Ribosomes are the most essential macromolecules in cells, as they serve as production lines for every single protein. Here, we address the demand to study ribosomes in living human cells by applying time-resolved microscopy. We show that oxazole yellow iodide (YO-PRO-1 dye) intercalates tRNA and rRNA with a determined equilibrium constant of 3.01 ± 1.43 × 10 M. Fluorescence correlation spectroscopy (FCS) is used to measure both the rotational (∼14 ms) and translational (∼4 μm s) diffusion coefficients of the 60S ribosomes directly within living human cells. Furthermore, we apply the empirical length-scale dependent viscosity model to calculate the hydrodynamic radius of 60S ribosomes, equal to ∼15 nm, for the first time determined inside living cells. The FCS in YO-PRO-1 stained cells is used to assess ribosome abundance changes, exemplified in rapamycin-treated HeLa cells, highlighting its potential for dynamic ribosome characterization within the cellular environment.
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