While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.< Leer menos