Advanced light microscopy methods have become invaluable research tools to investigate the molecular basis of complex biological mechanisms and their related disorders. To visualize cellular structures and molecular dynamics with increasing spatiotemporal resolution, researchers have developed a plethora of fluorescence-based imaging modalities and instrumentations as well as analysis tools. The Advanced Medical Bioimaging Core Facility of the Charité Universitätsmedizin (AMBIO) was established in September 2015 with the overarching aim of providing the infrastructure necessary for biomedical researchers with diverse backgrounds to efficiently perform cutting-edge biomedical imaging projects. The core AMBIO unit at the central Charité campus harbors four fully complementary advanced imaging systems (~ 3 MEuro) that offer highly flexible imaging modalities to investigate a large variety of biomedical samples. By offering support during all phases of the complex bioimaging process, including initial consulting on the imaging strategy and sample preparation, image acquisition, data processing and quantitative analysis, AMBIO will guarantee sustainable access to and efficient usage of high-end bioimaging instrumentation within the Charité research community and associated research institutions.