Image processing analysis of stem cell antigens
This thesis aims to investigate the automation of an image processing driven analysis of antigen distributions in the membrane of early human Haematopoietic StemIProgenitor Cells (HSPCs ) imaged by Laser Scanning Confocal Microscopy (LSCM). LSCM experiments generated a vast amount of images of both single and dual labelled HSPCs. Special focus was given to the analysis of colocalised antigen distributions, as colocalisation may involve functional relationships. However, quantitative methods are also investigated to characterise both single and dual labelled antigen distributions. Firstly, novel segmentation algorithms are developed and assessed for their performances in automatically achieving fast fluorescence signal identification. Special attention is given to global histogram-based thresholding methods due to their potential use in real time applications. A new approach to fluorescence quantification is proposed and tested. Secondly, visualisation techniques are developed in order to further assist the analysis of the antigen distributions in cell membranes. They include 3D reconstruction of the fluorescence, newly proposed 2D Antigen Density Maps (ADMs) and new 3D graphs of the spatial distributions (sphere models). Thirdly, original methods to quantitatively characterise the fluorescence distributions are developed. They are applied to both single and dual/colocalised distributions. For the latest, specific approaches are investigated and applied to colocalised CD34/CD164 distributions and to colocalised CD34[sup]class I CD34[sup]class II and CD34[sup]c1ass I CD34[sup]class III epitopes distributions (two combinations of the three known different isoforms of the CD34 molecule, a major clinical marker for HSPCs). The visualisation tools revealed that HSPC membrane antigens are often clustered within membrane domains. Three main types of clusters were identified: small clusters, large patch-like clusters and newly identified meridian-shaped crest-like (MSCL) clusters. Quantitative analysis of antigen distributions showed heterogeneous distributions of the various measured features (such as polarity or colocalisation patterns) within the HSPC populations analysed. Finally, the proposed methodology to characterise membrane antigen distributions is discussed, and its potential application to other biomedical studies is commented. The potential extensions of the innovative linear diffusion-based MultiScale Analysis (MSA) algorithm to other applications are outlined. Visual and quantitative analyses of antigen membrane distributions are eventually used to generate hypotheses on the potential, yet unknown roles of these early antigens and are discussed in the context of haematopoietic theories.