Figure 4.

Multiplane, multiplexed diffusion maps of Dendra2-Pol II expressed in U2OS cells. (A and B) Average intensity maps of Dentra2-Pol II time image series (left) and corresponding ACFs (right). 10 k images were acquired at different z positions (A, z₊ = 0.0 μm; B, z₈ = 5.6 μm) with a spacing of ∼0.8 μm and all eight planes were sequentially imaged with a total time of 5 min. The regions outside the cell nucleus showed zero correlation (flat ACFs in A shown in blue). (C) Representative diffusion maps for Dendra2-Pol II in the cell nucleus at different z positions (see Movie S2). (D) 3D orthogonal view of diffusion maps along x, y, and z axes. (E) Cumulative probability distribution of diffusion coefficient in serum-starved (cyan) and stimulated (magenta) environments (average from four cells; >13,400 data points for serum-stimulated and >4800 data points for serum-starved conditions). All imaging performed on a sCMOS camera with effective pixel size = 160 nm. ACF analysis was performed with 3 × 3 pixel binning. To see this figure in color, go online.