Atomic Force Microscopy-infrared Spectroscopy Research Articles

Evaluation of the nanostructure of calcium silicate hydrate based on atomic force microscopy-infrared spectroscopy experiments

Abstract Calcium silicate hydrate (C–S–H) is the main product of cement hydration, which forms the microstructure of cement via the stacking of basic nanocrystals or gel units, and has a substantial influence on the mechanical performance of cement. Tetrahedron chains of silicon oxide form the main nanoscale structure of basic C–S–H units. Evaluation on the nanostructure of these tetrahedron chains facilitates to understand the source of cement strength. This article first introduced the atomic force microscopy-infrared spectroscopy (AFM-IR) technique into evaluating the nanostructure of C–S–H. The nano infrared spectroscopy of stacking C–S–H nanograins and tetrahedron spatial distribution mapping was obtained. The results demonstrate that the relative quantity of tobermorite-like and jennite-like units in C–S–H nanograins can be analyzed by AFM-IR. The stacking between C–S–H particles is facilitated to a large extent by silicate ( SiO 4 2 − {\text{SiO}}_{4}^{2-} ) tetrahedron chains formed of three tetrahedrons bridged by two oxygen atoms (i.e., Q2 chains), and there are Q2 chains acting as bridges between C–S–H particles. The proportions of different types of Q2 chains available for facilitating C–S–H particle stacking vary at the nanoscale. AFM-IR spatial mapping demonstrate that the orientations of these Q2 chains are not evenly distributed. These findings provide experimental information of the stacking C–S–H gaps.

Polymerization-Induced Hierarchical Self-Assembly: From Monomer to Complex Colloidal Molecules and Beyond.

The nanoscale hierarchical design that draws inspiration from nature's biomaterials allows the enhancement of material performance and enables multifarious applications. Self-assembly of block copolymers represents one of these artificial techniques that provide an elegant bottom-up strategy for the synthesis of soft colloidal hierarchies. Fast-growing polymerization-induced self-assembly (PISA) renders a one-step process for the polymer synthesis and in situ self-assembly at high concentrations. Nevertheless, it is exceedingly challenging for the fabrication of hierarchical colloids via aqueous PISA, simply because most monomers produce kinetically trapped spheres except for a few PISA-suitable monomers. We demonstrate here a sequential one-pot synthesis of hierarchically self-assembled polymer colloids with diverse morphologies via aqueous PISA that overcomes the limitation. Complex formation of water-immiscible monomers with cyclodextrin via "host-guest" inclusion, followed by sequential aqueous polymerization, provides a linear triblock terpolymer that can in situ self-assemble into hierarchical nanostructures. To access polymer colloids with different morphologies, three types of linear triblock terpolymers were synthesized through this methodology, which allows the preparation of AXn-type colloidal molecules (CMs), core-shell-corona micelles, and raspberry-like nanoparticles. Furthermore, the phase separations between polymer blocks in nanostructures were revealed by transmission electron microscopy and atomic force microscopy-infrared spectroscopy. The proposed mechanism explained how the interfacial tensions and glass transition temperatures of the core-forming blocks affect the morphologies. Overall, this study provides a scalable method of the production of CMs and other hierarchical structures. It can be applied to different block copolymer formulations to enrich the complexity of morphology and enable diverse functions of nano-objects.

Unravelling the Structural Organization of Individual α-Synuclein Oligomers Grown in the Presence of Phospholipids

Parkinson's disease (PD) is a severe neurological disorder that affects more than 1 million people in the U.S. alone. A hallmark of PD is the formation of intracellular α-synuclein (α-Syn) protein aggregates called Lewy bodies (LBs). Although this protein does not have a particular localization in the central neural system, α-Syn aggregates are primarily found in certain areas of the midbrain, hypothalamus, and thalamus. Microscopic analysis of LBs reveals fragments of lipid-rich membranes, organelles, and vesicles. These and other pieces of experimental evidence suggest that α-Syn aggregation can be triggered by lipids. In this study, we used atomic force microscope infrared spectroscopy (AFM-IR) to investigate the structural organization of individual α-Syn oligomers grown in the presence of two different phospholipids vesicles. AFM-IR is a modern optical nanoscopy technique that has single-molecule sensitivity and subdiffraction spatial resolution. Our results show that α-Syn oligomers grown in the presence of phosphatidylcholine have a distinctly different structure than oligomers grown in the presence of phosphatidylserine. We infer that this occurs because of specific charges adopted by lipids, which in turn governs protein aggregation. We also found that the protein to phospholipid ratio has a substantial impact on the structure of α-Syn oligomers. These findings demonstrate that α-Syn is far more complex than expected from the perspective of the structural organization of oligomeric species.

Surface Functionalization of Ti3C2Tx MXene Nanosheets with Catechols: Implication for Colloidal Processing.

Precise tailoring of two-dimensional nanosheets with organic molecules is critical to passivate the surface and control the reactivity, which is essential for a wide range of applications. Herein, we introduce catechols to functionalize exfoliated MXenes (Ti3C2Tx) in a colloidal suspension. Catechols react spontaneously with Ti3C2Tx surfaces, where binding is initiated from a charge-transfer complex as confirmed by density functional theory (DFT) and UV-vis. Ti3C2Tx sheet interlayer spacing is increased by catechol functionalization, as confirmed by X-ray diffraction (XRD), while Raman and atomic force microscopy-infrared spectroscopy (AFM-IR) measurements indicate binding of catechols at the Ti3C2Tx surface occurs through metal-oxygen bonds, which is supported by DFT calculations. Finally, we demonstrate immobilization of a fluorescent dye on the surface of MXene. Our results establish a strategy for tailoring MXene surfaces via aqueous functionalization with catechols, whereby colloidal stability can be modified and further functionality can be introduced, which could provide excellent anchoring points to grow polymer brushes and tune specific properties.

Structural Insight in the Interfacial Effect in Ferroelectric Polymer Nanocomposites.

Both experimental results and theoretical models suggest the decisive role of the filler-matrix interfaces on the dielectric, piezoelectric, pyroelectric, and electrocaloric properties of ferroelectric polymer nanocomposites. However, there remains a lack of direct structural evidence to support the so-called interfacial effect in dielectric nanocomposites. Here, a chemical mapping of the interfacial coupling between the nanofiller and the polymer matrix in ferroelectric polymer nanocomposites by combining atomic force microscopy-infrared spectroscopy (AFM-IR) with first-principles calculations and phase-field simulations is provided. The addition of ceramic fillers into a ferroelectric polymer leads to augmentation of the local conformational disorder in the vicinity of the interface, resulting in the local stabilization of the all-trans conformation (i.e., the polar β phase). The formation of highly polar and inhomogeneous interfacial regions, which is further enhanced with a decrease of the filler size, has been identified experimentally and verified by phase-field simulations and density functional theory (DFT) calculations. This work offers unprecedented structural insights into the configurational disorder-induced interfacial effect and will enable rational design and molecular engineering of the filler-matrix interfaces of electroactive polymer nanocomposites to boost their collective properties.

Nanoscale Molecular Characterization of Hair Cuticle Cells Using Integrated Atomic Force Microscopy-Infrared Laser Spectroscopy.

The hair cuticle provides significant protection from external sources, as well as giving rise to many of its bulk properties, e.g., friction, shine, etc. that are important in many industries. In this work, atomic force microscopy-infrared spectroscopy (AFM-IR) has been used to investigate the nanometer-scale topography and chemical structure of human hair cuticles in two spectral regions. AFM-IR combines atomic force microscopy with a tunable infrared laser and circumvents the diffraction limit that has impaired traditional infrared spectroscopy, facilitating surface-selective spectroscopy at ultra-spatial resolution. This high resolution was exploited to probe the protein secondary structures and lipid content, as well as specific amino acid residues, e.g., cystine, within individual cuticle cells. Characterization across the top of individual cells showed large inhomogeneity in protein and lipid contributions that suggested significant changes to physical properties on approaching the hair edge. Additionally, the exposed layered sub-structure of individual cuticle cells allowed their chemical compositions to be assessed. The variation of protein, lipid, and cystine composition in the observed layers, as well as the measured dimensions of each, correspond closely to that of the epicuticle, A-layer, exocuticle, and endocuticle layers of the cuticle cell sub-structure, confirming previous findings, and demonstrate the potential of AFM-IR for nanoscale chemical characterization within biological substrates.

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20-100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Graphene Oxide-Induced Interfacial Transcrystallization of Single-Fiber Milkweed/Polycaprolactone/Polyvinylchloride Composites.

Understanding the interfacial crystallization is crucial for semi-crystalline polymer/natural fiber composites because it links to the final properties. This work reports, for the first time, the interfacial crystallization of a miscible blend between polycaprolactone (PCL) and polyvinylchloride (PVC) with milkweed fibers. We have first described the morphology of the fibers and the chemical composition of waxes covered on its surface. Our findings show that the transcrystallization (TC) layer of PCL/PVC could appear at the interface by simply coating with a layer of graphene oxide (GO) on the milkweed fiber. In our study, atomic force microscopy–infrared spectroscopy analysis shows that the crystallinity of the blends is higher at the vicinity of the interface compared to that in the bulk. The kinetic of the interfacial crystallization in terms of spherulite morphology and crystal growth rates at the nanoscale is examined. X-ray photoelectron spectroscopy and high-resolution transmission electron microscopy were used to analyze the prepared GO and evaluate its relationship with the interfacial crystallization behavior of the blends.

Nanoscale Structural Characterization of Individual Viral Particles Using Atomic Force Microscopy Infrared Spectroscopy (AFM-IR) and Tip-Enhanced Raman Spectroscopy (TERS).

Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy-infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.

Microstructure analysis and mechanical performance of crumb rubber modified asphalt concrete using the dry process

Globally, the vast majority of waste tires are landfilled, with catastrophic ecological consequences and in particular, serious threats to human health (e.g. fire, pests and soil contamination). Because of the increasing environmental awareness, the use of crumb rubber modified asphalt has become an important recycling strategy for waste tires. The so-called crumb rubber (CR), which is the recycled rubber from tires, has become a common additive in hot mix asphalt mixture due to its improvement of the mechanical performances of asphalt mixtures. The purpose of this study is to investigate the effect of adding crumb rubber to asphalt mixtures using the dry process by relating mechanical performances with microstructural characterizations. It was possible to observe the influence of conditioning process (time during which the asphalt mixture is kept at a high temperature after mixing) on the mechanical performance of the mixtures that depend primarily on the properties of the crumb rubber used. Specifically, it is shown that the conditioning time has an influence on the Marshall test results as well as the viscosity measurements. By using the Environmental Scanning Electron Microscope (ESEM), the distribution of the crumb rubber within the mixture has been investigated. In particular, the distribution of the crumb rubber by changing the conditioning time is of interest. The results showed that crumb rubber is well dispersed in the asphalt mixture when the conditioning time is increased. The effect of crumb rubber on the microstructure using Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) indicated that the main chemical change takes place in the para domain and catana or the so-called bee structures diminish on the CR modified bitumen as a consequence of more conditioning time.

Structural Characterization of Individual α-Synuclein Oligomers Formed at Different Stages of Protein Aggregation by Atomic Force Microscopy-Infrared Spectroscopy.

Aberrant α-synuclein aggregation is strongly associated with the onset and development of Parkinson's disease (PD). Therefore, characterizing the structure of toxic intermediate oligomers plays an essential role in better understanding their neurotoxicity. Using atomic force microscopy-infrared spectroscopy (AFM-IR), we were able to reveal the structure of α-synuclein oligomers present at different stages of protein aggregation and establish a relationship between morphology and structure on the single oligomer level. We were also able to probe the secondary structure evolution of individual oligomers. Moreover, the IR spectra of individual oligomers suggest structural rearrangement that is necessary for oligomers with an antiparallel β-sheet to propagate into fibrils that have a parallel-β-sheet secondary structure. Detailed investigation of structural organization of α-synuclein oligomers reported in this study is critically important to understand the toxicity of these protein species. We also anticipate that this work will help developing approaches for oligomer detection and consequently presymptomatic diagnostic of PD.

Semiquantitative Atomic Force Microscopy-Infrared Spectroscopy Analysis of Chemical Gradients in Silicone Optical Waveguides

Crossing losses in silicone optical waveguides are related to the magnitude and spatial extent of the waveguide refractive index gradient. When processing conditions are altered, the refractive index gradient can vary substantially, even when the formulation remains constant. Controlling the refractive index gradient requires control of the concentration of small molecules present within the core and clad layers. Developing a fundamental understanding of how small molecule migration drives changes in crossing loss requires the ability to examine chemical functionality over small length scales, which is a natural fit for atomic force microscopy-infrared spectroscopy (AFM-IR). In this work, AFM-IR spectra from model bilayer stacks are initially examined to understand molecular migration that occurs from heating the core and clad layers. The results of these model studies are then applied to photopatterned waveguide builds, where structure-function relationships are constructed between values of crossing loss and the concentration of C-H and O-H functionalities present in the core and clad layers. Results show that small molecule evaporation and migration are competing processes that need to be controlled to minimize crossing loss.

Direct Observation of Bound Water on Cotton Surfaces by Atomic Force Microscopy and Atomic Force Microscopy–Infrared Spectroscopy

A wet cotton rag becomes stiff after natural drying. We propose a model for this hardening phenomenon, which explains that the stiffness of cotton is caused by a cross-linked network between single...

Nanoscale chemical and mechanical heterogeneity of human dentin characterized by AFM-IR and bimodal AFM

Human dentin, as an important calcified tissue in the body, plays significant roles in withstanding masticatory forces and has a complex hierarchical organization. Understanding the composition and ultrastructure of dentin is critical for elucidating mechanisms of biomineralization under healthy and pathological states. Here, atomic force microscope infrared spectroscopy (AFM-IR) and AFM-based amplitude modulation-frequency modulation (AM-FM) techniques were utilized to detect the heterogeneity in chemical composition and mechanical properties between peritubular and intertubular dentin at the nanoscale. AFM-IR spectra collected from peritubular and intertubular dentin contained similar vibrational bands in the amide regions (I, II and III), suggesting that collagen may exist in both structures. A distinctive band at 1336 cm−1 indicative of SO stretching vibrations was detected only in peritubular dentin. AFM-IR imaging showed an uneven distribution of chemical components at different locations, confirming the heterogeneity of dentin. The Young’s modulus of peritubular dentin was higher, and was associated to a higher mineral content. This study demonstrated distinctive chemical and mechanical properties of peritubular dentin, implying the different development and mineralization processes between peritubular and intertubular dentin. AFM-IR is useful to provide compositional information on the heterogeneity of human dentin, helping to understand the mineral deposition mechanisms of dentin.

Field Manipulation of Infrared Absorption Properties in Thin Films

Infrared (IR) spectroscopy is a sensitive technique for fast and comprehensive analyses of organic and hybrid organic–inorganic materials, surfaces, and thin films. For interpretation of reflection and absorption IR spectra of thin films, vibrational bands in unpolarized IR spectra are often directly inspected. This approach can lead to misinterpretations. Herein, the influence of anisotropy and oscillator strength on the frequency and shape of observed vibrational bands in IR spectra of thin films is investigated. IR ellipsometric, polarization‐dependent reflectance spectra and polarization‐dependent atomic force microscope–IR spectroscopy (AFM–IR) nanopolarimetric absorption measurements of an about 84 nm thick anisotropic polyimide film (PI‐2611) are compared. The s‐ and p‐polarized spectra are used for separation of in‐plane and out‐of‐plane absorption components. The far‐field spectra are interpreted by analytical modeling. In detail, the frequency range of bands related to antiphase and in‐phase C═O stretching vibrations of the imide rings is investigated. For the antiphase C═O vibration as a relatively strong oscillator, the manipulation of absorption by the incident radiation‐induced electric fields in direction normal to the surface can be revealed in p‐polarized reflectance spectra and is identifiable in the p‐polarized IR nanopolarimetric absorption spectra.

Chemical analysis of tree growth in epoxy resin using AFM-IR spectroscopy

Recent advances have allowed submicron features of electrical trees to be imaged. Until recently it has not been possible to analyze chemical aging on the same scale. Here, the use of atomic force microscopy — infrared spectroscopy (AFM-IR) to characterize chemical degradation around a needle tip, and in the vicinity of electrical tree branches grown in epoxy resin is explored. The spectral signatures associated with the degradation of epoxies during electrical treeing are successfully identified with 50 nm resolution. This allows identification of chemical degradation in unprecedented detail. The method of polishing and grinding to prepare the samples was found to remove degraded material from exposed channels. This issue was overcome by not fully exposing the tree, but bringing it close enough to the surface that the AFM-IR was able to see within the channel. A short tree examined immediately after initiation showed no chemical degradation, indicating that its growth was not a chemically active process, suggesting electromechanical fracturing is more likely. Mature channels showed no singular chemical signature. Carbonyl groups were identified in all mature channels and were particularly concentrated at the branching points studied, suggesting higher levels of degradation during branching or accumulation at points of bifurcation. It is believed that these reactions are precipitated by hot electron impacts during discharges. AFM-IR has provided unprecedented detail concerning chemical damage incurred during the treeing process and can provide vital information to inform models of aging.

Detection of Antimicrobial Resistance-Related Changes in Biochemical Composition of Staphylococcus aureus by Means of Atomic Force Microscopy-Infrared Spectroscopy

The development of antimicrobial resistance (AMR) resulting from widespread antibiotic usage is occurring at an alarming pace, much faster than our understanding of the mechanisms behind resistance. Knowledge about resistance-related phenotypic and genotypic changes is critical for the development of new drugs. Here, we identify changes in the chemical composition of Staphylococcus aureus associated with the development of resistance to last resort drugs, vancomycin and daptomycin, using a novel, single cell, nanoscale technique, atomic force microscopy-infrared spectroscopy (AFM-IR), combined with chemometric analysis. We utilized paired clinical isolates, with the parent (susceptible) strain isolated prior to treatment and the daughter (resistant) strain obtained from the same patient after drug admission and clinical failure. We observed an increase in the amount of nonintracellular carbohydrates, indicating thickening or changes in the packing of the cell wall, as well as changes in the phospholipid content in relation to vancomycin resistance and daptomycin nonsusceptibility, respectively.

Nanoscale Structural Organization of Insulin Fibril Polymorphs Revealed by Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR).

Spontaneous aggregation of misfolded proteins typically results in the formation of morphologically and structurally different amyloid fibrils, protein aggregates that are strongly associated with various neurodegenerative disorders. Elucidation of the structural organization of amyloid aggregates is crucial to understanding their role in the onset and progression of these diseases. Using atomic force microscopy-infrared spectroscopy (AFM-IR), we investigated the structural organization of insulin fibrils. We found that insulin aggregation results in the formation of two structurally different fibril polymorphs. One polymorph has a β-sheet core surrounded by primarily unordered protein secondary structure. This polymorph has β-sheet-rich surface, whereas the surface of the other fibril polymorph is primarily composed of unordered protein. Using AFM-IR, we also revealed the structural organization of the insulin oligomers. Finally, we discovered a new pathway for amyloid fibril formation that is based on a fusion of several oligomers into a single fibril structure.

Brilliant mid-infrared ellipsometry and polarimetry of thin films: Toward laboratory applications with laser based techniques

Tunable quantum cascade lasers (QCLs) have recently been introduced as mid-infrared (mid-IR) sources for spectroscopic ellipsometric and polarimetric setups. QCLs, with their unique properties with respect to coherence and brilliance in either pulsed or continuous-wave operation, are opening up numerous new possibilities for laboratory and industrial applications. In this review, the authors will focus on thin-film characterization techniques like ellipsometric and nanopolarimetric methods and summarize related state-of-the-art techniques in this rapidly developing field. These methods are highly relevant for optical, electronical, and biomedical applications and allow detailed structural analyses regarding band properties, spectra–structure correlations, and material anisotropy. Compared to classical Fourier-transform-IR spectroscopy, thin-film sensitivity can be achieved at high spectral and spatial resolution (<0.5 cm−1, <150 μm). Measurement times are reducible by several orders of magnitude into the millisecond and microsecond range with laser-based polarimetric setups involving modulation or single-shot concepts. Thus, mid-IR ellipsometric and polarimetric hyperspectral imaging can be performed on the time scale of minutes. For mid-IR ellipsometric imaging, thickness and structure information become simultaneously accessible at spatial resolutions of a few 100 μm and possibly even at the micrometer scale by the integration of microscopic concepts. With the atomic force microscopy-infrared spectroscopy based nanopolarimetric approach, anisotropy in the absorption properties can be investigated with lateral resolutions beyond the diffraction limit, reaching a few 10 nm.

High Resolution Nanoscale Probing of Bacteriophages in an Inhalable Dry Powder Formulation for Pulmonary Infections.

Use of powder phage formulations for the treatment of multiple-drug-resistant pulmonary infections is gaining attention. To achieve therapeutic benefits, it is critical for phages to remain stable in the formulation. Assessment of phage stability relies on plaque assay (bioactivity), which requires powder samples to be reconstituted in liquid. The purpose of this study was to develop an innovative approach using photothermal-induced resonance-enhanced atomic force microscopy infrared spectroscopy (AFM-IR) to assess the presence of phages and investigate their protein conformation in the solid state. Staphylococcal phage S83 was spray-dried with lactose and sodium stearate using spray-drying. The phage powder recrystallized at 60% relative humidity (RH), so it was stored and handled below this RH. For the AFM-IR measurements, spray-dried Staphylococcal phage Sa83 powder was embedded in resin, followed by microtome sectioning. AFM-IR spectra collected from different regions within the microtomed sections revealed the presence of phage proteins with amide I and amide II bands at 1640 and 1550 cm-1, respectively. The phages were confirmed to be stable, as the plaque assay showed negligible titer reduction after spray-drying. Our results thus demonstrated the utility of AFM-IR for characterization of nanosized phages present in extremely low quantity in spray-dried particles. These biologically active phages were shown to retain their physical and chemical integrity in the spray-dried particles.