Detection of silver nanoparticles inside marine diatom Thalassiosira pseudonana by electron microscopy and focused ion beam

In the following article an electron/ion microscopy study will be presented which investigates the uptake of silver nanoparticles (AgNPs) by the marine diatom Thalassiosira pseudonana, a primary producer aquatic species. This organism has a characteristic silica exoskeleton that may represent a barrier for the uptake of some chemical pollutants, including nanoparticles (NPs), but that presents a technical challenge when attempting to use electron-microscopy (EM) methods to study NP uptake. Here we present a convenient method to detect the NPs interacting with the diatom cell. It is based on a fixation procedure involving critical point drying which, without prior slicing of the cell, allows its inspection using transmission electron microscopy. Employing a combination of electron and ion microscopy techniques to selectively cut the cell where the NPs were detected, we are able to demonstrate and visualize for the first time the presence of AgNPs inside the cell membrane

Scanning electron microscope images of a diatom exposed to AgNPs using the signal from the surface (A) and in transmission (B).

<p>(C) shows the EDX map of carbon (blue), silicon (yellow) and silver (red) of the area indicated by the dashed squares in (B) assigned to the organic part, the exoskeleton and AgNPs respectively (scale bar common to the pictures (A) and (B)).</p

Scanning electron microscope images of a second diatom exposed to AgNPs, using the signals from the surface (A) and in transmission (B).

<p>(C) and (D) show magnifications of the same area of the cell indicated with the dashed squares, where it is possible to detect NPs in the surface (i.e. blue arrow) or potential internalized particles (i.e. red arrow) (scale bars common to pictures (A), (B) and (C), (D) respectively).</p

Scanning electron microscope images of the nanostructured pores in the shell of two diatoms corresponding to the control (A) and AgNPs exposed (B) samples respectively (scale bar common in both pictures).

<p>Scanning electron microscope images of the nanostructured pores in the shell of two diatoms corresponding to the control (A) and AgNPs exposed (B) samples respectively (scale bar common in both pictures).</p

Sequence of the cut and detection of Ag content in the section from the same cell shown in <b>Figure 3</b>.

<p>(A), the cell after the deposition of the Pt protective layer. (B), the cell after the cut; (C) an enlargement of the section of the cell with an enhanced contrast. (D) and (E), the Ag and Os EDX maps respectively over the Secondary Electron signal collected by the EDX detector. (F) EDX spectra from the background (blue) and the bright spots (red) corresponding to the blue and red square area shown in the panel (D). The bright spots are also marked by the red arrow in panel (C).</p

Effects of Silver Nanoparticles in Diatom <i>Thalassiosira pseudonana</i> and Cyanobacterium <i>Synechococcus sp.</i>

The aim of the present study was to investigate the effect of silver nanoparticles (AgNP) of different sizes toward two primary producer aquatic species. <i>Thalassiosira pseudonana</i> and <i>Synechococcus sp.</i> have been selected as representative models for the lower trophic organisms in marine and freshwater habitats, respectively. Time-dependent cellular growth was measured upon exposure to both AgNP and silver nitrate (AgNO₃). In addition, AgNP behavior in freshwater and marine waters has been followed by CPS disc centrifuge, in the time frame of AgNP exposure studies, and the kinetic release of silver from AgNP of different sizes was measured by dialysis and inductively coupled plasma mass spectrometry (ICP-MS). The combination and interpretation of all these data suggest that a shared effect of AgNP and released silver was responsible for the toxicity in both organisms. Furthermore, the toxic effects induced by AgNP exposure in the present study seem to result from a mixture of parameters including aggregated state, size of the AgNP, stability of the preparation, and speciation of the released silver