Microscopy imaging methods for the detection of silver and titanium nanoparticles within cells

Scientific evaluation of potential environmental hazards resulting from man-made nanomaterials has been hampered by the inability to optimally detect cell-associated nanoparticles. We have successfully imaged TiO(2) nanoparticles in ARPE-19 cells using different light microscope modalities commonly available to investigators including fluorescence, dark field, phase, interference, and confocal. In this report, we describe different optical and lighting conditions necessary for optimal nanoparticle imaging in ARPE-19 cells.Microscopic examinations involved an E-800 Nikon microscope connected to a xenon light source along with special dark field objectives. For microscopy analyses, ARPE-19 cells were fixed in situ in cultured chamber slides or collected from T-25 flasks and then fixed in suspension. At the lowest concentrations of TiO(2) (0.1-0.3 μg/mL), it was possible to detect as few as 5-10 nanoparticles per cell due to intense light scattering by TiO(2). The degree of brightness detected indicated that the uptake of nanoparticles within ARPE-19 cells could be monitored using dark field microscopy. This report details how wide-field microscopy can be effectively used to detect nanoparticle uptake as well as to assess cellular health in ARPE-19 cell cultures.