Porous hydrogels provide an excellent environment for cell growth and tissue regeneration, with high permeability for oxygen, nutrients, and other water-soluble metabolites through their high water-content matrix. The ability to image three-dimensional (3D) cell growth is crucial for understanding and studying various cellular activities in 3D context, particularly for designing new tissue engineering scaffold, but it is still challenging to study cell-biomaterial interfaces with high resolution imaging. We demonstrate using focused ion beam (FIB) milling, electron imaging, and associated microanalysis techniques that novel 3D characterizations can be performed effectively on cells growing inside 3D hydrogel scaffold. With FIB-tomography, the porous microstructures were revealed at nanometer resolution, and the cells grown inside. The results provide a unique 3D measurement of hydrogel porosity, as compared with those from porosimetry, and offer crucial insights into material factors affecting cell proliferation at specific regions within the scaffold. We also proved that high throughput correlative imaging of cell growth is viable through a silicon membrane based environment. The proposed approaches, together with the protocols developed, provide a unique platform for analysis of the microstructures of novel biomaterials, and for exploration of their interactions with the cells as well. Biotechnol. Bioeng. 2013; 110: 318–326. © 2012 Wiley Periodicals, Inc.