The rapid developments in in-vivo molecular imaging modalities such as fluorescence and bioluminescence imaging enables the live imaging of gene expression, cell fate and protein interactions. Combined with detailed structural imaging modalities such as magnetic resonance (MR), the biochemical onset of disease and therapy can be monitored in combination with structural and functional consequences over time. This presentation discusses a number of image analysis challenges emerging from longitudinal pre-clinical molecular imaging studies that have been addressed within the ENCITE project. Three steps towards a quantitative 3D analysis of follow-up small animal imaging are presented: whole-body registration, change visualization in follow-up data and fusion of optical and 3D structural imaging data. Several application examples are presented in the context of cell tracking and translational cancer research.