Methods for topographical analysis of intra-nuclear BrdUrd-tagged fluorescence

The observation of BrdUrd staining in the nuclei of cells from exponentially growing populations reveals different typical replicating patterns. We propose a methodological approach to order and characterize BrdUrd intranuclear distributions. First, visual ordering of the patterns is assessed using a spectral analysis coupled to a k-nearest neighbors clustering technique. Subsequently, nine topographical features are introduced to characterize the spatial distribution of BrdUrd-tagged fluorescence in the nuclei of proliferating cells. These topographical features are based on a structural approach. The localization of fluorescence spots is expressed in terms of the normalized distance from the nuclear border and its standard deviation. These topographical features are simple to calculate and easy to relate to visual experience.