Micrographs of two vesicle populations with different vesicle densities in solution.

<p>Vesicle abundance is higher in (a) than in (b). The lower abundance of vesicles (b) is preferred for better performance of the computer vision algorithms.</p

Histograms of vesicle diameter size distribution (a) and vesicle isoperimetric quotient (b).

<p>Data samples for both histograms were taken from one randomly selected sample and their shapes are representative of all samples.</p

Vesicle measurements from three experiments; the experimental parameters for each experiment (time is the duration of incubation at recording; the number of chambers is the same as the number of samples).

<p>Relative standard deviation (RSD) measures the variation between chambers of the same populations. Each population had multiple chambers; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113405#pone-0113405-t001" target="_blank">Table 1</a> contains the mean vesicle quantity per chamber mean vesicle diameter and mean vesicle isoperimetric quotient (IPQ).</p><p>Vesicle measurements from three experiments; the experimental parameters for each experiment (time is the duration of incubation at recording; the number of chambers is the same as the number of samples).</p

Scheme of the perfusion chambers (a).

<p>Silicone model with four perfusion chambers named C1–C4 (b). The side view is given in the figure and the depth of the chambers is 0.5 mm. A single chamber with two tracks (P1 and P2) locations of two tracks recorded in each chamber (c). The two tracks together cover approximately 3% of the chamber area.</p

Changes in samples from different vesicle populations over time (from 3 to 60 minutes).

<p>Each experiment was conducted using a new initial vesicle population and each sample point represents a single chamber. Vesicle quantities (a), mean projected diameter sizes (b), and mean isoperimetric quotients (c). The box plot consists of mean minimal and maximal values 25th 50th and 75th percentile.</p

Light micrograph of vesicles in a micrograph (a), vesicles with overlaid segmentation (b), mask with segmented vesicles separated from the background in which each vesicle is marked with a number (c).

<p>Light micrograph of vesicles in a micrograph (a), vesicles with overlaid segmentation (b), mask with segmented vesicles separated from the background in which each vesicle is marked with a number (c).</p

FTIR microscopy reveals distinct biomolecular profile of crustacean digestive glands upon subtoxic exposure to ZnO nanoparticles

<p>Biomolecular profiling with Fourier-Transform InfraRed Microscopy was performed to distinguish the Zn²⁺-mediated effects on the crustacean (<i>Porcellio scaber</i>) digestive glands from the ones elicited by the ZnO nanoparticles (NPs). The exposure to ZnO NPs or ZnCl₂ (1500 and 4000 µg Zn/g of dry food) activated different types of metabolic pathways: some were found in the case of both substances, some only in the case of ZnCl₂, and some only upon exposure to ZnO NPs. Both the ZnO NPs and the ZnCl₂ increased the protein (∼1312 cm⁻¹; 1720–1485 cm⁻¹/3000–2830 cm⁻¹) and RNA concentration (∼1115 cm⁻¹). At the highest exposure concentration of ZnCl₂, where the effects occurred also at the organismal level, some additional changes were found that were not detected upon the ZnO NP exposure. These included changed carbohydrate (most likely glycogen) concentrations (∼1043 cm⁻¹) and the desaturation of cell membrane lipids (∼3014 cm⁻¹). The activation of novel metabolic pathways, as evidenced by changed proteins’ structure (at 1274 cm⁻¹), was found only in the case of ZnO NPs. This proves that Zn²⁺ are not the only inducers of the response to ZnO NPs. Low bioavailable fraction of Zn²⁺ in the digestive glands exposed to ZnO NPs further supports the role of particles in the ZnO NP-generated effects. This study provides the evidence that ZnO NPs induce their own metabolic responses in the subtoxic range.</p

Cellular Internalization of Dissolved Cobalt Ions from Ingested CoFe₂O₄ Nanoparticles: In Vivo Experimental Evidence

With a model invertebrate animal, we have assessed the fate of magnetic nanoparticles in biologically relevant media, i.e., digestive juices. The toxic potential and the internalization of such nanoparticles by nontarget cells were also examined. The aim of this study was to provide experimental evidence on the formation of Co²⁺, Fe²⁺, and Fe³⁺ ions from CoFe₂O₄ nanoparticles in the digestive juices of a model organism. Standard toxicological parameters were assessed. Cell membrane stability was tested with a modified method for measurement of its quality. Proton-induced X-ray emission and low energy synchrotron radiation X-ray fluorescence were used to study internalization and distribution of Co and Fe. Co²⁺ ions were found to be more toxic than nanoparticles. We confirmed that Co²⁺ ions accumulate in the hepatopancreas, but Fe^<i>n</i>+ ions or CoFe₂O₄ nanoparticles are not retained in vivo. A model biological system with a terrestrial isopod is suited to studies of the potential dissolution of ions and other products from metal-containing nanoparticles in biologically complex media

Cellular Internalization of Dissolved Cobalt Ions from Ingested CoFe₂O₄ Nanoparticles: In Vivo Experimental Evidence

With a model invertebrate animal, we have assessed the fate of magnetic nanoparticles in biologically relevant media, i.e., digestive juices. The toxic potential and the internalization of such nanoparticles by nontarget cells were also examined. The aim of this study was to provide experimental evidence on the formation of Co²⁺, Fe²⁺, and Fe³⁺ ions from CoFe₂O₄ nanoparticles in the digestive juices of a model organism. Standard toxicological parameters were assessed. Cell membrane stability was tested with a modified method for measurement of its quality. Proton-induced X-ray emission and low energy synchrotron radiation X-ray fluorescence were used to study internalization and distribution of Co and Fe. Co²⁺ ions were found to be more toxic than nanoparticles. We confirmed that Co²⁺ ions accumulate in the hepatopancreas, but Fe^<i>n</i>+ ions or CoFe₂O₄ nanoparticles are not retained in vivo. A model biological system with a terrestrial isopod is suited to studies of the potential dissolution of ions and other products from metal-containing nanoparticles in biologically complex media