A multiple technical approach to the study of apoptotic cell micronuclei

Apoptotic micronuclei have been studied, in different cell types, from a morphologic and functional point of view. Conventional electron microscopy, in various staining conditions, selective cytochemistry for DNA, and freeze fracture for the analysis of chromatin fiber organization and size were performed. In situ TdT and Pol I immunofluorescent techniques were carried out to detect double- and single-strand DNA breaking points by confocal laser scanning microscopy. Apoptotic cell ultrathin cryosections were also performed and were analysed by field emission in lens scanning electron microscopy. Double/single strand massively cleaved DNA was detected in micronuclei, with a highly supercoiled, uniformly packed, very dense arrangement.     

Cryo-electron diffraction as a tool to study local variations in the lipid organization of human stratum corneum

The human skin provides the body with a barrier against transepidermal water loss and the penetration of harmful agents (e.g. microbes) from outside. This barrier function is produced mainly by the outermost, nonviable layer of the epidermis, the stratum corneum (SC). The SC consists of terminally differentiated corneocytes surrounded by a continuous intercellular lipid domain, which contains mostly ceramides, cholesterol and free fatty acids. Small- and wide-angle X-ray diffraction studies have elucidated the lamellar and lateral lipid organizations in these domains. However, these techniques require bulk quantities of SC, as a result of which local structure information on the lipids cannot be obtained. Insights to these local lipid arrangements are important when new transdermal drug delivery systems have to be developed. Therefore, the technique of electron diffraction arose as a tool to study the lateral packing of the lipids in the intercellular domains of SC, locally. In a previous study, the suitability of electron diffraction was demonstrated using a lipid model system that resembled the lipid composition of the SC. The spacings calculated from the electron diffraction patterns were in good agreement with the spacings revealed by wide-angle X-ray diffraction. The results presented here succeed this previous study. We improved the microscope settings and developed a new preparation method to study ex vivo human SC by cryo-electron diffraction. The method is based on the conventional tape-stripping method and offers the possibility to study depth-related changes in the lipid organization of human SC. Diffraction patterns of both hexagonal and orthorhombic lipid lattices have been recorded with spacings that resembled those found in human SC by wide-angle X-ray diffraction. After lipid extraction, such diffraction patterns could no longer be detected in the samples.     

Light microscope based analysis of three-dimensional structure: Applications to the study of Drosophila salivary gland nuclei. I. Data collection and analysis

Many biological structures of interest are large enough that they may be viewed by light microscope methods, yet they are sufficiently complicated that interpretation of what is seen is quite difficult. The salivary gland nuclei from Dipterans are an example of this. Previous attempts at determining the path of the giant chromosomes in these nuclei have depended on the laborious construction of models by hand. A unified Computer Aided Modelling and Analysis system (CAMA) has been implemented, allowing data collection and analysis of structures visible by light microscopy. This system is extendible to the analysis of electron micrographs of serial sections or of other data consisting of images present in a stack.     

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 β4 integrin, which revealed an increase of β4 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.