Image analysis techniques for visualizing the spatial organization of DNA replication sites in the mammalian cell nucleus using multi-channel confocal microscopy

Confocal images of double labeled DNA replication sites in mouse 3T3 cells were analyzed using a set of image processing algorithms to investigate the spatial organization of replication sites at different times. Since confocal microscopy images have about a 1:10 ratio of horizontal (X-Y) versus vertical (Z) scale, extending traditional 2D image processing techniques to 3D rarely gives satisfactory results. By using 2D image segmentation on the individual slices to obtain replication site contours and combining this high-level 2D data using a modified 3D connected component labeling algorithm with weak connectivity in the Z direction, we were able to reconstruct the network of 3D replication sites. Instead of using the equivalent of traditional 2D 8-connectivity in 3D, we used a metric which evaluated the amount of overlap between successive contours of a replication site to decide whether the two contours belong to the same replication site. This method is applicable in analyzing the images of replication sites as they are bead like structures. Once the data structures describing the individual sites are obtained, several pattern recognition techniques are applied to elucidate 3D higher order assembly of replication sites. In one method, amount of site overlap between the two channels were calculated in an attempt to quantify the closeness of sites at different times. Another approach consisted of grouping the replication sites using a pre-set distance between sites required for a set of sites to be included in the same group.