Atomic Force Microscopy Images Research Articles

Insights into the writing process of the mask-free nanoprinting fluid force microscopy technology

Platelets are activated immediately when contacting with non-physiological surfaces. Minimization of surface-induced platelet activation is important not only for platelet storage but also for other blood-contacting devices and implants. Chemical surface modification tunes the response of cells to contacting surfaces, but it requires a long process involving many regulatory challenges to transfer into a marketable product. Biophysical modification overcomes these limitations by modifying only the surface topography of already approved materials. The available large and random structures on platelet storage bags do not cause a significant impact on platelets because of their smallest size (only 1–3 μm) compared to other cells. We have recently demonstrated the feasibility of the mask-free nanoprint fluid force microscope (FluidFM) technology for writing dot-grid and hexanol structures. Here, we demonstrated that the technique allows the fabrication of nanostructures of varying features including grid, circle, triangle, and Pacman-like structures. Characteristics of nanostructures including height, width, and cross-line were analyzed and compared using atomic force microscopy imaging. Based on the results, we identified several technical issues, such as the printing direction and shape of structures that directly altered nanofeatures during printing. Importantly, both geometry and interspace governed the degree of platelet adhesion, especially, the structures with triangular shapes and small interspaces prevent platelet adhesion better than others. We confirmed that FluidFM is a powerful technique to precisely fabricate a variety of desired nanostructures for the development of platelet/blood-contacting devices if technical issues during printing are well controlled.

Effect of enzymatic modification on the structure and rheological properties of diluted alkali-soluble pectin fraction rich in RG-I

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP’s richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.

IrO2-ZrO2-SiO2 ternary oxide composites- based DSAs: Activity toward oxygen evolution reaction with long-term stability

BackgroundHigher purity of copper is produced by Electrowinning (EW) in the cathode. A major challenge in this technique is the anodic structure that causes in the energy consumption, corrosion resistance, and contamination of the copper produced in the cathode. MethodsTernary metallic oxide (IrO2-ZrO2-SiO2) composites with the different molar percentages of IrO2: SiO2 (10:60, 15:55, and 20:50%) were prepared on titanium substrate by the sol-gel technique, and then thermal decomposition to study the impact of IrO2 and SiO2 contents on the electrocatalyst performances of the dimensionally stable anodes (DSAs). The physicochemical properties of the DSAs were determined by X-ray diffraction (XRD), Field emission scanning electron microscope (FE-SEM), Atomic force microscopy (AFM), and electrochemical measurements. Significant findingsFE-SEM and AFM images display the impact of SiO2 on the crystallization and crystal growth of IrO2, leading to the formation of finer oxide particles and the porous morphology of the ternary oxide coatings. The DSA composed of 50 % SiO2 and 20 % IrO2 (IZS (20–30–50)) with a more porous surface area represented high apparent electrocatalytic activity toward the oxygen evolution reaction with long-term stability as analyzed under cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and accelerated lifetime (ALT) plots.

Step height measurement of monoatomic silicon crystal lattice steps with a commercial atomic force microscope

Abstract Precise control of advanced materials relies on accurate dimensional metrology at the sub-nanometre scale. At this scale, the accuracy of scanning probe microscopy (SPM) has been limited by the lack of traceable transfer standard artefacts with calibration structures of suitable dimensions. With the adoption in 2019 of the silicon crystal lattice spacing as a secondary realization of the metre in the International System of Units (SI), SPM users have direct access to a realization of the SI metre at the sub-nanometre level by means of the step height of self-assembled monatomic lattice steps that can form on the surface of silicon crystals. A key challenge of successfully adopting this pathway is establishing the need for established protocols to minimize measurement errors and artifacts in routine laboratory use.
In this study, step height measurements of monoatomic lattice steps in an ordinal/staircase structure on a Si(111) crystal surface have been derived from images acquired with a commercially available, research-level atomic force microscope (AFM). Measurement results derived from AFM images using three different SPM image processing and analysis software packages are compared. Significant sources of measurement uncertainty are identified; principally the contribution from the dependence on scan direction. The calibration of the
AFM derived from this measurment was used to traceably measure the sub-nanometre lattice steps on a silicon carbide crystal surface to demonstrate the viability of this calibration pathway.

REPROGRAMMING FILAMENTOUS FD VIRUSES TO CAPTURE COPPER IONS.

C-terminal truncated variants (A, VA, NVA, ANVA, FANVA and GFANVA) of our recently identified Cu(II) specific peptide "HGFANVA" were displayed on filamentous fd phages. Wild type fd-tet and engineered virus variants were treated with 100 mM Cu(II) solution at a final phage concentration of 1011vir/ml and 1012vir/ml. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) imaging before Cu(II) exposure showed»6-8 nm thick filamentous virus layer formation. Cu(II) treatment resulted in aggregated bundle-like assemblies with mineral deposition. HGFANVA phage formed aggregates with an excessive mineral coverage. As the virus concentration was 10-fold decreased, nanowire-like assemblies were observed for shorter peptide variants A, NVA and ANVA. Wild type fd phages did not show any mineral formation. Energy dispersive X-ray spectroscopy (EDX) analyses revealed the presence of C and N peaks on phage organic material. Cu peak was only detected for engineered viruses. Metal ion binding of viruses was next investigated by enzyme-linked immunosorbent assay (ELISA) analyses. Engineered viruses were able to bind Cu(II) forming mineralized intertwined structures although no His (H) unit was displayed. Such genetically reprogrammed virus based biological materials can be further applied for bioremediation studies to achieve a circular economy.

Engineered microbubbles decorated with red emitting carbon nanoparticles for efficient delivery and imaging

Altering the route of uptake by the cells is an attractive strategy to overcome drug-receptor adaptation problems. Carbon nanoparticles (CNPs) with emission beyond tissue autofluorescence for imaging biological tissues were used to study the phenomenon of uptake by the cells. In this regard, red-emitting carbon nanoparticles (CNPs) were synthesized and incorporated onto lipid microbubbles (MBs). The CNPs showed red emissions in the range of 640 nm upon excitation with 480 nm wavelength of light. Atomic force microscopic and confocal microscopic images showed the successful loading of CNPs onto the MB. Carbon nanoparticle loaded microbubbles (CNP-MBs) were treated with NIH 3 T3 cells at different concentrations. Confocal microscopic imaging studies confirm the presence of CNPs inside the treated cells. Cytotoxicity studies revealed that the CNPs showed minimal toxicity towards cells after loading onto MBs. The CNPs are usually taken up by the cells through the clathrin-mediated (CME) pathway, but when loaded onto MBs, the mechanism of uptake of CNPs is altered, and the uptake by the cells was observed even in the presence of inhibitors for the CME pathway. Loading CNPs onto MBs resulted in the uptake of CNPs by the cell through micropinocytosis and sonophoresis in the presence of ultrasound. The in vivo uptake CNP-MBs were performed in Danio rerio (Zebrafish larvae). This study provides insights into altering the uptake pathway through reformulation by loading nanoparticles onto MBs.

Atomic force microscopy simulations for CO-functionalized tips with deep learning

Atomic force microscopy (AFM) operating in the frequency modulation mode with a metal tip functionalized with a CO molecule is able to image the internal structure of molecules with an unprecedented resolution. The interpretation of these images is often difficult, making the support of theoretical simulations important. Current simulation methods, particularly the most accurate ones, require expertise and resources to perform ab initio calculations for the necessary inputs (i.e charge density and electrostatic potential of the molecule). Here, we propose a computationally inexpensive and fast alternative to the physical simulation of these AFM images based on a conditional generative adversarial network (CGAN), that avoids all force calculations, and uses as the only input a 2D ball–and–stick depiction of the molecule. We discuss the performance of the model when trained with different subsets extracted from the previously published QUAM-AFM database. Our CGAN reproduces accurately the intramolecular contrast observed in the simulated images for quasi–planar molecules, but has limitations for molecules with a substantial internal corrugation, due to the strictly 2D character of the input.

Rheological, thermal, and structural properties of heat-induced gluten gel: Effects of starch with varying degrees of debranching

This study evaluated the effects of starch with varying degree of debranching on the rheological, thermal, and structural properties of heat-induced gluten gel. As the duration of starch debranching treatment increased from 0 to 8 h, the viscoelasticity of the gel containing debranched starch (DBS) improved. Compared with the gluten gel (G), the gel strength of the G + DBS (8 h) sample increased by 65.2 %. The degradation temperature of gluten was minimally affected by DBS, while the weight loss rate increased by 4.4 %. Furthermore, the α-helical structure of gluten decreased, concomitant with an increase in β-sheet content. Notably, DBS treated for 8 h exhibited more hydrogen bonds with the tyrosine of gluten and triggered disulfide bridge conformation to transition from g-g-g to t-g-g, thereby reducing the stability of the molecular conformation of gluten proteins, as evidenced by the decreased height and width of the molecular chains observed in atomic force microscopy images. Overall, the composite gel structure induced by DBS exhibited a more continuous and homogeneous owing to the improved compatibility between DBS and gluten proteins, favoring the formation of a robust gel. These findings provide valuable insights for utilizing DBS to enhance gluten gel properties.

Nonclassical solid-state organic crystallization via particle migration and disintegration

Crystallization processes of organic molecules remain inadequately understood compared to their inorganic counterparts. Recent discoveries challenging classical theories of crystallization underscore the complexity of organic crystallization mechanism. We herein report the growth of rod-shaped diethyl 2,5-dihydroxyterephthalate (DDT) crystals at room temperature. Utilizing in-situ atomic force microscope (AFM) imaging, we observed multifaceted crystal growth which includes coalescence, ripening, and the long-range migration of micro-sized DDT particles. Additionally, we noted their simultaneous disintegration into nanoclusters before integrating into larger crystals. This study offers fresh insights into the non-classical crystallization processes of organic molecules.

Synthesis of Wrinkled MoS2 Thin Films Using a Two-Step Method Consisting of Magnetron Sputtering and Sulfurization in a Confined Space

Considering the increasing need for sustainable and economical energy storage solutions, the integration of layered materials such as MoS2 into these systems represents an important step toward enhancing energy sustainability and efficiency. Exploring environmentally responsible fabrication techniques, this study assesses wrinkled MoS2 thin films synthesized from distinct Mo and MoS2 targets, followed by sulfurization conducted in a graphite box. We utilized magnetron sputtering to deposit precursor Mo and MoS2 films on Si substrates, achieving thicknesses below 20 nm. This novel approach decreases sulfur by up to tenfold during sulfurization due to the confined space technique, contributing also to avoiding the formation of toxic gases such as SO2 or the necessity of using H2S, aligning with sustainable materials development. Thinner MoS2 layers were obtained post-sulfurization from the MoS2 precursors, as shown by X-ray reflectometry. Raman spectroscopy and grazing X-ray diffraction analyses confirmed the amorphous nature of the as-deposited films. Post-sulfurization, both types of films exhibited crystalline hexagonal MoS2 phases, with the sulfurized Mo showing a polycrystalline nature with a (100) orientation and sulfurized MoS2 displaying a (00L) preferred orientation. The X-ray photoelectron spectroscopy results supported a Mo:S ratio of 1:2 on the surface of the films obtained using the MoS2 precursor films, confirming the stoichiometry obtained by means of energy dispersive X-ray spectroscopy. Scanning electron microscopy and atomic force microscopy images revealed micrometer-sized clusters potentially formed during rapid cooling post-sulfurization, with an increased average roughness. These results open the way for the further exploration of wrinkled MoS2 thin films in advanced energy storage technologies.

Influence of the DEA Concentration on Structural and Optical Properties of Nanodot PbS Thin Films Growth by Chemical Solution Deposition: Unveiling Dual Optical Absorption Edges

The present paper presents a straightforward method for producing thin film layers of sulfide quantum dots (PbS-QDs) on a glass substrate using chemical solution deposition (CSD) assisted by dipcoating technique. The deposited PbS-QDs films were subjected to a comprehensive analysis using atomic force microscopy (AFM), energy dispersive X-ray (EDX) scanning electron microscopy (SEM), X-ray diffraction (XRD), UV–vis–IR absorption, and photoluminescence spectroscopic (PL) techniques to investigate the effects of varying concentrations of diethanol amine (DEA) on their morphology, crystal structure, elemental composition, light absorption, and emission characteristics. The spherical shape of the PbS-QDs was confirmed by AFM and SEM images with average sizes around 100 and 50 nm, respectively. The energy dispersive X-ray (EDX) analysis provides evidence the existence of Pb and S elements within the PbS matrix. X-ray diffraction (XRD) results validate that the deposited films exhibit high crystallinity, with a preferential orientation along the (111) plane and a face-centered cubic lattice structure of PbS. The crystallite size of PbS is measured to be 46.6 nm. Based on the optical absorption measurements, we have determined the size range of PbS nanocrystals to be between 4.3 and 11.5 nm. The optical studies reveal the presence of two optical absorption edges within the visible and infrared spectrum, two direct band gap energy, two cut-off wavelengths, two confinement energy, two Urbach energy tail, and dual emission peaks of PbS-QDs at room temperature. The analysis reveals the presence of two distinct band gap energies, one in the visible range (1.3–2.28 eV) and the other in the infrared range (0.65–0.88 eV), which can be attributed to the formation of two distinct sizes of quantum dots situated in two different layers. The first layer, deposited directly on the glass substrate, comprises quantum dots with an average size of approximately 5.2 nm, while the second layer contains quantum dots with an average size of about 9.5 nm. This ability to tune the band gap of PbS in the visible range up to the IR band (0.65–2.28 eV) is a critical feature that holds the potential for the development of innovative optoelectronic devices.

Nanoscale visualization of high-angle misorientations in quartz-rich rocks using SEM-EBSD and Atomic Force Microscopy

High-angle misorientations can significantly influence material properties. In this study, optical microscopy, Scanning Electron Microscope-Electron Backscatter Diffraction (SEM-EBSD), and Atomic Force Microscopy (AFM) have been used to investigate high-angle misorientations in quartz-bearing crustal rocks. Thin sections of high-grade quartzofeldspathic rocks were subjected to chemical mechanical polishing (CMP) with colloidal silica. In quartz, high-angle misorientations like random high angle grain boundaries (RHAGBs) and Dauphiné twin boundaries (DTBs) could be discriminated using EBSD techniques but not optical microscopy. In nanoscale AFM images, indented channels are observed along RHAGBs but not DTBs; these result from material removal during CMP, indicating lower compactness of RHAGBs compared to DTBs. Along any RHAGB, EBSD reveals different misorientations across segments between consecutive RHAGB-DTB intersections. Grains adjacent to these RHAGB segments have angles between their c-axes varying from 61-66° with parallel {101‾2} planes, and 81–84° with parallel {112‾2} planes, respectively. These symmetries represent the Japan and Sardinian twin laws of quartz, indicating that the RHAGB segments become low-energy twin boundaries, thereby reducing the overall surface energy of the aggregate. Finally, these results suggest that apart from surface topography quantification and high-resolution nanoscale imaging, AFM in conjunction with SEM-EBSD can be used for precisely locating sites for TEM study.

Image-driven machine learning for automatic characterization of grain size and distribution in smart vanadium dioxide thin films

This study focuses on characterizing grain size and distribution in vanadium dioxide (VO2) thin films using Atomic Force Microscope (AFM) images. Traditional segmentation methods face challenges in accurately detecting grains, and this paper addresses these limitations by employing machine learning-based classification approaches, defining three classes: grains, joints or dividing lines, and the background. Various classification methods, including supervised, unsupervised, and thresholding-based approaches, were investigated, demonstrating the superiority of machine learning-based methods over traditional segmentation techniques. Deep Learning (DL) models, such as Convolutional Neural Networks (CNN), Deep Belief Networks (DBN), and Deep Boltzmann Machines (DBM), achieved the best results in accuracy and precision. The DBN algorithm, in particular, outperformed other methods with a 93.66% accuracy in grain detection. This could be attributed to its probabilistic modeling and effective handling of challenging pixels, making it a powerful tool for accurate grain size and distribution analysis.

How Does the Antibiotic Amphotericin B Enter Membranes and What Does It Do There?

Amphotericin B is a popular antifungal antibiotic, but the exact way it works is still a matter of debate. Here, we used monolayers composed of phosphatidylcholine with ergosterol as a model of fungal lipid membranes to study drug incorporation from the aqueous phase and analyze the molecular reorganization of membranes underlying the biological activity of the antibiotic. The results show that the internalization of antibiotic molecules into membranes occurs only in the presence of ergosterol in the lipid phase. Comparison of images of solid-supported monolayers obtained by atomic force microscopy and lifetime imaging fluorescence microscopy shows the formation of intramembrane clusters of various sizes in the lipid phase, consisting mainly of antibiotic dimers and relatively large membrane pores (∼15 nm in diameter). The results reveal multiple modes of action of amphotericin B, acting simultaneously, each of which adversely affects the structural properties of the lipid membranes and their physiological functionality.

Reconfigurable aqueous 3D printing with adaptive dual locks.

Using aqueous two-phase systems (ATPSs) for three-dimensional (3D) printed complex structures has attracted considerable attention in the field of biomedicine. In this study, we present an unusual approach to constructing reconfigurable 3D printed structures within an aqueous environment. Inspired by biological systems, we introduce both specific and nonspecific interactions to anchor functionalized nanoparticles to the water-water interface, thereby imparting adaptive dual locks of structural integrity and permeability to the 3D printed liquid structures. Using state-of-the-art in situ liquid-liquid interfacial atomic force microscopy imaging, we successfully demonstrate various morphologies of interfacial films formed at the ATPS interface. In addition, by incorporating d-glucose or sodium alginate into the systems, the dual locks can be easily manipulated. Our study paves a pathway for 3D printing multiresponsive all-aqueous systems with controllable structures and permeability, showing promising implications for the development of smart drug delivery systems and in vivo reactions.

Designing reduced graphene oxide decorated Ni doped δ-MnO2 nanocomposites for supercapacitor applications

Reduced Graphene Oxide (rGO) decorated δ-MnO2 and Ni-doped δ-MnO2 samples have been synthesized by a microwave-assisted co-precipitation technique which is low cost, simple and fast reaction method. The structural and morphological properties of synthesized samples have been characterized using x-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). XRD result indicates that all samples have monoclinic crystal structure with δ phase of MnO2. The morphologies of MnO2 transformed into perforated porous nanoflakes with Ni doping and rGO composition in MnO2. AFM images confirmed that surface roughness for 3 % Ni-doped MnO2 samples is higher due to the chemical adsorption of samples. Raman spectroscopy results show the presence of vibrational and Raman active modes of MnO2.X-ray photoelectronic spectroscopy revealed the presence of mixed valencies of Mn such as Mn2+, Mn3+, and Mn4+ in prepared nanocomposites while Ni shows 2+ valency. The specific capacitance (Cs) of pure δ -MnO2 samples were 68 Fg-1 and rGO decorated 3 % Ni-doped MnO2 composition 694 Fg-1 at 10 mVs−1 respectively. EIS data of the rGO decorated Ni-doped MnO2 composition samples confirm that the porous nanoflake-shaped rGO decorated 3 % Ni-doped MnO2 composite sample has a low resistance value among all samples. These results reveal that rGO-decorated 3 % Ni-doped MnO2 nanoflakes should be a promising material for designing and fabricating the electrode material for high-performance supercapacitors.

Epitaxial AlN film with improved quality on Si (111) substrates realized by boron pretreatment via MOCVD

This study investigated the growth of AlN epitaxial films on 2-in. Si(111) via metal-organic chemical vapor deposition. By introducing triethylboron (TEB) during trimethylaluminum pretreatment, a nearly crack free AlN epilayer with a thickness of 500 nm was acquired. The x-ray diffraction rocking curves of AlN (002) and (102) exhibited full width at half maximum values of 0.22° and 0.36°, respectively. Atomic force microscopy image analysis showed that after the introduction of TEB, larger grains appeared on the surface of Si(111) substrate, promoting the 3D growth pattern of the subsequent AlN buffer layer. Laytec reflection curves depicted the morphological transition from 3D to 2D growth mode during AlN deposition. At the same time, the curvature value was significantly reduced by 20 km−1, and the Raman spectrum peak of E2(high) shifted from 648.7 to 652.5 cm−1, indicating that the surface tensile stress was greatly reduced, effectively suppressing the crack problem of AlN on Si(111).

Study of AlN Epitaxial Growth on Si (111) Substrate Using Pulsed Metal–Organic Chemical Vapour Deposition

A dense and smooth aluminium nitride thin film grown on a silicon (111) substrates using pulsed metal–organic chemical vapor deposition is presented. The influence of the pulsed cycle numbers on the surface morphology and crystalline quality of the aluminium nitride films are discussed in detail. It was found that 70 cycle numbers produced the most optimized aluminium nitride films. Field emission scanning electron microscopy and atomic force microscopy images show a dense and smooth morphology with a root-mean-square-roughness of 2.13 nm. The narrowest FWHM of the X-ray rocking curve for the AlN 0002 and 10–12 reflections are 2756 arcsec and 3450 arcsec, respectively. Furthermore, reciprocal space mapping reveals an in-plane tensile strain of 0.28%, which was induced by the heteroepitaxial growth on the silicon (111) substrate. This work provides an alternative approach to grow aluminium nitride for possible application in optoelectronic and power devices.

Morphology Observation of Two-Dimensional Monolayers of Model Proteins on Water Surface as Revealed by Dropping Method.

We have investigated the morphology of two-dimensional monolayers of gramicidin-D (GD) and alamethicin (Al) formed on the water surface by the dropping method (DM) using surface tension measurement (STm), Brewster angle microscopy (BAM), and atomic force microscopy (AFM). Dynamic light scattering (DLS) revealed that GD in alcoholic solutions formed a dimeric helical structure. According to the CD and NMR spectroscopies, GD molecules existed in dimer form in methanol and lipid membrane environments. The STm results and BAM images revealed that the GD dimer monolayer was in a liquid expanded (LE) state, whereas the Al monolayer was in a liquid condensed (LC) state. The limiting molecular area (A0) was 6.2 ± 0.5 nm2 for the GD-dimer and 3.6 ± 0.5 nm2 for the Al molecule. The AFM images also showed that the molecular long axes of both the GD-dimer and Al were horizontal to the water surface. The stability of each monolayer was confirmed by the time dependence of the surface pressure (π) observed using the STm method. The DM monolayer preparation method for GD-dimer and Al peptide molecules is a useful technique for revealing how the model biological membrane's components assemble in two dimensions on the water surface.

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.