Darkfield-Confocal Microscopy Detection of Nanoscale Particle Internalization by Human Lung Cells Public Deposited

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  • Zucker, Robert M
    • Other Affiliation: Toxicology Assessment Division, NHEERL, U.S. EPA, Research Triangle Park, NC, USA
  • Samet, James M
    • Other Affiliation: Environmental Public Health Division, NHEERL, U.S. EPA, Chapel Hill, NC, USA
  • Hofer, Thomas PJ
    • Other Affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health, Clinical Cooperation Group Inflammatory Lung Diseases, Gauting, Germany
  • Gibbs-Flournoy, Eugene A
    • Affiliation: School of Medicine, Curriculum in Toxicology
  • Bromberg, Philip A
    • Affiliation: School of Medicine, Center for Environmental Medicine, Asthma and Lung Biology
  • Abstract Background Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane. Results Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO2 particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependant internalization process. Conclusions DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels.
Date of publication
  • 21247485
  • doi:10.1186/1743-8977-8-2
Resource type
  • Article
Rights statement
  • In Copyright
Rights holder
  • Eugene A Gibbs-Flournoy et al.; licensee BioMed Central Ltd.
Journal title
  • Particle and Fibre Toxicology
Journal volume
  • 8
Journal issue
  • 1
Page start
  • 2
  • English
Is the article or chapter peer-reviewed?
  • Yes
  • 1743-8977
Bibliographic citation
  • Particle and Fibre Toxicology. 2011 Jan 19;8(1):2
  • Open Access
  • BioMed Central Ltd

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