Mechanisms of Apoptotic and Necrotic Cell Death
Time Resolved Multi-Parameter Analysis of Membrane Related Processes
FONDECYT 3030065, Post-Doc, 04/2003 – 04/2006, CECS, Valdivia, Chile
PI: S Härtel, Tutor: F Barros
ABSTRACT
The main topic addressed in the scope of the present project FONDECYT 3030065 concerned the development of IMAGE PROCESSING ROUTINES which tackled current questions in the fields of cell death and differentiation (NECROBIOLOGY) and MEMBRANE STRUCTURE, DYNAMICS & FUNCTION. Special interest was focused on the development of image processing routines for conventional bright field microscopy and for time resolved three dimensional confocal laser scanning microscopy. The combination of image processing routines with digital microscopic data enables the investigator to address diverse questions in biological systems on a three dimensional level. The developed image processing units point towards a time resolved 3-D feature classification for the coupling of individual parameters (derived from single objects/cells) with parameter-pattern (statistical information) of the embedding macro-systems. This methodology is summarized as �Time Resolved 3-D Cytometry� and covered (i) the acquisition and correction of microscopic data, (ii) the detection and segmentation of regions of interest (ROI) in three dimensional image stacks, (iii) the extraction of features, (iv) feature classification, and, (v) the presentation of information. Since similar pattern exist in different scales in time and space, the conceptual work in one area can be extrapolated to different fields. This also explains the interdisciplinary collaboration in this field ranging from domain formation in lipid membranes, to cell biology, embryology, botanic, and finally to extra-terrestrial physics. Selected topics in image processing were addressed by undergraduate students from the Universidad Austral de Chile (UACh) and from the Centro de Formación Técnica in the scope of their thesis. The routines have converged into a modular, object orientated software package which runs on Windows, Mac, or Unix PCs. In addition, a first version of an internet based tool for online image processing has been designed in collaboration with a local software company Xperts (www.xperts.cl) and included into our web-page for scientific image analysis (SCIAN, www.scian.cl).
Different results that have been published in the course of this project contributed new insight into the implication the homeostasis of cation pumps on volume increase during necrotic cell death, revealed different kinetics of morphologic and texture dependent features of nuclear structures in human cornea epithelial cells, dissected selective effects of ultraviolet irradiation on spontaneous and induced apoptosis in freshly extracted mice thymocytes, and reported that phosphohydrolytic enzymes provide laterally immiscible lipid domains with the capacity to mediate and transduce information at the membrane level from a nm to the µm scale. The results also contributed to a novel method to quantify surface bound phosphatase activities in mycorrhizal trees, and quantified triploidization rates in rainbow trout.
In addition to the published results, the present project has stimulated future research oriented projects like FONDECYT 1060890 (starting in April 2006) or the human frontier science program HFSP (application 20.03.06), and stimulated the formulation of technically innovative projects (Chile-Innova, application 01.04.06).