Ambient atomic resolution atomic force microscopy with qPlus sensors: Part 1.

Atomic force microscopy (AFM) is an enormous tool to observe nature in highest resolution and understand fundamental processes like friction and tribology on the nanoscale. Atomic resolution in highest quality was possible only in well-controlled environments like ultrahigh vacuum (UHV) or controlled buffer environments (liquid conditions) and more specified for long-term high-resolution analysis at low temperatures (∼4 K) in UHV where drift is nearly completely absent. Atomic resolution in these environments is possible and is widely used. However, in uncontrolled environments like air, with all its pollutants and aerosols, unspecified thin liquid films as thin as a single molecular water-layer of 200 pm or thicker condensation films with thicknesses up to hundred nanometer, have been a problem for highest resolution since the invention of the AFM. The goal of true atomic resolution on hydrophilic as well as hydrophobic samples was reached recently. In this manuscript we want to review the concept of ambient AFM with atomic resolution. The reader will be introduced to the phenomenology in ambient conditions and the problems will be explained and analyzed while a method for scan parameter optimization will be explained. Recently developed concepts and techniques how to reach atomic resolution in air and ultra-thin liquid films will be shown and explained in detail, using several examples. Microsc. Res. Tech. 80:50-65, 2017. © 2016 Wiley Periodicals, Inc.

Influence of PEGylation on nanoparticle mobility in different models of the extracellular matrix.

Nanoparticle transport inside the extracellular matrix (ECM) is a crucial factor affecting the therapeutic success. In this work, two in vitro ECM models - a neutrally charged collagen I network with an effective pore size of 0.47µm and Matrigel, a basement membrane matrix with strong negative charge and effective pore size of 0.14µm - were assessed for barrier function in the context of diffusing nanoparticles. Nanoparticles with a size of 120nm were coated with poly(ethylene glycol) (PEG) of different molecular weights - 2, 5 and 20kDa - over a range of gradually increasing coating densities - precisely 0.2, 2, 8 and 20PEG/nm2. The PEG corona was imaged in its native state without any drying process by atomic force microscopy, revealing that the experimentally determined arrangement of PEG at the surface did not match with what was theoretically expected. In a systematic investigation of nanoparticle mobility via fluorescence recovery after photobleaching, increasing both PEG MW and PEGylation density gradually improved diffusion properties predominately in collagen. Due to its smaller pore size and electrostatic obstruction, diffusion coefficients were about ten times lower in Matrigel than in the collagen network and an extension of the PEG MW and density did not necessarily lead to better diffusing particles. Consequently, collagen gels were revealed to be a poor model for nanoparticle mobility assessment, as neither their pore size nor their electrostatic properties reflect the expected in vivo conditions. In Matrigel, diffusion proceeded according to a sigmoidal increase with gradually increasing PEG densities showing threshold zeta potentials of 11.6mV (PEG2kDa) and 13.8mV (PEG5kDa), below which particles were regarded as mobile. Irrespective of the molecular weight particles with a PEGylation density lower than 2PEG/nm2 were defined as immobile and those with a PEG coverage of more than 8PEG/nm2 as mobile.

Atomic Resolution of Calcium and Oxygen Sublattices of Calcite in Ambient Conditions by Atomic Force Microscopy Using qPlus Sensors with Sapphire Tips.

Characterization and imaging at the atomic scale with atomic force microscopy in biocompatible environments is an ongoing challenge. We demonstrate atomically resolved imaging of the calcite (101̅4) surface plane using stiff quartz cantilevers ("qPlus sensors", stiffness k = 1280 N/m) equipped with sapphire tips in ambient conditions without any surface preparation. With 10 atoms in one surface unit cell, calcite has a highly complex surface structure comprising three different chemical elements (Ca, C, and O). We obtain true atomic resolution of calcite in air at relative humidity ranging from 20% to 40%, imaging atomic steps and single atomic defects. We observe a great durability of sapphire tips with their Mohs hardness of 9, only one step below diamond. Depending on the state of the sapphire tip, we resolve either the calcium or the oxygen sublattice. We determine the tip termination by comparing the experimental images with simulations and discuss the possibility of chemical tip identification in air. The main challenges for imaging arise from the presence of water layers, which form on almost all surfaces and have the potential to dissolve the crystal surface. Frequency shift versus distance spectra show the presence of at least three ordered hydration layers. The measured height of the first hydration layer corresponds well to X-ray diffraction data and molecular dynamic simulations, namely, ∼220 pm. For the following hydration layers we measure ∼380 pm for the second and third layer, ending up in a total hydration layer thickness of at least 1 nm. Understanding the influence of water layers and their structure is important for surface segregation, surface reactions including reconstructions, healing of defects, and corrosion.

Oligolayer-coated nanoparticles: impact of surface topography at the nanobio interface.

Layer-by-layer coating of nanoparticles with a layer number in the single-digit range has gained increasing attention in the field of nanomedicinal research. However, the impact of using various polyelectrolytes on oligolayer formation and, more importantly, their influence on the interaction with the biological system has not often been considered in the past. Hence, we investigated the polyelectrolyte deposition profiles and resulting surface topographies of up to three polyelectrolyte layers on a flat gold sensor surface using three different polycations, namely, poly(ethylene imine) (PEI), poly(allylamine hydrochloride) (PAH), and poly(diallylammonium chloride) (PD), each in combination with poly(styrenesulfonate) (PSS). Surface plasmon resonance spectroscopy and atomic force microscopy revealed that the PEI/PSS pair in particular showed a so-called overshoot phenomenon, which is associated with partial polyelectrolyte desorption from the surface. This is also reflected by a significant increase in the surface roughness. Then, after having transferred the oligolayer assembly onto nanoparticles of ∼32 nm, we realized that quite similar surface topographies must have emerged on a curved gold surface. A major finding was that the extent of surface roughness contributes significantly to the fashion by which the oligolayer-coated nanoparticles interact with serum proteins and associate with cells. For example, for the PEI/PSS system, both the surface roughness and protein adsorption increased by a factor of ∼12 from the second to third coating layer and, at the same time, the cell association massively decreased to only one-third. Our study shows that surface roughness, along with other particle properties such as size, shape, zeta potential, and hydrophobicity, is another decisive factor for nanoparticles in a biological context, which has indeed been discussed previously but has not to date been investigated for oligolayers.

Influence of matrix and filler fraction on biofilm formation on the surface of experimental resin-based composites.

The aim of this study was to investigate the impact of resin matrix chemistry and filler fraction on biofilm formation on the surface of experimental resin-based composites (RBCs). Specimens were prepared from eight experimental RBC formulations differing in resin matrix blend (BisGMA/TEGDMA in a 7:3 wt% ratio or UDMA/aliphatic dimethacrylate in a 1:1 wt% ratio) and filler fraction (no fillers; 65 wt% dental glass with an average diameter of 7 or 0.7 µm or 65 wt% SiO2 with an average diameter of 20 nm). Surface roughness, surface free energy, and chemical surface composition were determined; surface topography was visualized using atomic force microscopy. Biofilm formation was simulated under continuous flow conditions for a 48 h period using a monospecies Streptococcus mutans and a multispecies biofilm model. In the monospecies biofilm model, the impact of the filler fraction overruled the influence of the resin matrix, indicating lowest biofilm formation on RBCs with nano-scaled filler particles and those manufactured from the neat resin blends. The multispecies model suggested a more pronounced effect of the resin matrix blend, as significantly higher biofilm formation was identified on RBCs with a UDMA/dimethacrylate matrix blend than on those including a BisGMA/TEGDMA matrix blend but analogous filler fractions. Although significant differences in surface properties between the various materials were identified, correlations between the surface properties and biofilm formation were poor, which highlights the relevance of surface topography and chemistry. These results may help to tailor novel RBC formulations which feature reduced biofilm formation on their surface.

Atomically resolved graphitic surfaces in air by atomic force microscopy.

Imaging at the atomic scale using atomic force microscopy in biocompatible environments is an ongoing challenge. We demonstrate atomic resolution of graphite and hydrogen-intercalated graphene on SiC in air. The main challenges arise from the overall surface cleanliness and the water layers which form on almost all surfaces. To further investigate the influence of the water layers, we compare data taken with a hydrophilic bulk-silicon tip to a hydrophobic bulk-sapphire tip. While atomic resolution can be achieved with both tip materials at moderate interaction forces, there are strong differences in force versus distance spectra which relate to the water layers on the tips and samples. Imaging at very low tip-sample interaction forces results in the observation of large terraces of a naturally occurring stripe structure on the hydrogen-intercalated graphene. This structure has been previously reported on graphitic surfaces that are not covered with disordered adsorbates in ambient conditions (i.e., on graphite and bilayer graphene on SiC, but not on monolayer graphene on SiC). Both these observations indicate that hydrogen-intercalated graphene is close to an ideal graphene sample in ambient environments.

Streptococcus mutans biofilm formation and release of fluoride from experimental resin-based composites depending on surface treatment and S-PRG filler particle fraction.

PURPOSE: To evaluate fluoride release and biofilm formation on resin-based composites (RBCs) including surface pre-reacted glass ionomer (S-PRG) filler particles. MATERIALS AND METHODS: Specimens were prepared from experimental RBCs including different fractions of S-PRG fillers (0/10/30/50/70% w/v). RBCs were light cured against mylar strips (MYL), and 50% of the specimens were additionally polished to a high gloss (POL). Surface roughness (SR), surface free energy (SFE) and fluoride release were determined. Streptococcus mutans biofilm formation (SMBF) was simulated for 48 h and 120 h; adherent viable biomass was assessed using an MTT-based assay. RESULTS: The highest SR was identified for POL specimens manufactured from the RBC with a filler fraction of 70%. For all specimens and surface treatments, polishing caused an increase in surface free energy. For both MYL and POL specimens, increasing the filler fraction coincided with an increased release of fluoride; a higher release of fluoride was identified for POL specimens with filler fractions of 50% and 70% in comparison to their MYL counterparts. Release of fluoride was lower after 120 h than after 48 h. No differences in SMBF were identified between MYL and POL specimens with identical filler fractions after 48 h of biofilm formation; with increasing filler fractions, a tendency towards decreasing SMBF was observed. After 120 h, less SMBF was identified for POL specimens with filler fractions of 30%, 50% and 70% in comparison to corresponding MYL specimens. CONCLUSION: The inclusion of S-PRG fillers and an effective surface treatment may reduce biofilm formation on RBCs.

Observation of 4 nm pitch stripe domains formed by exposing graphene to ambient air.

We study epitaxial graphene on the 6H-SiC(0001) surface under ambient conditions using frequency-modulation atomic force microscopy. We observe large terraces with a self-assembled stripe structure within a highly adsorbate covered surface on top of the graphene. To identify the origin of the structure, we compare the experimental data on graphene with calculations and experiments on graphite that predict the formation of a solid-gas monolayer in the solid-liquid interface of hydrophobic surfaces.