AbstractThe cell nucleus is a three-dimensional, dynamic organelle that is organized into many subnuclear bodies, such as chromatin and nucleoli. The structure and function of these bodies is maintained by diffusion and interactions between related factors as well as dynamic and structural changes. Recent studies using fluorescent microscopic techniques suggest that protein factors can access and are freely mobile in mitotic chromosomes, despite their densely packed structure. However, the physicochemical properties of the chromosome itself during cell division are not yet fully understood. Physical parameters, such as the refractive index (RI), volume of the mitotic chromosome, and diffusion coefficients of fluorescent probes inside the chromosome were quantified using an approach combining label-free optical diffraction tomography with complementary confocal laser scanning microscopy and fluorescence correlation spectroscopy. Variance in these parameters correlated among various osmotic conditions, suggesting that changes in RI are consistent with those in the diffusion coefficient for mitotic chromosomes and cytosol. Serial RI tomography images of chromosomes in live cells during mitosis were compared with three-dimensional confocal micrographs to demonstrate that compaction and decompaction of chromosomes induced by osmotic change were characterized by linked changes in chromosome RI, volume, and the mobility of fluorescent proteins.
Direct Measurement of Association and Dissociation Rates of DNA Binding in Live Cells by Fluorescence Correlation Spectroscopy