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Three-Dimensional Analysis of the Arrangement of Compact Chromatin in the Nucleus of G0 Rat Lymphocytes.

López-Velázquez G,  Márquez J.*,  Ubaldo E.,  Corkidi G.*,  Echeverría O.,  Vázquez-Nin G.

Facultad de Ciencias and *Centro de Instrumentos, UNAM, México D.F.

Full paper published in Histochemistry and Cell Biology, 105:153-161, 1996.

ABSTRACT :   Three dimensional reconstructions of chromatin bulk and individual clumps
were obtained to study the arrangement of compact chromatine of  G0  lymphocites. Serial  ultra-micro sections of rat spleen were contrasted with preferential procedures for DNA and transmision electron microscopy (TEM) photographs were digitized for image processing in a IBM PC-compatible microcomputer with an image processing and acquisition board (PLATES 1-3).  Two and three-dimensional analysis software was developped by us for this application.  Acquisition and pre-processing were performed with a comercial package software.   Computer 3D reconstructions showed continuos layers of compact chromatin in contact with the nuclear envelope (PLATES 4-9).  This arrangement prevented automatic analysis of individual chromatin bodies.  Qualitative simmilarities were found in the ensemble of compact chromatine for different lymphocites.  Using computer Mathematical Morphology Filtering techniques, the continuous bulk was divided into individual components.  Operator assistance was needed to separate peripheral chromatin in each image, since most of the internal components are in contact with the nuclear envelope.  Digital reconstruction and 3D processing shown that the number of contact points with the envelope and the number of individual chromatine bodies are similar or correspond to a haploid number of chromosomes.  It was also observed that the nucleolus-asociated chromatine is contained in the largest body, and when two nucleoli are present, their associated chromatin bodies contact the nuclear envelope in diametrically opposed areas.  This particular feature was also observed in rat hepathocites.  In conclusion, (a) there is no one-to-one correspondance between individual compact chromatin components and entire chromosomes, nor a pair of them; (b) compact chromatine arrangements are not identical in each G0  lymphocites, but repeated patterns do exist, with small morphological changes; and (c) different cell types of individuals of the same species share common morphological features.  

  

BibTeX entry:

@article{LopezCROMA96, author = {G. L{\'{o}}pez-Velazquez and J. Marquez and E. Ubaldo and G. Corkidi and O. Echeverr{\'{\i}}a and G. Vazquez-Nin}, journal = {Histochemistry and Cell Biology}, number = {105}, pages = {53-161}, title = {Three-Dimensional Analysis of the Arrangement of Compact Chromatin in the Nucleus of G0 Rat Lymphocytes.}, year = {1996}, url ={"http://www.tsi.enst.fr/~marquez/CELL/lympho.html"} }


From the 3D Image Processing point of view . . .

Computer reconstructions and 3D processing proved to be valuable tools in this analysis, and allowed to set specific protocols of morpho-analysis from serial TEM micrographs. Elemental alignement for translation along the centroid axis was integrated in the image processing software, but manual, interactive alignement and edition were needed during acquisition.  Several morphometric parameters were also considered for a later study:  size, irregularity and excentricity of chromatin bodies, cartographic projection of chromatin domains, as observed at the envelope surface, and individal component display rendering, for comparative examination.   Similar techniques for preparation of  TEM image slices, morphoanalisis and 3D visualization have been applied in the study of nucleus-cytoplasm interfaces in dopaminergic neurons from Parkinson Desease patients.   Geometry distorsion correction techniques (image un-warping) were employed to correct residual rotational misalignement, and mechanical and electron beam heating deformations.  These techniques may still be improved to minimize manual interaction, at least for rotational alignement, by using the principal axes of the moment of inertia.  Beside other quantities, isotropy measures can then be performed.

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> Word 3.0 paper wo figs. (small corrections not included)

>  References for this research
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To contact the authors:

López-Velázquez Gabriel   Laboratory of  Electron Microscopy, Department of Biology, Faculty of Sciences, UNAM
Márquez Jorge   Center of Instruments UNAM - Dept. IMAGE - Ecole Nationale Supérieure des Télécommunications
Corkidi Gabriel    Center of Instruments UNAM
Vázquez-Nin Gerardo
 Laboratory of Electron Microscopy, Department of Biology, Faculty of Sciences, UNAM


page by Jorge Marquez   January 25, 1998.