Segmentation and shape tracking of overlapping macrophages in fluorescent microscopy images

This thesis investigates the complex movement of migrating cells and small organisms. Whilst special emphasis has been devoted to macrophages observed with fluorescent confocal microscopy, the segmentation, tracking and shape analysis techniques developed here can be extended to various other applications, not exclusively of cells. With the acquisition conditions and particular resolution, macrophages exhibit very complex shapes. In some cases, the cells overlap, complicating the analysis considerably. A software framework is proposed to detect, track and analyse the shape and movement of macrophages. The framework, called macrosight, includes the following: (i) a novel 2D matrix with multiscale angle variation, called the anglegram, based on the angles between the points of the boundary of an object through which its general shape can be characterised. For the case of the macrophages, the anglegram helped identify the intersection of overlapping cell boundaries. (ii) A cell shape tracking algorithm providing a measured evolution of shape parameters and categorisation of shape types. This was based on metrics extracted from the anglegram and used for non-overlapping cells. (iii) An algorithm to assess the changes of direction derived from cell-cell contact. The algorithm may assist in the identification of cell-cell interactions, which may influence migration patterns. The anglegram showed promising results identifying two types of junctions in the boundaries of segmented objects, both in synthetic and real data. Tests on synthetic data showed that the method outperformed the classic junction detection technique by Harris. The framework can be used to explore the influence of cell-cell contact in movement patterns. The early experiments presented provided statistically significant evidence of such influence, opening the path for additional information of the tracks being included to further investigate on a larger number of interactions. This thesis provides preliminary studies of macrophages’ shapes and interactions. Future developments incorporating the techniques proposed in this thesis would provide a precise analysis of cell movement in biological studies, which could lead to pioneering insights concerning cell migration