Biophysical properties of cells such as intracellular mass density and cell mechanics are known to be involved in a wide range of homeostatic functions and pathological alterations. An optical readout that can be used to quantify such properties is the refractive index (RI) distribution. It has been recently reported that the nucleus, initially presumed to be the organelle with the highest dry mass density (ρ) within the cell, has in fact a lower RI andρthan its surrounding cytoplasm. These studies have either been conducted in suspended cells, or cells adhered on 2D substrates, neither of which reflects the situationin vivowhere cells are surrounded by the extracellular matrix (ECM). To better approximate the 3D situation, we encapsulated cells in 3D covalently-crosslinked alginate hydrogels with varying stiffness, and imaged the 3D RI distribution of cells, using a combined optical diffraction tomography (ODT)-epifluorescence microscope. Unexpectedly, the nuclei of cells in 3D displayed a higherρthan the cytoplasm, in contrast to 2D cultures. Using a Brillouin-epifluorescence microscope we subsequently showed that in addition to higherρ, the nuclei also had a higher longitudinal modulus (M) and viscosity (η) compared to the cytoplasm. Furthermore, increasing the stiffness of the hydrogel resulted in higherMfor both the nuclei and cytoplasm of cells in stiff 3D alginate compared to cells in compliant 3D alginate. The ability to quantify intracellular biophysical properties with non-invasive techniques will improve our understanding of biological processes such as dormancy, apoptosis, cell growth or stem cell differentiation.