The cell is three-dimensionally and dynamically organized into cellular compartments, including the endoplasmic reticulum, mitochondria, vesicles, and nucleus, which have high relative molecular density. The structure and functions of these compartments and organelles may be deduced from the diffusion and interaction of related biomolecules. Among these cellular components, various protein molecules can freely access the nucleolus or mitotic chromosome through Brownian diffusion, even though they have a densely packed structure. However, physicochemical properties of the nucleolus and chromosomes, such as molecular density and volume, are not yet fully understood under changing cellular conditions. Many studies have been conducted based on high-resolution imaging and analysis techniques using fluorescence. However, there are limitations in imaging only fluorescently labeled molecules, and cytotoxicity occurs during three-dimensional imaging. Alternatively, the recently developed label-free three-dimensional optical diffraction tomography (ODT) imaging technique can divide various organelles in cells into volumes and analyze them by refractive index, although specific molecules cannot be observed. A previous study established an analytical method that provides comprehensive insights into the physical properties of the nucleolus and mitotic chromosome by utilizing the advantages of ODT and fluorescence techniques, such as fluorescence correlation spectroscopy and confocal laser scanning microscopy. This review article summarizes a recent study and discusses the future aspects of the ODT for cellular compartments.