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LIGHT MICROSCOPY DETECTION OF NANOSCALE PARTICLE INTERNALIZATION BY HUMAN LUNG CELLS
Citation:
GIBBS-FLOURNOY, E. A., R. M. ZUCKER, W. Cheng, W. Hofer, P. A. BROMBERG, AND J. M. SAMET. LIGHT MICROSCOPY DETECTION OF NANOSCALE PARTICLE INTERNALIZATION BY HUMAN LUNG CELLS. Presented at American Thoracic Society Meeting, New Orleans, LA, May 14 - 19, 2010.
Impact/Purpose:
Ultrafine particulate matter (PM) is reported to be more strongly correlated with adverse health effects relative to larger particle size fractions.
Description:
RATIONALE. Ultrafine particulate matter (PM) is reported to be more strongly correlated with adverse health effects relative to larger particle size fractions. These epidemiological findings are supported by toxicological studies suggesting that particle size is inversely associated with potency. The industrial proliferation of nanomaterials has added to the concern over the potential toxicity of nanoscaled particles (NP). Study of particle dosimetry, deposition, uptake and clearance hinges on the ability to visualize the particles at cellular and subcellular levels. Regarding NP, there is great interest in determining whether particles of varying composition can enter the cell through active or passive processes and to study any resulting effects. Unfortunately, the small size of these nanoparticles is typically beyond the limits of resolution of conventional light microscopy. The use of electron microscopy for NP detection in cells is costly, laborious, limited to particles that are sufficiently electron dense, and is incompatible with live cell imaging. To overcome these limitations, we evaluated the potential of a novel combination of confocal and darkfield (CFIDF) microscopy for the efficient detection of environmentally relevant NP in human lung cells. METHODS. Human alveolar macrophages (HAM) obtained by bronchoalveolar lavage using an IRE approved protocol, or BEAS 2B cells were exposed to 25nm Ti02 NP, 140 nm fluorescent nanodiamond crystals, or 100 nm fluorescein-conjugated polystyrene beads for periods ranging from 5 min to 24 hrs. Cells were fixed in 4.5% parafomaldehyde, stained with DAPI, Nile Red, andlor LysoTracker Red and mounted on slides using #1.5 coverslips. Z-stack images were acquired and processed on an inverted Nikon C1Si Spectral Confocal Imaging System equipped with a darkfield condenser and a 60x or 100x lens using 404, 488, and/or 561 nm lasers for excitation RESULTS. Control experiments using fluorescent polystyrene beads demonstrated spatial colocalization of fluorescence (confocal) and scattered transmitted light (darkfield). CF/DF microscopy analysis showed time-dependent internalization of Ti02, nanodiamond, and polystyrene beads in HAM and BEAS cells. Optical sectioning of the cells confirmed localization ofNP clustered in foci coincident with the cytoplasm ofthe cells. CONCLUSIONS. These findings demonstrate the utility of CFIDF microscopy in the study of NP within human lung cells. This technique offers significant advantages over electron microscopy including reduced cost, compatibility with live cell analysis, and the ability to detect environmentally relevant carbonaceous particles that may not be suitable to electron microscopy. THIS ABSTRACT OF A PROPOSED PRESENTAnON DOES NOT NECESSARILY REFLECT EPA POLICY.