Biological processes occur in space and time, but current experimental methods for systems biology are limited in their ability to resolve this spatiotemporal complexity of life. In addition, traditional “omics” methods often suffer from limited sensitivity and need to average over populations of cells at the expense of cell to cell variation. Next-generation systems biology therefore requires methods that can capture data and build models in four dimensions, three-dimensional space and time, and needs to address dynamic events in single living cells. In fact, recent advances in automated fluorescence microscopy, cell microarray platforms, highly specific probes, quantitative image analysis and data mining provide a powerful emerging technology platform to enable systems biology of the living cell. These imaging technologies, here referred to as “Systems microscopy”, will be a cornerstone for next-generation systems biology to elucidate and understand complex and dynamic molecular, sub-cellular and cellular networks. As a paradigm to enable systems biology at the cellular scale of biological organization, this NoE will have as its core biological theme two basic but complex cellular processes that are highly relevant to human cancer: cell division and cell migration. Methods, strategies and tools established here will be applicable to many disease-associated processes and will be instrumental for obtaining a systems level understanding of the molecular mechanisms underlying human diseases as manifested at the living cell level.Through close multidisciplinary collaborations in our programme of joint activities this NoE will develop a powerful enabling platform for next-generation systems biology and will apply these tools to understand cellular systems underlying human cancer. This provides a unique opportunity for Europe to acquire a global lead in systems microscopy.