Jean Paul Rigaut

An empirical formulation relating boundary lengths to resolution in specimens showing ‘non-ideally fractal’ dimensions

The concept of fractal dimension offers an elegant basis for the understanding of changes in boundary lengths due to different resolutions. However, in many cases, the usual log‐log ‘fractal plot’ does not quite fit the observed data, with most biological structures for instance.

Automated image segmentation by mathematical morphology and fractal geometry

A new method for automated image segmentation is presented. It makes use of a combination of principles derived from mathematical morphology and fractal geometry.

Reflectance in situ hybridization (RISH): detection, by confocal reflectance laser microscopy, of gold-labelled riboprobes in breast cancer cell lines and histological specimens

A method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens.

Analysis by confocal scanning laser microscopy imaging of the spatial distribution of intermediate filaments in foetal and adult rat liver cells.

Confocal scanning laser microscopy has been used to make three‐dimensional observations of the spatial distribution of cytoskeleton intermediate filaments in rat liver hepatocytes, at various stages during foetal development and in the adult. Single and double immuno‐labelling with fluorescein and Texas Red fluorescence have been used to study the intracellular spatial distribution of C18 cytokeratin and vimentin. Simultaneous confocal imaging with double‐fluorescence emission requires an image processing step for the correction of ‘contamination’ effects due to the overlap between fluorescein and Texas Red emission spectra.

Improvement in the Specificity of the Silver Staining Technique for Ag NOR-ASSOCIATED Acidic Proteins in Paraffin Sections

Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiffs reagent improves the specificity of Goodpasture and Blooms AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.

Computer simulation modelling and visualization of 3D architecture of biological tissues

Recent technical improvements, such as 3D microscopy imaging, have shown the necessity of studying 3D biological tissue architecture during carcinogenesis. In the present paper a computer simulation model is developed allowing the visualization of the microscopic biological tissue architecture during the development of metaplastic and dysplastic lesions.The static part of the model allows the simulation of the normal, metaplastic and dysplastic architecture of an external epithelium. This model is associated to a knowledge base which contains only data on the nasal epithelium. The latter has been well studied by numerous authors and its lesional states are well known. An inference engine allows the initialization of the static model parameters. A statistical comparison between simulated epithelia and real epithelia is achieved by adjusting the parameter values during the simulation.The dynamic part of the model allows the simulation of a growth process on a 3D representation based on the static model. The main hypothesis is that nasal epithelium is submitted to a continuous transformation from normal to cancer through metaplasia and dysplasia. The evolution of each cell (represented by its nucleus) depends on its local environment and also on its heritage from its mother-cell.Simulation of tissue renewal of the nasal pseudostratified epithelium has been achieved. The evolution from normal to hyperplasia has been simulated. After modification of the cell cycle modelling, the simulation of the development of metaplastic foci has been obtained.

Combined analysis of in situ hybridization, cell cycle and structural markers using reflectance and immunofluorescence confocal microscopy

SummaryA method for the simultaneous detection of mRNA by reflectance in situ hybridization (RISH), cell cycle and structural markers by immunofluorescence using confocal laser scanning microscopy is presented. The mRNA expression of two ras-related genes rhoB and rhoC was analysed in human breast cancer cell lines and human histological specimens (breast cancer tissues and skin biopsies). In breast cancer cell lines, the conditions were optimized to detect RNA-RNA hybrids and DNA synthesis after pulse-labelling with bromodeoxyuridine. Endonuclease-exonuclease digestion, which allows the accessibility to specific antibodies of halogenated pyrimidine molecules, was carried out following ISH. Finally, cytokeratin or vimentin staining was performed. The detection of signals, arising from 1-nm colloidal gold particles without silver enhancement, by reflectance confocal laser scanning microscopy is described. Bromodeoxybiridine DNA markers and cytokeratin/vimentin staining were detected concomitantly using different fluorochromes. To allow comparative expression of two related genes, the mRNA of rhoB and rhoC were detected using digoxigenin- or biotin-labelled riboprobes and, after 3-D imaging, a detailed analysis by optical horizontal (x, y) and vertical (x, z) sectioning was undertaken. The subsequent bromodeoxyuridine detection procedure permitted to us explore the specific transcription of these two genes during S and non-S phases. This method allows the identification and localization of several subcellular components in cells within a complex tissue structure and makes it possible to analyse further transcript localization in relation to the function of the encoded protein and to the cell cycle.

Spatial distribution of cytoskeleton intermediate filaments during fetal rat hepatocyte differentiation

The construction of the liver parenchyma throughout fetal development depends on the elaboration of intercellular contacts between epithelial cells and between epithelial and mesenchymal cells. During this time, the spatial distribution of cytokeratins in hepatocytes shows a striking evolution as demonstrated by confocal microscopy and image analysis.

The ‘corpuscle’ stereological problem—re-evaluation using slab fragment volumes and application to the correction of DNA histograms from sections of spherical nuclei

A new category of stereological size distribution unfolding models is introduced. It is based on the use of the volumes of particle slab fragments, in addition to their profile dimensions. When spheres are cut by a slab of known (constant) thickness, an estimation of discrete sphere sizes from section data is then possible, as only one parent sphere solution exists for any slab fragment, given the latters projection size and volume. The unfolding algorithm consists in sequentially testing a set of equations: only one of the solutions satisfies various constraints on bounds. A precise determination of the section thickness is required. Truncation parameters, instead of being troublesome inputs as in classical unfolding models, become valuable outputs.

Rates of regression and progression of dysplastic lesions in the nasal mucosa in nickel workers: a Markov model approach

Nasal epithelial dysplasia is considered a precancerous state. From 1976 through 1989, regular screening for such lesions has been performed among workers at the Falconbridge nickel refinery in Kristiansand. The longitudinal data thus obtained have been evaluated to ascertain to what extent, if any, pre-existing dysplasia can regress when exposure to nickel is reduced. A total of 418 pairs of observations were available from 243 workers. Interpretation of the data is complicated by the fact that dysplasia may remain undetected in small biopsies and the probability of detection of existing dysplasia was, therefore, incorporated into the two-state Markov model. Transition probability rates were estimated by maximum likelihood. The results suggest that regression of dysplasia has taken place and that regression rates increased with time. This finding probably reflects a decreased exposure resulting from a combination of a reduction in airborne nickel, improved personal hygiene and allocation of workers with dysplasia to work in areas with lower nickel exposure. Our results indicate that the chance of developing carcinomas related to nickel exposure is reduced. There are, however, indications that dysplasias continue to develop at a low rate.