abberior dyes & labels
Methods in cell biology
Quantitative live and super-resolution microscopy of mitotic chromosomes
Walther, N., & Ellenberg, J.
FCS-calibrated confocal imaging, Mitotic chromosomes, Absolute protein number, Subchromosomal protein localization
Microscopy is a powerful tool to investigate the dynamic molecular processes of mitosis in living cells. Recent advances in genome editing made it possible to tag all endogenous alleles of a protein of interest with a fluorescent marker and routinely create homozygous human knock-in cell lines for mitotic proteins. This opens the exciting possibility to perform quantitative and dynamic proteomic measurements in single dividing cells. However, commonly used wide field or confocal fluorescence imaging lack the quantitative power to determine the absolute number of proteins and do not provide sufficient resolution to localize single copies of proteins precisely within mitotic structures to map their molecular architecture. To overcome these limitations, we here combine two different advanced microscopy methods to determine the concentrations and absolute copy numbers as well as the subchromosomal distributions of mitotic chromosome structure proteins. The first method, quantitative 4D imaging, integrates confocal time-lapse microscopy with fluorescence correlation spectroscopy (FCS) in live cells to determine the absolute numbers of fluorescently tagged proteins in cellular compartments. The second method prepares cells fixed in specific mitotic stages for stimulated emission depletion (STED) microscopy, enabling the acquisition of super-resolved 3D volumes of entire dividing cells.