Fish are ideally suited for in vivo-imaging due to their transparency at early stages combined with a large genetic toolbox. Key challenges to further advance imaging are fluorophore selection, immobilization of the specimen and approaches to eliminate pigmentation. We addressed all three and identified the fluorophores and anaesthesia of choice by high throughput time-lapse imaging. Our results indicate that eGFP and mCherry are the best conservative choices for in vivo-fluorescence experiments, when availability of well-established antibodies and nanobodies matters. Still, mVenusNB and mGFPmut2 delivered highest absolute fluorescence intensities in vivo. Immobilization is of key importance during extended in vivo imaging. Here, traditional approaches are outperformed by mRNA injection of α-Bungarotoxin which allows a complete and reversible, transient immobilization. In combination with fully transparent juvenile and adult fish established by the targeted inactivation of both, oca2 and pnp4a via CRISPR/Cas9-mediated gene editing in medaka we could dramatically improve the state-of-the art imaging conditions in post-embryonic fish, now enabling light-sheet microscopy of the growing retina, brain, gills and inner organs in the absence of side effects caused by anaesthetic drugs or pigmentation.