Nanoparticle dynamics in living cells and <i>in vitro</i> assessed by RICS.

<p>(A) A time series of fluorescent COOH-PS [YO] NPs that at room temperature freely diffuse in water was acquired by confocal microscopy (frame size: 512×512 pixels; pixel size: 0.03 mm; scan speed: 6.4 ms/pixel). (A) shows one image of the time series image stack. (B) Two-dimensional spatial autocorrelation of the image stack displaying the region for 32 pixel shifts in negative and positive directions. Numbers along the color code bar indicate the correlation values. (C) Using a region of interest (ROI, 64x64 pixels) analysis, which scans sub-regions within the original image, a map for the diffusion coefficient is generated along with a diffusion coefficient <i>(D)</i> color map. A corresponding scale indicating the hydrodynamic radius (<i>r</i>_H) is also shown. (D) Image of a living HEp-2 cell incubated with fluorescent COOH-PS [YO] NPs for 1 hour. The white box indicates the region were subsequently a confocal time series image stack was acquired (frame size: 512×512 pixels; pixel size: 0.03 mm; scan speed: 6.4 ms/pixel). One image of the resulting image stack is shown in (E). (F) Two-dimensional spatial autocorrelation of the image stack (as described in B). (G) Diffusion coefficient distribution map of the region shown in (E) along with a diffusion coefficient <i>(D)</i> color map and a scale indicating the corresponding apparent hydrodynamic radius (<i>r</i>_H) corrected for the apparent viscosity of 30 nm particles in the nucleus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062018#pone.0062018-Bulinski1" target="_blank">[52]</a>. (H) A confocal time series image stack was acquired in the cytoplasm of a COOH-PS [YO] NP-treated HEp-2 cell (frame size: 512×512 pixels; pixel size: 0.03 mm; scan speed: 6.4 ms/pixel). One image of the resulting time series stack is shown. Note that the selected region also contains a region outside the cell (m, medium). (I) Two-dimensional spatial autocorrelation of the image stack (as described in B). Diffusion coefficient distribution maps of the region shown in (H) along with the respective diffusion coefficient <i>(D)</i> color maps were generated for the diffusion coefficient ranges between 0 and 2.7 µm2s-1 (J), or between 0 and 0.48 µm2s-1 (K). Black areas within the diffusion maps indicate regions not covered by the diffusion coefficient range displayed or areas with fits of insufficient quality. Bar, 10 µm.