We demonstrate a method to control the distance between a custom probe and a sample on a μm to nm scale. The method relies on the closed-loop feedback on the angular deflection of an in-contact AFM microcantilever. High performance in stability and accuracy is achieved in this method by taking advantage of the small mechanical feedback path between surface and probe. We describe how internal error sources that find their origin in the microcantilever and feedback can be minimized to achieve an accurate and precise control up to 3 nm. In particular, we investigated how hysteresis effects in the feedback caused by friction forces between tip and substrate can be minimized. By applying a short calibration procedure, distance control from contact to several micrometers probe-sample distance can be obtained with an absolute nanometer-scale accuracy. The method presented is compatible with any probe that can be fixed on a microcantilever chip and can be easily built into existing AFM systems.

We demonstrate a method to accurately control the distance between a custom probe and a sample on a {\mu}m to nm scale. The method relies on the closed-loop feedback on the angular deflection of an in-contact AFM microcantilever. High performance in stability and accuracy is achieved in this method by taking advantage of the small mechanical feedback path between surface and probe. We describe how internal error sources that find their origin in the microcantilever and feedback can be minimized to achieve an accurate and precise control up to 3 nm. In particular, we investigated how hysteresis effects in the feedback caused by friction forces between tip and substrate, can be minimized. By applying a short calibration procedure, distance control from contact to several micrometers probe-sample distance can be obtained with an absolute nanometer-scale accuracy. The method presented is compatible with any probe that can be fixed on a microcantilever chip and can be easily built into existing AFM systems.

An integrated intra‐laser‐cavity microparticle sensor based on a dual‐wavelength distributed‐feedback channel waveguide laser in ytterbium‐doped amorphous aluminum oxide on a silicon substrate is demonstrated. Real‐time detection and accurate size measurement of single micro‐particles with diameters ranging between 1 µm and 20 µm are achieved, which represent the typical sizes of many fungal and bacterial pathogens as well as a large variety of human cells. A limit of detection of ∼500 nm is deduced. The sensing principle relies on measuring changes in the frequency difference between the two longitudinal laser modes as the evanescent field of the dual‐wavelength laser interacts with micro‐sized particles on the surface of the waveguide. Improvement in sensitivity far down to the nanometer range can be expected upon stabilizing the pump power, minimizing back reflections, and optimizing the grating geometry to increase the evanescent fraction of the guided modes.

An integrated intra-laser-cavity microparticle sensor based on a dual-phase-shift, dual-wavelength distributed-feedback channel waveguide laser in Al2O3:Yb3+ is presented. Real-time detection and accurate size measurement of single microparticles with diameters ranging between 1 μm and 20 μm are achieved, which represent the typical sizes of many fungal and bacterial pathogens as well as a large variety of human cells. The sensing principle relies on measuring changes in the frequency difference between the two longitudinal laser modes, as the evanescent field of the dual-wavelength laser interacts with micro-sized particles on the surface of the waveguide.

Plasma treatment is a method to modify the physicochemical properties of biomaterials, which consequently may affect interactions with cells. Based on the rationale that physical cues on the surface of culture substrates and implants, such as surface roughness, have proven to alter cell behaviour, we used electrospinning to fabricate fibrous three-dimensional scaffolds made of a poly (ethylene oxide terephthalate)/poly (butylene terephthalate) copolymer to mimic the physical microenvironment of extracellular matrix and applied radio-frequency oxygen plasma treatment to create nanoscale roughness. Scanning electron microscopy (SEM) analysis revealed a fibre diameter of 5.49 ± 0.96 µm for as-spun meshes. Atomic force microscopy (AFM) measurements determined an exponential increase of surface roughness with plasma treatment time. An increase in hydrophilicity after plasma treatment was observed, which was associated with higher oxygen content in plasma treated scaffolds compared to untreated ones. A more pronounced adsorption of bovine serum albumin occurred on scaffolds treated with plasma for 15 and 30 min compared to untreated fibres. Clinically relevant human mesenchymal stromal cells (hMSCs) were cultured on untreated, 15 and 30 min treated scaffolds. SEM analysis confirmed cell attachment and a pronounced spindle-like morphology on all scaffolds. No significant differences were observed between different scaffolds regarding the amount of DNA, metabolic activity and alkaline phosphatase (ALP) activity after 7 days of culture. The amount of ALP positive cells increased between 7 and 21 days of culture on both untreated and 30 min treated meshes. In addition, ALP staining of cells on plasma treated meshes appeared more pronounced than on untreated meshes after 21 days of culture. Quantitative polymerase chain reaction showed significant upregulation of bone sialoprotein and osteonectin expression on oxygen plasma treated fibres compared to untreated fibres in basic culture medium after 7 days of culture, while no differences were observed in the expression of other osteogenic markers. At 21 days, no osteocalcin protein could be detected by ELISA at any of the substrates. In conclusion, this study shows that oxygen plasma treatment can successfully be applied to modify the nanoscale surface properties of polymeric electrospun fibre meshes, which in turn may positively affect osteogenic differentiation of hMSCs.

Atomic force microscopy (AFM) is widely used to measure morphological and mechanical properties of biological materials at the nanoscale. AFM is able to visualize and measure these properties in different environmental conditions. However, these conditions can influence the results considerably, rendering their interpretation a matter of some subtlety. We demonstrate this by imaging ~10 nm diameter α-synuclein amyloid fibrils, focusing specifically on the structure of the C-terminal part of the protein monomers incorporated into fibrils. Despite these influences leading to variations in fibril heights, we have shown that by maintaining careful control of AFM settings we can quantitatively compare the morphological parameters of fibrils imaged in air or in buffer conditions. From this comparison we were able to deduce the semiflexible character of this C-terminal region. Fibril height differences measured in air and liquid indicate that the C-terminal region collapses onto the fibril core upon drying. The fibril heights decrease upon increasing ion concentration in solution, suggesting that the C-terminal tails collapse into more compact structures as a result of charge screening. Finally, PeakForce QNM measurements show an apparent heterogeneity of C-terminal packing along the fibril length.

Recently several atomic force microscopy (AFM)-based surface property mapping techniques like pulsed force microscopy (PFM), harmonic force microscopy or Peakforce QNM (R) have been introduced to measure the nano-and micro-mechanical properties of materials. These modes all work at different operating frequencies. However, complex materials are known to display viscoelastic behavior, a combination of solid and fluid-like responses, depending on the frequency at which the sample is probed. In this report, we show that the frequency-dependent mechanical behavior of complex materials, such as polymer blends that are frequently used as calibration samples, is clearly measurable with AFM. Although this frequency-dependent mechanical behavior is an established observation, we demonstrate that the new high frequency mapping techniques enable AFM-based rheology with nanoscale spatial resolution over a much broader frequency range compared to previous AFM-based studies. We further highlight that it is essential to account for the frequency-dependent variation in mechanical properties when using these thin polymer samples as calibration materials for elasticity measurements by high-frequency surface property mapping techniques. These results have significant implications for the accurate interpretation of the nanomechanical properties of polymers or complex biological samples. The calibration sample is composed of a blend of soft and hard polymers, consisting of low-density polyethylene (LDPE) islands in a polystyrene (PS) surrounding, with a stiffness of 0.2 GPa and 2 GPa respectively. The spring constant of the AFM cantilever was selected to match the stiffness of LDPE. From 260 Hz to 1100 Hz the sample was imaged with the PFM method. At low frequencies (0.5-35 Hz), single-point nanoindentation was performed. In addition to the material's stiffness, the relative heights of the LDPE islands (with respect to the PS) were determined as a function of the frequency. At the lower operation frequencies for PFM, the islands exhibited lower heights than when measured with tapping mode at 120 kHz. Both spring constants and heights at the different frequencies clearly show a frequency-dependent behavior.

We demonstrate an integrated optical particle sensor based on a dual-wavelength distributed-feedback waveguide laser. Micro-particles were detected down to a size of 1 μm, which represents the typical size of many fungal and bacterial pathogens.

Current therapeutic approaches under development for Alzheimer disease, including γ-secretase modulating therapy, aim at increasing the production of Aβ(1-38) and Aβ(1-40) at the cost of longer Aβ peptides. Here, we consider the aggregation of Aβ(1-38) and Aβ(1-43) in addition to Aβ(1-40) and Aβ(1-42), in particular their behavior in mixtures representing the complex in vivo Aβ pool. We demonstrate that Aβ(1-38) and Aβ(1-43) aggregate similar to Aβ(1-40) and Aβ(1-42), respectively, but display a variation in the kinetics of assembly and toxicity due to differences in short timescale conformational plasticity. In biologically relevant mixtures of Aβ, Aβ(1-38) and Aβ(1-43) significantly affect the behaviors of Aβ(1-40) and Aβ(1-42). The short timescale conformational flexibility of Aβ(1-38) is suggested to be responsible for enhancing toxicity of Aβ(1-40) while exerting a cyto-protective effect on Aβ(1-42). Our results indicate that the complex in vivo Aβ peptide array and variations thereof is critical in Alzheimer disease, which can influence the selection of current and new therapeutic strategies.

Poly(ε-caprolactone) (PCL) fibers ranging from 250 to 700 nm in diameter were produced by electrospinning a polymer tetrahydrofuran/N,N-dimethylformamide solution. The mechanical properties of the fibrous scaffolds and individual fibers were measured by different methods. The Young's moduli of the scaffolds were determined using macro-tensile testing equipment, whereas single fibers were mechanically tested using a nanoscale three-point bending method, based on atomic force microscopy and force spectroscopy analyses. The modulus obtained by tensile-testing eight different fiber scaffolds was 3.8±0.8 MPa. Assuming that PCL fibers can be described by the bending model of isotropic materials, a Young's modulus of 3.7±0.7 GPa was determined for single fibers. The difference of three orders of magnitude observed in the moduli of fiber scaffolds vs. single fibers can be explained by the lacunar and random structure of the scaffolds.

We report on the use of three different atomic force spectroscopy modalities to determine the nanomechanical properties of amyloid fibrils of the human α-synuclein protein. α-Synuclein forms fibrillar nanostructures of approximately 10 nm diameter and lengths ranging from 100 nm to several microns, which have been associated with Parkinson's disease. Atomic force microscopy (AFM) has been used to image the morphology of these protein fibrils deposited on a flat surface. For nanomechanical measurements, we used single-point nanoindentation, in which the AFM tip as the indenter is moved vertically to the fibril surface and back while the force is being recorded. We also used two recently developed AFM surface property mapping techniques: Harmonic force microscopy (HarmoniX) and Peakforce QNM. These modalities allow extraction of mechanical parameters of the surface with a lateral resolution and speed comparable to tapping-mode AFM imaging. Based on this phenomenological study, the elastic moduli of the α-synuclein fibrils determined using these three different modalities are within the range 1.3-2.1 GPa. We discuss the relative merits of these three methods for the determination of the elastic properties of protein fibrils, particularly considering the differences and difficulties of each method.

The mechanical properties of individual collagen fibrils of approximately 200 nm in diameter were determined using a slightly adapted AFM system. Single collagen fibrils immersed in PBS buffer were attached between an AFM cantilever and a glass surface to perform tensile tests at different strain rates and stress relaxation measurements. The stress–strain behavior of collagen fibrils immersed in PBS buffer comprises a toe region up to a stress of 5 MPa, followed by the heel and linear region at higher stresses. Hysteresis and strain-rate dependent stress–strain behavior of collagen fibrils were observed, which suggest that single collagen fibrils have viscoelastic properties. The stress relaxation process of individual collagen fibrils could be best fitted using a two-term Prony series. Furthermore, the influence of different cross-linking agents on the mechanical properties of single collagen fibrils was investigated. Based on these results, we propose that sliding of microfibrils with respect to each other plays a role in the viscoelastic behavior of collagen fibrils in addition to the sliding of collagen molecules with respect to each other. Our finding provides a better insight into the relationship between the structure and mechanical properties of collagen and the micro-mechanical behavior of tissues.

The amyloid peptides Aβ40 and Aβ42 of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aβ42 seems more pathogenic than Aβ40, the reason for this is not well understood. We show here that small alterations in the Aβ42:Aβ40 ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ42:Aβ40 ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti‐amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non‐toxic intermediates. The amyloid peptides Aβ40 and Aβ42 of Alzheimer's disease contribute differentially to the disease process. The findings show here that small changes in the Aβ42/40 ratio have significant effects on the neurotoxicity of Aβ mixtures.

Aliphatic polyesters, e.g. poly(e-caprolactone) (PCL), poly(lactide)s, poly(glycolide) and their copolymers, have shown a great potential for biomedical and pharmaceutical applications. They may be applied in the field of tissue engineering or tissue regeneration as scaffold materials, on account of their remarkable compatibility with physiological medium. Moreover, due to the hydrolytic and enzymatic instability of their ester bonds, they are degraded in a physiological environment into non-toxic products, which make them outstanding candidates for shortto medium-term applications.

Micromechanical properties of single elastic fibers and fibrillin–microfibrils, isolated from equine ligamentum nuchae using chemical and enzymatic methods, were determined with atomic force microscopy (AFM). Young's moduli of single elastic fibers immersed in water, devoid of or containing fibrillin–microfibrils, were determined using bending tests. Bending freely suspended elastic fibers on a micro-channeled substrate by a tip-less AFM cantilever generated a force versus displacement curve from which Young's moduli were calculated. For single elastic fibers, Young's moduli in the range of 0.3–1.5MPa were determined, values not significantly affected by the presence of fibrillin–microfibrils. To further understand the role of fibrillin–microfibrils in vertebrate elastic fibers, layers of fibrillin–microfibrils were subjected to nano-indentation tests. From the slope of the force versus indentation curves, Young's moduli ranging between 0.56 and 0.74MPa were calculated. The results suggest that fibrillin–microfibrils are not essential for the mechanical properties of single vertebrate elastic fibers.