Citation:

Zucker, R. AND W. Boyes. Combination of Dark-Field and Confocal Microscopy for the Optical Detection of Silver and Titanium Nanoparticles in Mammalian Cells. Edition 2nd, Chapter 28, Ferrari, Enrico and Solovier, Mikhail (ed.), Nanoparticles in Biology and Medicine. Second Edition. Springer, Heidelberg, Germany, 2118:395-414, (2020). https://doi.org/10.1007/978-1-0716-0319-2_28

Impact/Purpose:

Nanomaterials may be hazards to humans, other organisms and the environment (1). Nanoparticles (NPs) are typically defined as having a size in at least one dimension between 1 nm and 100 nm. Such nanomaterials can present health risks due to their composition, reactivity as well as their small size (2). Detecting nanoparticles in solutions, tissues or cells remains difficult. The availability of methods suitable for routine identification and characterization of biological objects such as cells containing nanomaterials would benefit the understanding of potential risks posed by nanomaterials. Traditional methods used for studying nanoparticles and their effects at the cellular level rely on transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), dynamic light scattering (DLS) and fluorescence microscopy (FM), the latter for studying fluorescent nanoparticles. Unfortunately these methods are less suitable toxicological investigations and FM is only suitable for use with fluorescent materials, is limited to certain particle size and carries the risk of sample photo bleaching. TEM and SEM are labor-intensive and time-consuming and are not suitable for field applications. DLS can be used to determine a hydrodynamic diameter of pure nanomaterials in aqueous suspensions but is not suitable for studying nanoparticle interactions with cells or the identification and localization of nanoparticles within cells More traditional light microscopy methods could have been very suitable for studying NPs and especially for environmental and toxicology applicators, but the detection of NPs is usually beyond the resolution diffraction limit of conventional light microscopes (~200 nm).

Description:

We describe here two optical microscopy techniques - dark-field confocal light scanning microscope (DF-CLSM) and dark-field wide-field confocal microscopy (DF-WFCM), that can be used to study interaction between nanoparticle and cells in 3D space. Dark field microscopy can detect very small structures below the diffraction limit of conventional light microscopes, whilst 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 setupprimarilysetup 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 nanoparticles uptake by cells and how to assess some aspects of cellular health in in vitro cell cultures exposed to nanoparticles.

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