Submicronic-Scale Mechanochemical Characterization of Oxygen-Enriched Materials.

Conventional techniques that measure the concentration of light elements in metallic materials lack high-resolution performance due to their intrinsic limitation of sensitivity. In that context, scanning microwave microscopy has the potential to significantly enhance the quantification of element distribution due to its ability to perform a tomographic investigation of the sample. Scanning microwave microscopy associates the local electromagnetic measurement and the nanoscale resolution of an atomic force microscope. This technique allows the simultaneous characterization of oxygen concentration as well as local mechanical properties by microwave phase shift and amplitude signal, respectively. The technique was calibrated by comparison with nuclear reaction analysis and nanoindentation measurement. We demonstrated the reliability of the scanning microwave technique by studying thin oxygen-enriched layers on a Ti-6Al-4V alloy. This innovative approach opens novel possibilities for the indirect quantification of light chemical element diffusion in metallic materials. This technique is applicable to the control and optimization of industrial processes.

Infrared nanospectroscopic imaging of DNA molecules on mica surface.

Significant efforts have been done in last two decades to develop nanoscale spectroscopy techniques owning to their great potential for single-molecule structural detection and in addition, to resolve open questions in heterogeneous biological systems, such as protein-DNA complexes. Applying IR-AFM technique has become a powerful leverage for obtaining simultaneous absorption spectra with a nanoscale spatial resolution for studied proteins, however the AFM-IR investigation of DNA molecules on surface, as a benchmark for a nucleoprotein complexes nanocharacterization, has remained elusive. Herein, we demonstrate methodological approach for acquisition of AFM-IR mapping modalities with corresponding absorption spectra based on two different DNA deposition protocols on spermidine and Ni2+ pretreated mica surface. The nanoscale IR absorbance of distinctly formed DNA morphologies on mica are demonstrated through series of AFM-IR absorption maps with corresponding IR spectrum. Our results thus demonstrate the sensitivity of AFM-IR nanospectroscopy for a nucleic acid research with an open potential to be employed in further investigation of nucleoprotein complexes.

Imaging Artificial Membranes Using High-Speed Atomic Force Microscopy.

Supported lipid bilayers represent a very attractive way to mimic biological membranes, especially to investigate molecular mechanisms associated with the lateral segregation of membrane components. Observation of these model membranes with high-speed atomic force microscopy (HS-AFM) allows the capture of both topography and dynamics of membrane components, with a spatial resolution in the nanometer range and image capture time of less than 1 s. In this context, we have developed new protocols adapted for HS-AFM to form supported lipid bilayers on small mica disks using the vesicle fusion or Langmuir-Blodgett methods. In this chapter we describe in detail the protocols to fabricate supported artificial bilayers as well as the main guidelines for HS-AFM imaging of such samples.

Microwave Spectroscopic Detection of Human Hsp70 Protein on Annealed Gold Nanostructures on ITO Glass Strips.

Conductive indium-tin oxide (ITO) and non-conductive glass substrates were successfully modified with embedded gold nanoparticles (AuNPs) formed by controlled thermal annealing at 550 °C for 8 h in a preselected oven. The authors characterized the formation of AuNPs using two microscopic techniques: scanning electron microscopy (SEM) and atomic force microscopy (AFM). The analytical performances of the nanostructured-glasses were compared regarding biosensing of Hsp70, an ATP-driven molecular chaperone. In this work, the human heat-shock protein (Hsp70), was chosen as a model biomarker of body stress disorders for microwave spectroscopic investigations. It was found that microwave screening at 4 GHz allowed for the first time the detection of 12 ng/µL/cm² of Hsp70.

Spontaneous non-canonical assembly of CcmK hexameric components from ß-carboxysome shells of cyanobacteria.

CcmK proteins are major constituents of icosahedral shells of ß-carboxysomes, a bacterial microcompartment that plays a key role for CO2 fixation in nature. Supported by the characterization of bidimensional (2D) layers of packed CcmK hexamers in crystal and electron microscopy structures, CcmK are assumed to be the major components of icosahedral flat facets. Here, we reassessed the validity of this model by studying CcmK isoforms from Synechocystis sp. PCC6803. Native mass spectrometry studies confirmed that CcmK are hexamers in solution. Interestingly, potential pre-assembled intermediates were also detected with CcmK2. Atomic-force microscopy (AFM) imaging under quasi-physiological conditions confirmed the formation of canonical flat sheets with CcmK4. Conversely, CcmK2 formed both canonical and striped-patterned patches, while CcmK1 assembled into remarkable supra-hexameric curved honeycomb-like mosaics. Mutational studies ascribed the propensity of CcmK1 to form round assemblies to a combination of two features shared by at least one CcmK isoform in most ß-cyanobacteria: a displacement of an α helical portion towards the hexamer edge, where a potential phosphate binding funnel forms between packed hexamers, and the presence of a short C-terminal extension in CcmK1. All-atom molecular dynamics supported a contribution of phosphate molecules sandwiched between hexamers to bend CcmK1 assemblies. Formation of supra-hexameric curved structures could be reproduced in coarse-grained simulations, provided that adhesion forces to the support were weak. Apart from uncovering unprecedented CcmK self-assembly features, our data suggest the possibility that transitions between curved and flat assemblies, following cargo maturation, could be important for the biogenesis of ß-carboxysomes, possibly also of other BMC.