Darkfield-Confocal Microscopy detection of nanoscale particle internalization by human lung cells

Abstract

BackgroundConcerns 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.ResultsValidation 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.ConclusionsDF-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.

Analytical approaches to support current understanding of exposure, uptake and distributions of engineered nanoparticles by aquatic and terrestrial organisms

“…Using dark field optics, noble metal nanoparticles, such as Ag and Au in particular can be detected in biological matrices due to their specific optical properties (strong localised surface plasmon resonance) (Stebounova et al 2011). The interfacing of dark field imaging with confocal microscopy can now also allow effective 3-D imaging of unlabelled ENPs in cells and tissues as an approach that clearly can be relevant to ecotoxicology studies (Gibbs-Flournoy et al 2011).…”

Nanotechnology: Toxicologic Pathology

“…For example, caveolin-mediated endocytosis may facilitate NP transport into the cell, within the cell, and then exocytosis back out of the cell, a process that is sometimes known as transcytosis. Because such transcytosis pathways are capable of transporting NPs across cells, caveolin-mediated NP transport may be a means for traversing the BBB to directly access the brain (Gibbs-Flournoy et al 2011; Kreuter 2013; Wagner et al 2012; Wohlfart, Gelperina, and Kreuter 2012). Additional endocytic pathways can also be used for traversing the BBB (Bhaskar et al 2010; Wagner et al 2012).…”

“…The confocal microscope can also demonstrate NPs using fluorescent, reflective, or transmitted light, depending upon the features of the NP. For example, fluorescent NPs such as quantum dots can be imaged in the fluorescent microscope, while MWCNTs can be demonstrated using either transmitted light or reflected light (Gibbs-Flournoy et al 2011; Porter et al 2010). These features allow the localization of NPs within cells or even within nuclei (Porter et al 2012; Figure 8).…”

CdTe quantum dots induce activation of human platelets: implications for nanoparticle hemocompatibility

“…Ultramicroscopic study of QD-induced platelet activation QDs are beyond the diffraction limit of resolution for conventional microscopy (∼200 nm). 32 Therefore, to study in depth the structure of QD-induced platelet-platelet interactions, TEM was used. TEM images of platelets are shown in Figure 4.…”

“…48 Most of these studies were performed using LTA. We have shown that LTA may not be sensitive enough for studying platelet-NP interactions, and a more [29][30][31][32][33] may be preferred. In keeping with this notion, we detected QD-induced platelet aggregation in PRP using QCM-D but not LTA.…”