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DEVELOPEMENT OF A 3D-IMAGE ANALYSIS SYSTEM FOR TRANSMISION ELECTRON MICROSCOPY:     
Application to the Study of Dopaminergic Neuron Nuclei from the
Substantia Nigra in the Parkinson Disease

Jorge Márquez,  Philippe Anglade,  Etienne Hirsch,  Yves Agid

Laboratoire de Médecine Expérimentale - INSERM U289
Hôpital de la Salpêtrière, 47 Bd del'Hôpital 74013 PARIS

DEA de Génie Biologique et Médical Université Paris XII.    Année 1993-1994.

Abstract

The subject of this research combines the domains of Biomedical Imaging, image processing and the study of degenerative pathologies of the Central Nervous System.

The objectif of the present study, from the biological point of view, was to make evident, in the Parkinson disease, the presence of compensation phenomena of neural death, at the level of the nuclei of dopaminergic neurons from the substantia nigra pars compacta (SNpc). To this end, three morphological characteristiques of nuclei were analyzed:  the compacity of the chromatin (genetic activity index), the size and shape of lthe nucleole (protein synthesis index), and the surface of the nuclear enveloppe (that is, the index of exchanges number/rate between the nucleus and the cytoplasm).

To accomplish this objectif, we have developped a system and an image analysis methodology for two and three dimensions (2D et 3D), which enable, to process images of ultrathin slice series from electron microscopy, and reconstruct the three-dimensional nuclei of dopaminergic neurons from the SNpc, and quantify their morphological parameters in witness (healthy subjects) and parkinson patients.

During experimental work, we have acquired, for each witness subject (n=3) and each parkinson patient (n=3), the serial slices of at least four nuclei (making a total of 27 nuclei).   Images constituting the biological material to analyze, were obtained from serial slices micrographs taken at a transmision electron microscope. These images were digitized and processed for 3D reconstruction using a comercial imaging system (IMAGENIA from BIOCOM) and a 3D system that we have previously designed and adapted fro this project. New software tools were intedrated to both analysis systems on a IBM-PC 386 compatible computer.

Concerning 2D imaging, we implemented a methodology specific to image processing for micrographs of ultrathin serial slices, obtaining a set of tools enabling correction of problems characterizing such images (inhomogeneities, distortions,...), as well as conversion and formating tools for 3D reconstruction.

The 3D technique that we employed consisted in building a discrete representation of digitized objects, using their discrete external surface (or boundary), using volume elements (voxels) and their faces.  This technique allowed to perform, on the objects to be analyzed, all processings required for a 3D representation, to obtain morphological parameters of objects of interest, and to visualize their structure and spatial arrangement.

The methodology and the system we developped are at present being validated and applied to analysis of morphological modifications of dopaminergic-neuron nuclei from the SNpc in Parkinson disease.  Observation of ultrathin slices in the TEM, and first 3D reconstructions have shown a disminution of the volume measured on the nucleole and a diminution of invaginations of the nucleus-cytoplasme interfaces.  Nevertheless, no meaningful morphological modification was observed at the level of the chromatin, whose intrincate structure have not been already decomposed in sub-elements, in able to study them.  Preliminar results, if verified by quantitative study, would suggest the abscence of compensation phenomena of neural death, at the level of the cellular bodies of dopaminergic neurons from the SNpc in patients with Parkinson disease .

In summary, preliminar biological results show that quantitative evaluation of these modifications, effectuated on several neural nuclei, by using the 3D analysis system, will permit to make evident morphological changes in neurons spared by the pathology.

The system and the 2D-3D methodology and system that we have developped, shall be employed in other biological problems when three-dimensional reconstruction of serial slices is to be done from electron microscopy or other imaging techniques.  It has been previewed to port the system to more powerful equipements, such as a workstation.

This DEA (master) internship has been done at the INSERM U0289 laboratory, of the Hôpital de la Salpêtrière, Paris, with Drs. Philippe Anglade and Etienne Hirsch as advisors.

Keywords:  Parkinson disease, neural nucleus, electron microscopy, image processing, 3D reconstruction.

>>>>>>  To have a copy (in french):   marquez@ima.enst.fr


BibTeX entry:

@mastersthesis{JM:DEA94, author = {Jorge Marquez}, type = "Master Thesis, Dipl{\^o}me d'Etudes Approfondies", school = {Universit{\'{e}} Cr{\'{e}}teil-Paris XII}, title = {D{\'{e}}veloppement d'un système d'analyse d'images 3D pour la microscopie {\'{e}}lectronique: Application à l'{\'{e}}tude des noyaux des neurones dopaminergiques de la substantia nigra dans la maladie de parkinson.}, month = sep, year = {1994}, url = "http://www.tsi.enst.fr/~marquez/CELL/parkinson.html", }


TABLE DES MATIERES (TABLE CONTENTS)

RESUME

1. INTRODUCTION

2  MATERIEL

3 RESULTATS  I:

DEVELOPPEMENT D'UNE METHODOLOGIE POUR L'ANALYSE D'IMAGES DES NOYAUX NEURONAUX EN 3D

4 RESULTATS II:

APPLICATION DU SYSTEME D'ANALYSE 3D, A L'ETUDE DES NOYAUX DES NEURONES DOPAMINERGIQUES DE LA SUBSTANTIA NIGRA PARS COMPACTA

5 DISCUSSION

6 CONCLUSIONS ET PERSPECTIVES

7 BIBLIOGRAPHIE

8  ANNEXES

9  LISTE DES FIGURES


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