Complementary Imaging of Silver Nanoparticle Interactions with Green Algae: Dark-Field Microscopy, Electron Microscopy, and Nanoscale Secondary Ion Mass Spectrometry.

Increasing consumer use of engineered nanomaterials has led to significantly increased efforts to understand their potential impact on the environment and living organisms. Currently, no individual technique can provide all the necessary information such as their size, distribution, and chemistry in complex biological systems. Consequently, there is a need to develop complementary instrumental imaging approaches that provide enhanced understanding of these "bio-nano" interactions to overcome the limitations of individual techniques. Here we used a multimodal imaging approach incorporating dark-field light microscopy, high-resolution electron microscopy, and nanoscale secondary ion mass spectrometry (NanoSIMS). The aim was to gain insight into the bio-nano interactions of surface-functionalized silver nanoparticles (Ag-NPs) with the green algae Raphidocelis subcapitata, by combining the fidelity, spatial resolution, and elemental identification offered by the three techniques, respectively. Each technique revealed that Ag-NPs interact with the green algae with a dependence on the size (10 nm vs 60 nm) and surface functionality (tannic acid vs branched polyethylenimine, bPEI) of the NPs. Dark-field light microscopy revealed the presence of strong light scatterers on the algal cell surface, and SEM imaging confirmed their nanoparticulate nature and localization at nanoscale resolution. NanoSIMS imaging confirmed their chemical identity as Ag, with the majority of signal concentrated at the cell surface. Furthermore, SEM and NanoSIMS provided evidence of 10 nm bPEI Ag-NP internalization at higher concentrations (40 μg/L), correlating with the highest toxicity observed from these NPs. This multimodal approach thus demonstrated an effective approach to complement dose-response studies in nano-(eco)-toxicological investigations.