Combination of Dark-Field and Confocal Microscopy for the Optical Detection of Silver and Titanium Nanoparticles in Mammalian Cells.

We describe here two optical microscopy techniques-dark-field confocal light scanning microscopy (DF-CLSM) and dark-field wide-field confocal microscopy (DF-WFCM), that can be used to study interaction between nanoparticles and cells in 3D space. Dark field microscopy can detect very small structures below the diffraction limit of conventional light microscopes, while a confocal setup provides vertical sectioning capabilities to render specimens in 3D. The use of DF-WFCM instead of DF-CLSM allows faster sample processing but yields lower resolution. We used a retinal pigment epithelial cell line ARPE-19 to illustrate different optical and lighting conditions necessary for optimal imaging of metal and metal oxide nanoparticles (TiO2 and Ag). Our experimental setup primarily involved an E-800 Nikon and Nikon Ni upright microscopes and a Nikon Ti2 microscope connected to a xenon light source along with special dark-field objectives. For confocal studies we used either Leica and Nikon inverted confocal microscopes. For microscopic 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 TiO2 or Ag tested (0.1-0.3 µg/mL), we were able to detect as few as 5-10 nanoparticles per cell due to intense light scattering by the particles. The degree of brightness detected indicated that the uptake of nanoparticles within ARPE-19 cells could be monitored using dark-field microscopy. Here we describe how to use wide-field microscopy to follow nanoparticle uptake by cells and how to assess some aspects of cellular health in in vitro cell cultures exposed to nanoparticles.

Detection of Silver and TiO2 Nanoparticles in Cells by Flow Cytometry.

Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. A FACSCalibur™ flow cytometer and a Stratedigm S-1000 flow cytometer were used to measure changes in light scatter from cells after incubation with either silver nanoparticles (AgNP) or TiO2 nanoparticles. Within the range of between 0.1 µg/mL and 30 µg/mL the nanoparticles caused a proportional increase of the side scatter and decrease of the forward scatter intensity signals. At the lowest concentrations of TiO2 (ranging between 0.1 µg/mL and 0.3 µg/mL), the flow cytometer can detect as few as 5-10 nanoparticles per cell. The influence of nanoparticles on the cell cycle was detected by nonionic detergent lysis of nanoparticle incubated cells that were stained with DAPI or propidium iodide (PI). Viability of nanoparticle treated cells was determined by PI exclusion. Surface plasmonic resonance (SPR) was detected primarily in the far-red fluorescence detection channels after excitation with a 488 nm laser.Our results suggest that the uptake of nanoparticles within cells can be monitored using flow cytometry. This uptake of nanoparticle data was confirmed by viewing the nanoparticles in the cells using dark-field microscopy. The flow cytometry detection of nanoparticles approach may help fill a critical need to assess the relationship between nanoparticle dose and cellular toxicity. Such experiments using nanoparticles could potentially be performed quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health.

Detection of TiO2 nanoparticles in cells by flow cytometry.

Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. A FACSCalibur™ flow cytometer was used to measure changes in light scatter from cells after incubation with TiO(2) nanoparticle. Both the side scatter and forward scatter changed substantially in response to the TiO(2). Between 0.1 and 30 µg/mL TiO(2), the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO(2) particles. At the lowest concentrations of TiO(2) (0.1-0.3 µg/mL), the flow cytometer apparently could detect as few as 5-10 nanoparticles per cell as shown using dark field microscopy. The influence of nanoparticles on the cell cycle was detected by nonionic detergent lysis of nanoparticle-incubated cells. Viability of nanoparticle-treated cells was determined by PI exclusion.These data suggest that the uptake of nanoparticles within cells can be monitored using flow cytometry and confirmed by dark field microscopy. This approach may help fill a critical need to assess the relationship between nanoparticle dose and cellular toxicity. Such experiments could potentially be performed quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health.

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.