ABSTRACT Understanding how cell-cell communication and collective cell phenomena shape cell function and cell fate decisions requires the ability to follow the detailed molecular processes as they take place inside live cells, with the cell remaining embedded in its natural setting of the tissue and organism of origin. Although tremendous progress has been made in imaging of single isolated cells, the requirements for probing crowded multi-cellular systems with high spatio-temporal resolution and down to single-molecule sensitivity present an enormous challenge for optical microscopy. Here we propose to tackle this challenge, using recent breakthroughs to i) increase the capability of extracting high-resolution information from weak fluorescence signals – using 3D interferometry; ii) image fragile/delicate biological samples using optimized configurations - based on selective- plan illumination; iii) maintain/recover high resolution information through optically inhomogenous samples – using adaptive optics. We hypothesize that successful synthesis of these three key technologies will create new approaches that cross into previously uncharted realms of combined spatio-temporal resolution, detection sensitivity, non-invasiveness and penetration depth. Based on these ideas we propose the following two specific aims: (1) To develop multi-color volumetric 3D interferometric imaging, based on reliable, artifact-free optical reconstructions, for increasing the ability to extract high-resolution information from weak fluorescence signals; (2) To achieve non-invasive, background-free, long-term 4D imaging, at ~100nm near-isotropic 3D spatial resolution, at millisecond acquisition times and over large and highly crowded (multi)cellular volumes. The new techniques will significantly increase our abilities to interrogate dynamic biological processes with molecular detail, in single isolated cells as well as in intact complex multi-cellular systems, thus having widespread and immediate impact across biomedical disciplines.