Project abstract How molecular organization and activity leads to tissue level outcomes is an open question in biology and biomedical research. Addressing this question is technically challenging because we cannot observe with molecular precision at the tissue scale. While optical microscopy is the method of choice to observe architecture and dynamics within living cells and organisms, it has severe limitations in spatiotemporal resolution and volumetric coverage. Thus, our most detailed observations of cellular dynamics and ultrastructure have been limited to single cells on coverslips, which were far removed from their physiological context. Here I propose to extend the capabilities of light-sheet fluorescence microscopy (LSFM), a technology that provides gentle and efficient 3D imaging, but only moderate resolution. We will combine LSFM with super-resolution methods to allow rapid imaging of subcellular dynamics away from coverslips. We will further explore ways to increase the volumetric acquisition rate of LSFM such that large samples can be rapidly explored. These new developments will be tied together by smart sampling strategies, which will enable autonomous exploration of large samples while applying the highest resolution only locally. This way, we expect that rare cellular events can be studied in subcellular detail in entire model organisms or organs that were rendered transparent through clearing. The proposed rapid volumetric imaging capabilities will enable imaging of all neurons in a small model organism such as C.elegans or Zebrafish embryos with up to 100 Hz volume rate. Such rapid pan-neuronal imaging may shed light on how a brain functions. While we expect that the potential for discovery with such microscope technology is immense, access to such advanced instrumentation is often limited. To address this, we will develop a modular light-sheet platform that can be adapted to a wide range of imaging tasks. We will further explore ways to simplify the microscope architecture and design less expensive variants to aid dissemination. The resulting new microscope technology will enable large volume imaging experiments that have been prohibited by either lack of spatial resolution or acquisition speed, and as such accelerate biological and biomedical research.