Combination of Dark-Field and Confocal Microscopy for the Optical Detection of Silver and Titanium Nanoparticles in Mammalian Cells
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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.
Key wordsNanoparticle Microscopy Dark-field Microscopy Toxicology Nanotoxicity Confocal Microscopy Organelles Fluorescence
The manuscript was edited by Enrico Ferrari and Mikhail Soloviev. Appreciation is given to Carl Blackman Eugene Gibbs, James Samet, Laura Degn, Sarah Hutchinson, and Kristen Sanders for their helpful discussions on the research included in the protocol.
Government Disclaimer: The research described in this chapter has been supported by the US Environmental Protection agency. It has been subjected to agency review and does not necessarily reflect the views of the agency, and no official endorsement should be inferred. The mention of trade names or commercial products does not constitute endorsement or recommendation for use.
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