ABSTRACT

Alliances between experimental and computational disciplines are increasingly important to meet the challenges of modern live sciences. In laboratories all over the world, the precise quantification of ´colocalization` with microscopic imaging techniques has become a frequent task. Colocalization verifies the presence of biological molecules, structures, or organelles at the same physical location. So far, colocalization has not been resolved satisfactorily on a mathematic-statistical level. We therefore focus this project on the development of a coherent and robust approach to quantify the colocalization of fluorescently labeled biological structures with confocal laser scanning microscopy. In contrast to all preceding methods which calculate colocalization coefficients for an image set with rather simple, global mathematical coefficients (based on the theories of Manders or Pearson), our approach develops a consequent bottom-up algorithm based on the calculation of local scaling functions which we term Colocalization Scaling Index Methods (C-SIM). These local C-SIM are defined within a given radius r for every position xi in the image matrices. Our approach will account for the strong interdependence in adjacent pixel regions which are induced by the physical properties of the experiment-specific point spread functions (PSF) in the diffraction limited detection volume of contemporary light microscopes. The properties of the following mathematical approaches for the local estimation of colocalization will be investigated: the Scaling Index Method SIM(xi|r), the Correlation Coefficient of Raw Images CCRI(xi|r), the Joint Moment of Standardized Images JMSI(xi|r), the auto- and cross-correlation functions G(X)(xi|r), and coefficients based on the theories of Pearson and Manders. Based in our experience with SIM-related techniques we have strong evidences to believe that the success of all C-SIM will critically depend on the optimal selection of the sensitive C-SIM radii. Preliminary results indicate that global and local auto- and cross-correlation functions G(X)(xi|r) possess promising properties to determine the size of interdependent pixel regions. These properties will be investigated in a detailed manner in order to guarantee an operator independent approach for the optimal selection of C-SIM radii.
An important question concerns the probabilistic evaluation of the calculated C-SIM maps. A final decision has to be made if a local colocalization is coincidental or not. In this context we will implement a novel approach based on a defined displacement algorithm (DDA) for the entire set of the C-SIM(xi|r) descriptors. Local DDAs provide the possibility to generate local probability density functions (PDF), facilitating the definition of a precise statistical probability (p-values) which separates true versus coincidental colocalization. Local DDAs will lead to a robust probabilistic model where the initial regional interdependency between the subjacent biological structures is preserved and every pixel is evaluated in respect to its vicinity.
Our probabilistic approach will be validated from a theoretical level (simulation) up to an experimental level which will lead us to the best possible description of colocalization with C-SIM(xi|r). Last but not least, C-SIM will be applied to current issues in cell biology, ranging from experiments with different cell cultures to experiments which include transgenic animals. The experimental applications will culminate to resolve colocalization in vivo on a three dimensional level in cells within developing organisms such as the translucent embryo of zebrafish (Danio rerio). Our approach has a strong transdisciplinary focus. It combines facets from physical, mathematical, computational, and biological sciences. It finally aims to a successive implementation of the developed routines in form of a free cross-platform runtime utility based on IDL Virtual Machine and to the implementation as an interactive internet-based tool on our web-page for interactive Scientific Image Analysis (SCIAN, www.scian.cl). With this approach, we are defending the spirit of public domain software and the benefits of the development will be accessible to the entire scientific community. It should be empathized that our web-page is developed and maintained in collaboration with a local software company, Xperts Ltda. (Ingenieria en Informática y Comunicaciones, Valdivia, www.xperts.cl), which has originated important mutual benefits and strengthens the transfer of scientific image processing technologies into different areas.

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).