Analysis of foci volume and arrangement within the cell nucleus from top line to bottom line: Foci count of the fourth experiment analyzed with NIS elements; average membrane distance of the foci in [%] compared to the maximum possible radius within the nucleus; average foci volume in arbitrary units; volume ratio of all combined foci to the nucleus volume; note that the two values in brackets faced a very high standard deviation and are—Due to the low foci count—Not used for any conclusion even though fitting well to the other values.

<p>Analysis of foci volume and arrangement within the cell nucleus from top line to bottom line: Foci count of the fourth experiment analyzed with NIS elements; average membrane distance of the foci in [%] compared to the maximum possible radius within the nucleus; average foci volume in arbitrary units; volume ratio of all combined foci to the nucleus volume; note that the two values in brackets faced a very high standard deviation and are—Due to the low foci count—Not used for any conclusion even though fitting well to the other values.</p

Transmission electron microscopy images show GNPs being included into the cytosol but excluded from the lumina of intracellular vesicles, microbodies, the golgi apparatus and the endoplasmic reticulum.

<p>Further GNPs are preferentially accumulated in groups of varying extend. A. Overview of a group of GNPs (black arrow) inside the cytosol (Cy). The extracellular space (ES) and the nucleus (Nu) do not carry any GNP accumulations. B. Locality in the cytosol with a local GNP accumulation in the vicinity of components of the intracellular membrane apparatus (IM). C. Insert showing the homogeneous size of SmartFlares in a cluster.</p

Localization microscopy images of a SkBr3 cells.

<p>Illumination at 561 nm transfected with 8 μl SmartFlare solution after 18 h incubation time with the characteristic ring like shape (A-C). Wide-field overview (A) and localization microscopy image (B) with points representing the loci of blinking events. Merged image (C) of wide-field overview (red) and detected GNP signals (yellow) of the pointillist localization microscopy image. Localization microscopy image of a cell treated by unmodified GNP for comparison (D). The scale bars equal 20 μm each.</p

Analysis of γH2AX foci (red) for different SmartFlare concentrations incubated for 18 h on SkBr3 cells (cell nuclei are stained in blue by DAPI) after irradiation of 4 Gy: control probe (left), and 8 μl SmartFlare transfected (right).

<p>The scale bars equal 20 μm.</p

Epifluorescence image of SkBr3 cells transfected with SmartFlares illuminated with a 671 nm laser.

<p>While Cy5 signals can be clearly seen in the cytoplasm of the cells, cells treated with no or unmodified GNP are almost not visible due to the very low auto-fluorescence at 671 nm (not shown). The scale bar equals 20 μm.</p

Comparison of the averaged detected foci count for control (orange), unmodified GNP (green) and SmartFlare probes (brown) at 0 Gy, 2 Gy and 4 Gy.

<p>The standard deviation is highlighted as black arrow line. A significance level of 0.05 was used for all statistical tests. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190183#pone.0190183.t002" target="_blank">Table 2</a> (below) the significance levels are presented which would be required that the significance disappears. These values have to be seen in relation to 0.05 if one compares the histograms in this figure.</p

3D image of fluorescently labelled γH2AX foci (pink) in a SkBr3 cell nucleus after irradiation of 4 Gy and incubation with 8 μl SmartFlare solution.

<p>The foci are represented with NIS-Elements. The cube’s edge length equals 20 μm. No special foci arrangement or distribution could be detected, independently from dose or GNP concentration.</p

Localization microscopy signal counts for unmodified GNP, SmartFlares.

<p>For each wavelength of 491 nm and 561 nm standard deviation and the error of the mean are given.</p