Nano-Raman mapping of a porous glass-ceramic SERS substrate in collection mode.

Porous glass-ceramics is an extremely important material to be used in combination with metallic nanolayers as a Surface-Enhanced Raman Scattering (SERS) substrate for biological and chemical analysis, demonstrating excellent biocompatibility and chemical inertness. These materials show their own Raman background signal lateral distribution, mostly from crystalline skeleton, which has to be considered. A nano-Raman setup using the optical fibre of a Scanning Near-Field Optical Microscope (SNOM), working in collection mode, is described and applied for mapping of such glass-ceramic. The collected Raman signal of Ti and P containing phase distribution in this near-field geometry reaches spatial resolution around 50 nm.

Low-frequency electromagnetic field effects on functional groups in human skin keratinocytes cells revealed by IR-SNOM.

Human HaCaT cells, exposed for 24 h to a 1 mT (rms) 50 Hz sinusoidal magnetic field in a temperature-regulated solenoid, suffer detectable changes in their biochemical properties and shapes. By using infrared wavelength-selective scanning near-field optical microscopy, we observed changes in the distribution of the inner chemical functional groups and in the cell morphology with a resolution of 80-100 nm.

SNOM and AFM microscopy techniques to study the effect of non-ionizing radiation on the morphological and biochemical properties of human keratinocytes cell line (HaCaT).

In this study we have employed atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM) techniques to study the effect of the interaction between human keratinocytes (HaCaT) and electromagnetic fields at low frequency. HaCaT cells were exposed to a sinusoidal magnetic field at a density of 50 Hz, 1 mT. AFM analysis revealed modification in shape and morphology in exposed cells with an increase in the areas of adhesion between cells. This latter finding was confirmed by SNOM indirect immunofluorescence analysis performed with a fluorescent antibody against the adhesion marker beta4 integrin, which revealed an increase of beta4 integrin segregation in the cell membrane of 50-Hz exposed cells, suggesting that a higher percentage of these cells shows a modified pattern of this adhesion marker.

Artificially induced unusual shape of erythrocytes: an atomic force microscopy study.

We used air operating atomic force microscopy (AFM) to study several morphological modifications of human erythrocytes, artificially produced by addition of exogenous agents including phospholipids, low ionic strength media and drugs. Most experiments were performed on unfixed samples to avoid treating red blood cells (RBCs) with chemical agents that can, in principle, induce morphological alteration. After detailed quantitative AFM characterization, the artificially produced abnormally shaped RBCs were compared with cells that occur with high incidence in blood pathologies. This morphological approach suggests a new strategy to describe and understand the biochemical and/or mechanical modifications responsible for the underlying pathologically induced changes and prove AFM to be a suitable tool to study erythrocyte deformation.

Spectroscopic scanning near-field optical microscopy with a free electron laser: CH2 bond imaging in diamond films.

Hydrogen chemistry in thin films and biological systems is one of the most difficult experimental problems in today's science and technology. We successfully tested a novel solution, based on the spectroscopic version of scanning near-field optical microscopy (SNOM). The tunable infrared radiation of the Vanderbilt free electron laser enabled us to reveal clearly hydrogen-decorated grain boundaries on nominally hydrogen-free diamond films. The images were obtained by SNOM detection of reflected 3.5 microm photons, corresponding to the C-H stretch absorption, and reached a lateral resolution of 0.2 microm, well below the lambda/2 (lambda = wavelength) limit of classical microscopy.

Three dimensional (3D) analysis of the morphological changes induced by 50 Hz magnetic field exposure on human lymphoblastoid cells (Raji).

Human Raji B lymphoid cells after exposure for 64 h to a 1 mT (rms) 50 Hz sinusoidal magnetic field showed a reorganization of membrane and cytoskeletal components. Atomic force microscopy in air revealed several modifications in 80% of the exposed cells, such as loss of microvilli-like structures followed by progressive appearance of membrane introflections. This change in plasma membrane morphology was also accompanied by a different actin distribution, as detected by phalloidin fluorescence. These observations support our previous hypothesis that electric and magnetic fields may modify the plasma membrane structure.

Photocurrent near-field microscopy of Schottky barriers.

We used a combination of internal photoemission and of near-field optical microscopy (SNOM) to study the lateral variations in solid interface properties such as energy barriers and electron-hole recombination. In particular we investigated the fully formed Pt-GaP, Au-GaAs, Au-SiNx-GaAs and PtSi-Si Schottky barriers. Our approach enabled us to measure large lateral variations in the photocurrent with spatial resolution on the nanometric scale. Due to the ability of SNOM to supply parallel topographic information, we observed photocurrent variations from zone to zone that only correlated in a few cases with local variations in surface morphology. We assigned the uncorrelated fluctuations to local variations in the interface stoichiometry, the presence of interface states induced by the metallic overlayer and to defect states at the junction. Furthermore, by tuning the photon energy and applied bias we were able to measure the surface distribution of the diffusion length.

Neurone decapping characterization by atomic force microscopy: a topological systematic analysis.

We tested a new approach to cell decapping on rat cerebellar neurones, and observed its effects on cell topography by atomic force microscopy (AFM). The results clearly demonstrate the effectiveness of our decapping approach, and also the ability of AFM to reveal fine details of the decapped cells. Specifically, varying the conditions and duration of the decapping process modifies the extent of the decapping. Such a method can be used to investigate the cytoplasm with surface sensitive techniques.

Atomic Force microscopy of neuron networks.

We imaged uncoated neuron networks by an atomic force microscope in the repulsive regime of contact mode. Images of granule cells and their axons have been clearly revealed with details smaller than 20 nm. The good stability of the sample and the mechanical reproducibility of the microscope allowed the imaging of a neuron culture area of several square microns. By combining tens of images, we were able to reconstruct a highly defined neuronal network. Furthermore, the images were very reproducible over repeated scanning acquisition, demonstrating the mechanical and thermal stability of the instrument-sample system.

Molecular structure of DNA by scanning tunneling microscopy.

Uncoated DNA molecules marked with an activated tris(l-aziridinyl) phosphine oxide (TAPO) solution were deposited on gold substrates and imaged in air with the use of a high-resolution scanning tunneling microscope (STM). Constant-current and gap-modulated STM images show clear evidence of the helicity of the DNA structure: pitch periodicity ranges from 25 to 35 angstroms, whereas the average diameter is 20 angstroms. Molecular structure within a single helix turn was also observed.