Abstract By generating three-dimensional (3D) microtissues or organoids that accurately mimic the in vivo cellular functions, the organ-on-a-chip technologies have been playing an increasingly important role in exploiting biology and screening pharmaceutical compounds. Their successful utility is accompanied by the high demand for on- site imaging of the microtissues’ morphology, function, and molecular signatures that allow us to understand their behaviors and responses. Clearly, to image the 3D microtissues, the imaging technique should ideally provide volumetric information, together with microscopic resolutions for cellular or even subcellular analyses. On-chip imaging of intact 3D microtissues within microfluidic devices is however, fundamentally hindered by intratissue optical scattering. The overall goal of this proposal thus seeks to innovate a methodology, where a meticulously designed microfluidic bioreactor platform will be developed to enable simultaneous expansion and clearing of intact microtissues directly on-chip for in situ, super-resolution, and volumetric characterizations, breaking limits in both imaging depth and optical diffraction with conventional fluorescence microscopy.