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Fig. 2 | Genome Biology

Fig. 2

From: Hi-D: nanoscale mapping of nuclear dynamics in single living cells

Fig. 2

Experimental validation of the Hi-D approach. a Exemplary frame of a simulated time series with low density (0.001/px3) of emitters undergoing Brownian motion convolved by a typical point spread function (left). The time series is subject to Hi-D and single-particle tracking estimating the trajectories of emitters. From the estimated trajectories, the MSD is computed and compared to the ground truth diffusion constant. The relative error in the determined diffusion constant is shown. b High density (0.02/px3) of emitters with patches of super-high density (0.035/px3) encircled for visualization, imitating regions of densely packed chromatin. Dashed lines show the optimal value, i.e., perfect agreement between estimation and ground truth. Red lines indicate the median value. Data from 10 independent simulations. Statistical significance assessed by a two-sample Kolmogorov-Smirnov test (***: p < 0.001). c MSD curves computed in fixed (n = 13) and living quiescent (− serum; n = 13) and serum stimulated (+ serum; n = 14) U2OS cells. Diffusion constants for the three average curves were derived by regression yielding D = (0.87 ± 0.1) · 10−3 μm2/s for quiescent, D = (2.6 ± 0.1) · 10−4 μm2/s for stimulated, and D = (6.1 ± 0.1) · 10−6 μm2/s for fixed cells. MSD curves show considerably higher MSD values for living cells and diffusion constants are two orders of magnitude higher for living cells thus confirming the detection of motion well above noise background. d Diffusion constants derived from a nucleus corrupted with varying levels of signal-to-noise ratio. Results are consistent up to a lower bound of ~ 20 dB. e Map of diffusion constants computed by Hi-D (left) and iMSD (right). Diffusion constants are color coded from their minimum to their maximum value (blue to yellow; for absolute values see f). Red arrows indicate regions of high mobility detected by both methods. f Diffusion constants shown in e and g corresponding values of the anomalous exponent computed by Hi-D (blue) and iMSD (red)

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