Co-localization of moving point like structures in confocal microscopy

Co-localization of fast-moving vesicular structures in sequential acquisition mode displays a challenge as conventional pixel-based metrics as the Pearson correlation coefficient or the Manders coefficients tend to perform poorly under these circumstances. To address this issue, we performed object-based co-localization analysis to sequentially scanned confocal images and time series and tested these object -based methods against well-established metrics. To generate data for validation, the lysosomes of MCF-7 cells were co-labeled with RFP and GFP. To further test the procedures, organelles that are known to be spatially well separated were co-stained.  Furthermore, to demonstrate the practical application of the object-based methods, cells with RFP labeled lysosomes were incubated with fluorescent carbon nanoparticles, suspected to enter the endo-lysosomal pathway.