Key words. Atomic force microscopy, β 4 integrin, extremely low frequency, scanning near-field optical microscopy. SummaryIn 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 β 4 integrin, which revealed an increase of β 4 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.

A multipurpose scanning near field optical microscope (SNOM) operating at ambient pressure is described with the aim of characterizing the inner parts of biological molecules and any semiconductor or metal microstructure. Therefore, in addition to the requirements of reliability and mechanical stability we have carefully considered analyzing a sample with all available geometries for input/output of photons, in order to get as much information as possible. The SNOM unit consists of two separable cylindrical supports; the lower one contains the sample holder mounted on top of a piezoelectric scanner which is contained in a motor controlled x-y-z stage. A piezo-modulated stretched optical fiber with a few tens of nanometer pinhole and a shear-force apparatus mounted inside the top cylinder allow for topography measurements. The reflectivity of the sample can be measured by applying different methods: the sample can be illuminated on top by an external source, as well as by the optical fiber used for the detection of the reflectivity signal. An aperture in the lower cylinder allows for illumination of the sample on the back: in this case the fiber collects the evanescent wave induced at the top of the sample. Another aperture in the lower cylinder allows measurement of the reflected light which includes a contribution due to the interaction with the fiber. Also photocurrent experiments can be easily performed by illuminating the sample with the fiber and detecting the transmitted signal using a current–voltage converter mounted inside the top cylinder. A video-camera that can reach 170 enlargements is mounted on the top cylinder for positioning the fiber on particular regions of the sample. Reflectivity and photocurrent measurements have been performed on uncoated neurons, CsI compound, Au/GaAs, and PtSi/Si systems, reaching a resolution well below the diffraction limit.

No abstract

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (lambda) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 micro m ( approximately lambda/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.