDC/NHS crosslinking chemistry. It is shown that the RGD-coated glass induces larger attachment forces on mesenchymal stem cell cultures (MSCs), compared to the bare glass substrates. These higher forces correlate well with the enhanced adhesion of the MSCs observed on RGD-coated substrates through conventional adhesion cell cultures and inverse centrifugation tests. The methodology based on the SCFS technique presented in this work constitutes a fast procedure for the screening of new peptides or their combinations to select candidates that may enhance the response of the organism to the implant of the functionalized biomaterials.

Atomic force microscopy-based assessment of multimechanical cellular properties for classification of graded bladder cancer cells and cancer early diagnosis using machine learning analysis.

Cellular mechanical properties (CMPs) have been frequently reported as biomarkers for cell cancerization to assist objective cytology, compared to the current subjective method dependent on cytomorphology. However, single or dual CMPs cannot always successfully distinguish every kind of malignant cell from its benign counterpart. In this work, we extract 4 CMPs of four different graded bladder cancer (BC) cell lines by AFM (atomic force microscopy)-based nanoindentation to generate a CMP database, which is used to train a cancerization-grade classifier by machine learning. The classifier is tested on 4 categories of BC cells at different cancer grades. The classification shows split-independent robustness and an accuracy of 91.25% with an AUC-ROC (ROC stands for receiver operating characteristic, and ROC curve is a graphical plot which illustrates the performance of a binary classifier system as its discrimination threshold is varied) value of 97.98%. Finally, we also compare our proposed method with traditional invasive diagnosis and noninvasive cancer diagnosis relying on cytomorphology, in terms of accuracy, sensitivity and specificity. Unlike former studies focusing on the discrimination between normal and cancerous cells, our study fulfills the classification of 4 graded cell lines at different cancerization stages, and thus provides a potential method for early detection of cancerization. STATEMENT OF SIGNIFICANCE: We measured four cellular mechanical properties (CMPs) of 4 graded bladder cancer (BC) cell lines using AFM (atomic force microscopy). We found that single or dual CMPs cannot fulfill the task of BC cell classification. Instead, we employ MLA (Machine Learning Algorithm)-based analysis whose inputs are BC CMPs. Compared with traditional cytomorphology-based prognoses, the non-invasive method proposed in this study has higher accuracy but with many fewer cellular properties as inputs. The proposed non-invasive prognosis is characterized with high sensitivity and specificity, and thus provides a potential tumor-grading means to identify cancer cells with different metastatic potential. Moreover, our study proposes an objective grading method based on quantitative characteristics desirable for avoiding misdiagnosis induced by ambiguous subjectivity.

Development of hidden Markov modeling method for molecular orientations and structure estimation from high-speed atomic force microscopy time-series images.

High-speed atomic force microscopy (HS-AFM) is a powerful technique for capturing the time-resolved behavior of biomolecules. However, structural information in HS-AFM images is limited to the surface geometry of a sample molecule. Inferring latent three-dimensional structures from the surface geometry is thus important for getting more insights into conformational dynamics of a target biomolecule. Existing methods for estimating the structures are based on the rigid-body fitting of candidate structures to each frame of HS-AFM images. Here, we extend the existing frame-by-frame rigid-body fitting analysis to multiple frames to exploit orientational correlations of a sample molecule between adjacent frames in HS-AFM data due to the interaction with the stage. In the method, we treat HS-AFM data as time-series data, and they are analyzed with the hidden Markov modeling. Using simulated HS-AFM images of the taste receptor type 1 as a test case, the proposed method shows a more robust estimation of molecular orientations than the frame-by-frame analysis. The method is applicable in integrative modeling of conformational dynamics using HS-AFM data.

Low cost and massively parallel force spectroscopy with fluid loading on a chip.

Current approaches for single molecule force spectroscopy are typically constrained by low throughput and high instrumentation cost. Herein, a low-cost, high throughput technique is demonstrated using microfluidics for multiplexed mechanical manipulation of up to ~4000 individual molecules via molecular fluid loading on-a-chip (FLO-Chip). The FLO-Chip consists of serially connected microchannels with varying width, allowing for simultaneous testing at multiple loading rates. Molecular force measurements are demonstrated by dissociating Biotin-Streptavidin and Digoxigenin-AntiDigoxigenin interactions along with unzipping of double stranded DNA of varying sequence under different dynamic loading rates and solution conditions. Rupture force results under varying loading rates and solution conditions are in good agreement with prior studies, verifying a versatile approach for single molecule biophysics and molecular mechanobiology. FLO-Chip enables straightforward, rapid, low-cost, and portable mechanical testing of single molecules that can be implemented on a wide range of microscopes to broaden access and may enable new applications of molecular force spectroscopy.

Simultaneous assessment of radial and axial myocyte mechanics by combining atomic force microscopy and carbon fibre techniques.

Cardiomyocytes sense and shape their mechanical environment, contributing to its dynamics by their passive and active mechanical properties. While axial forces generated by contracting cardiomyocytes have been amply investigated, the corresponding radial mechanics remain poorly characterized. Our aim is to simultaneously monitor passive and active forces, both axially and radially, in cardiomyocytes freshly isolated from adult mouse ventricles. To do so, we combine a carbon fibre (CF) set-up with a custom-made atomic force microscope (AFM). CF allows us to apply stretch and to record passive and active forces in the axial direction. The AFM, modified for frontal access to fit in CF, is used to characterize radial cell mechanics. We show that stretch increases the radial elastic modulus of cardiomyocytes. We further find that during contraction, cardiomyocytes generate radial forces that are reduced, but not abolished, when cells are forced to contract near isometrically. Radial forces may contribute to ventricular wall thickening during contraction, together with the dynamic re-orientation of cells and sheetlets in the myocardium. This new approach for characterizing cell mechanics allows one to obtain a more detailed picture of the balance of axial and radial mechanics in cardiomyocytes at rest, during stretch, and during contraction. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Overview of Popular Techniques of Raman Spectroscopy and Their Potential in the Study of Plant Tissues.

Raman spectroscopy is one of the main analytical techniques used in optical metrology. It is a vibration, marker-free technique that provides insight into the structure and composition of tissues and cells at the molecular level. Raman spectroscopy is an outstanding material identification technique. It provides spatial information of vibrations from complex biological samples which renders it a very accurate tool for the analysis of highly complex plant tissues. Raman spectra can be used as a fingerprint tool for a very wide range of compounds. Raman spectroscopy enables all the polymers that build the cell walls of plants to be tracked simultaneously; it facilitates the analysis of both the molecular composition and the molecular structure of cell walls. Due to its high sensitivity to even minute structural changes, this method is used for comparative tests. The introduction of new and improved Raman techniques by scientists as well as the constant technological development of the apparatus has resulted in an increased importance of Raman spectroscopy in the discovery and defining of tissues and the processes taking place in them.

Quantitative Assessment of Tip Effects in Single-Molecule High-Speed Atomic Force Microscopy Using DNA Origami Substrates.

High-speed atomic force microscopy (HS-AFM) is widely employed in the investigation of dynamic biomolecular processes at a single-molecule level. However, it remains an open and somewhat controversial question, how these processes are affected by the rapidly scanned AFM tip. While tip effects are commonly believed to be of minor importance in strongly binding systems, weaker interactions may significantly be disturbed. Herein, we quantitatively assess the role of tip effects in a strongly binding system using a DNA origami-based single-molecule assay. Despite its femtomolar dissociation constant, we find that HS-AFM imaging can disrupt monodentate binding of streptavidin (SAv) to biotin (Bt) even under gentle scanning conditions. To a lesser extent, this is also observed for the much stronger bidentate SAv-Bt complex. The presented DNA origami-based assay can be universally employed to quantify tip effects in strongly and weakly binding systems and to optimize the experimental settings for their reliable HS-AFM imaging.

Assessment of biophysical properties of Haemonchus contortus from different life cycle stages with atomic force microscopy.

Atomic force microscopy (AFM) was used in this work to investigate the ultrastructural and mechanical characteristics of Haemonchus contortus, the major gastrointestinal nematode that infects small ruminants worldwide. The biophysical characterization of this species is extremely important in order to reveal mechanisms of action of drugs and to classify its ultrastructure and biomechanical properties. High-resolution topographic images by AFM as well as data on biomechanical properties of cuticles were obtained at different stages of H. contortus. The results reveal details of the mechanical and structural properties of this nematode never observed before for nematodes parasite with other microscope techniques. Qualitative and quantitative reductions in the elasticity of the larvae stage egg were compared with those of the morulae stage, and the increased adhesion of unsheathed L3 were compared with the same stage of sheathed larvae. The results presented here open possibilities for understanding the mechanisms of drug and biomolecular actions that can be used to control infections caused by H. contortus.

Deciphering multivalent glycocluster-lectin interactions through AFM characterization of the self-assembled nanostructures.

Pseudomonas aeruginosa is a human opportunistic pathogen responsible for lung infections in cystic fibrosis patients. The emergence of resistant strains and its ability to form a biofilm seem to give a selective advantage to the bacterium and thus new therapeutic approaches are needed. To infect the lung, the bacterium uses several virulence factors, like LecA lectins. These proteins are involved in bacterial adhesion due to their specific interaction with carbohydrates of the host epithelial cells. The tetrameric LecA lectin specifically binds galactose residues. A new therapeutic approach is based on the development of highly affine synthetic glycoclusters able to selectively link with LecA to interfere with the natural carbohydrate-LecA interaction. In this study, we combined atomic force microscopy imaging and molecular dynamics simulations to visualize and understand the arrangements formed by LecA and five different glycoclusters. Our glycoclusters are small scaffolds characterized by a core and four branches, which terminate in a galactose residue. Depending on the nature of the core and the branches, the glycocluster-lectin interaction can be modulated and the affinity increased. We show that glycocluster-LecA arrangements highly depend on the glycocluster architecture: the core influences the rigidity of the geometry and the directionality of the branches, whereas the nature of the branch determines the compactness of the structure and the ease of binding.

Fast and high-resolution mapping of elastic properties of biomolecules and polymers with bimodal AFM.

Fast, high-resolution mapping of heterogeneous interfaces with a wide elastic modulus range is a major goal of atomic force microscopy (AFM). This goal becomes more challenging when the nanomechanical mapping involves biomolecules in their native environment. Over the years, several AFM-based methods have been developed to address this goal. However, none of these methods combine sub-nanometer spatial resolution, quantitative accuracy, fast data acquisition speed, wide elastic modulus range and operation in physiological solutions. Here, we present detailed procedures for generating high-resolution maps of the elastic properties of biomolecules and polymers using bimodal AFM. This requires the simultaneous excitation of the first two eigenmodes of the cantilever. An amplitude modulation (AM) feedback acting on the first mode controls the tip-sample distance, and a frequency modulation (FM) feedback acts on the second mode. The method is fast because the elastic modulus, deformation and topography images are obtained simultaneously. The method is efficient because only a single data point per pixel is needed to generate the aforementioned images. The main stages of the bimodal imaging are sample preparation, calibration of the instrument, tuning of the microscope and generation of the nanomechanical maps. In addition, with knowledge of the deformation, bimodal AFM enables reconstruction of the true topography of the surface. It takes ~9 h to complete the whole procedure.

Multifrequency Kelvin probe force microscopy on self assembled molecular layers and quantitative assessment of images' quality.

The application of single-pass multifrequency Kelvin probe force microscopy (KPFM) for topography and contact potential difference (CPD) measurements of organic self-assembled monolayers (SAM) is demonstrated. Four modes of mechanical and electrical cantilever excitation were tested in order to obtain the best possible resolution in the CPD measurements. The algorithm using maximum capacity of information channel for quantitative image quality assessment was proposed to compare and assess the quality of the recorded images and imaging modes. The improvement of the quality of CPD imaging in multiresonance operation was confirmed.

Calibration of colloidal probes with atomic force microscopy for micromechanical assessment.

Mechanical assessment of biological materials and tissue-engineered scaffolds is increasingly focusing at lower length scale levels. Amongst other techniques, atomic force microscopy (AFM) has gained popularity as an instrument to interrogate material properties, such as the indentation modulus, at the microscale via cantilever-based indentation tests equipped with colloidal probes. Current analysis approaches of the indentation modulus from such tests require the size and shape of the colloidal probe as well as the spring constant of the cantilever. To make this technique reproducible, there still exist the challenge of proper calibration and validation of such mechanical assessment. Here, we present a method to (a) fabricate and characterize cantilevers with colloidal probes and (b) provide a guide for estimating the spring constant and the sphere diameter that should be used for a given sample to achieve the highest possible measurement sensitivity. We validated our method by testing agarose samples with indentation moduli ranging over three orders of magnitude via AFM and compared these results with bulk compression tests. Our results show that quantitative measurements of indentation modulus is achieved over three orders of magnitude ranging from 1 kPa to 1000 kPa via AFM cantilever-based microindentation experiments. Therefore, our approach could be used for quantitative micromechanical measurements without the need to perform further validation via bulk compression experiments.

High-throughput assessment of mechanical properties of stem cell derived red blood cells, toward cellular downstream processing.

Stem cell products, including manufactured red blood cells, require efficient sorting and purification methods to remove components potentially harmful for clinical application. However, standard approaches for cellular downstream processing rely on the use of specific and expensive labels (e.g. FACS or MACS). Techniques relying on inherent mechanical and physical properties of cells offer high-throughput scalable alternatives but knowledge of the mechanical phenotype is required. Here, we characterized for the first time deformability and size changes in CD34+ cells, and expelled nuclei, during their differentiation process into red blood cells at days 11, 14, 18 and 21, using Real-Time Deformability Cytometry (RT-DC) and Atomic Force Microscopy (AFM). We found significant differences (p < 0.0001; standardised mixed model) between the deformability of nucleated and enucleated cells, while they remain within the same size range. Expelled nuclei are smaller thus could be removed by size-based separation. An average Young's elastic modulus was measured for nucleated cells, enucleated cells and nuclei (day 14) of 1.04 ± 0.47 kPa, 0.53 ± 0.12 kPa and 7.06 ± 4.07 kPa respectively. Our identification and quantification of significant differences (p < 0.0001; ANOVA) in CD34+ cells mechanical properties throughout the differentiation process could enable development of new routes for purification of manufactured red blood cells.

A cost-effective method to immobilize hydrated soft-tissue samples for atomic force microscopy.

Immobilizing hydrated soft tissue specimens for atomic force microscopy (AFM) is a challenge. Here, we describe a simple and very cost-effective immobilization method, based on the use of transglutaminase in an aqueous environment, and successfully apply it to AFM characterization of human native Wharton's Jelly (nWJ), the gelatinous connective tissue matrix of the umbilical cord. A side-by-side comparison with a widely used polyphenolic protein-based tissue adhesive (Corning Cell-Tak), which is known to bind strongly to virtually all inorganic and organic surfaces in aqueous environments, shows that both adhesives successfully immobilize nWJ in its physological hydrated state. The cost of transglutaminase, however, is over 3000-fold lower than that of Cell-Tak, making it a very attractive method for immobilizing soft tissues for AFM characterization.

Deformability Assessment of Waterborne Protozoa Using a Microfluidic-Enabled Force Microscopy Probe.

Many modern filtration technologies are incapable of the complete removal of Cryptosporidium oocysts from drinking-water. Consequently, Cryptosporidium-contaminated drinking-water supplies can severely implicate both water utilities and consumers. Existing methods for the detection of Cryptosporidium in drinking-water do not discern between non-pathogenic and pathogenic species, nor between viable and non-viable oocysts. Using FluidFM, a novel force spectroscopy method employing microchannelled cantilevers for single-cell level manipulation, we assessed the size and deformability properties of two species of Cryptosporidium that pose varying levels of risk to human health. A comparison of such characteristics demonstrated the ability of FluidFM to discern between Cryptosporidium muris and Cryptosporidium parvum with 86% efficiency, whilst using a measurement throughput which exceeded 50 discrete oocysts per hour. In addition, we measured the deformability properties for untreated and temperature-inactivated oocysts of the highly infective, human pathogenic C. parvum to assess whether deformability may be a marker of viability. Our results indicate that untreated and temperature-inactivated C. parvum oocysts had overlapping but significantly different deformability distributions.

Condensin Smc2-Smc4 Dimers Are Flexible and Dynamic.

Structural maintenance of chromosomes (SMC) protein complexes, including cohesin and condensin, play key roles in the regulation of higher-order chromosome organization. Even though SMC proteins are thought to mechanistically determine the function of the complexes, their native conformations and dynamics have remained unclear. Here, we probe the topology of Smc2-Smc4 dimers of the S. cerevisiae condensin complex with high-speed atomic force microscopy (AFM) in liquid. We show that the Smc2-Smc4 coiled coils are highly flexible polymers with a persistence length of only ∼ 4 nm. Moreover, we demonstrate that the SMC dimers can adopt various architectures that interconvert dynamically over time, and we find that the SMC head domains engage not only with each other, but also with the hinge domain situated at the other end of the ∼ 45-nm-long coiled coil. Our findings reveal structural properties that provide insights into the molecular mechanics of condensin complexes.

Simulation Assisted Analysis of the Intrinsic Stiffness for Short DNA Molecules Imaged with Scanning Atomic Force Microscopy.

Studying the mechanical properties of short segments of dsDNA can provide insight into various biophysical phenomena, from DNA looping to the organization of nucleosomes. Scanning atomic force microscopy (AFM) is able to acquire images of single DNA molecules with near-basepair resolution. From many images, one may use equilibrium statistical mechanics to quantify the intrinsic stiffness (or persistence length) of the DNA. However, this approach is highly dependent upon both the correct microscopic polymer model and a correct image analysis of DNA contours. These complications have led to significant debate over the flexibility of dsDNA at short length scales. We first show how to extract accurate measures of DNA contour lengths by calibrating to DNA traces of simulated AFM data. After this calibration, we show that DNA adsorbed on an aminopropyl-mica surface behaves as a worm-like chain (WLC) for contour lengths as small as ~20 nm. We also show that a DNA binding protein can modify the mechanics of the DNA from that of a WLC.