In vivo imaging across multiple scales is commonly associated with challenging compromises between the achievable contrast, imaging speed and spatial resolution. Optoacoustic (or photoacoustic) imaging is increasingly attracting the attention of the biomedical research community due to its excellent spatial and temporal resolution, centimeter scale penetration into living tissues, and versatile endogenous and exogenous optical absorption contrast. State-of-the-art implementations of multi-spectral optoacoustic tomography (MSOT) are based on multi-wavelength excitation of tissues to visualize specific molecules within opaque tissues. As a result, the MSOT technology can noninvasively deliver structural, functional, metabolic, and molecular information from living tissues. Our recent efforts in the field of optoacoustic functional and molecular imaging have established new technological platforms employing spherical matrix arrays, parallel acquisition hardware, GPU-based data processing and fast-tuning laser systems in order to enable acquisition and visualization of spectroscopic information from entire tissue volumes at video rates. This has set the stage for the so-called five dimensional (real-time three-dimensional multi-spectral) optoacoustic imaging that offers unparalleled capabilities among the existing bio-imaging modalities. Applications are explored in the areas of functional neuro-imaging, fast tracking of agent kinetics and biodistribution, cardiovascular research, monitoring of therapies and drug efficacy as well as targeted molecular imaging studies. Clinical translation roadmap will be finally discussed.