Intracellular imaging of nanoparticles: Is it an elemental mistake to believe what you see?
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In order to understand how nanoparticles (NPs <100 nm) interact with cellular systems, potentially causing adverse effects, it is important to be able to detect and localize them within cells. Due to the small size of NPs, transmission electron microscopy (TEM) is an appropriate technique to use for visualizing NPs inside cells, since light microscopy fails to resolve them at a single particle level. However, the presence of other cellular and non-cellular nano-sized structures in TEM cell samples, which may resemble NPs in size, morphology and electron density, can obstruct the precise intracellular identification of NPs. Therefore, elemental analysis is recommended to confirm the presence of NPs inside the cell. The present study highlights the necessity to perform elemental analysis, specifically energy filtering TEM, to confirm intracellular NP localization using the example of quantum dots (QDs). Recently, QDs have gained increased attention due to their fluorescent characteristics, and possible applications for biomedical imaging have been suggested. Nevertheless, potential adverse effects cannot be excluded and some studies point to a correlation between intracellular particle localization and toxic effects.
J774.A1 murine macrophage-like cells were exposed to NH2 polyethylene (PEG) QDs and elemental co-localization analysis of two elements present in the QDs (sulfur and cadmium) was performed on putative intracellular QDs with electron spectroscopic imaging (ESI). Both elements were shown on a single particle level and QDs were confirmed to be located inside intracellular vesicles. Nevertheless, ESI analysis showed that not all nano-sized structures, initially identified as QDs, were confirmed. This observation emphasizes the necessity to perform elemental analysis when investigating intracellular NP localization using TEM.
KeywordsIntracellular Localization Single Particle Level Electron Energy Loss Spectrum Electron Spectroscopic Imaging Laser Scanning Microscopy Analysis
electron spectroscopic imaging
laser scanning microscope
phosphate buffered saline
transmission electron microscope.
The tremendous application potential of nano-sized particles (NPs 1-100 nm; ISO/TS 27687:2008) is in sharp contrast to a growing number of critical reports regarding their potential toxicity. In order to correlate any toxic reaction with a NP type, it is indispensable to investigate if the particles are attached to the cell surface or if they enter cells. If NPs are found in cells, their localization in different compartments such as endosomes, lysosomes, mitochondria, the nucleus or the cytosol, may also provide some answers regarding their potential toxicity.
Transmission electron microscopy (TEM) offers adequate resolution to visualize NPs at a single particle level as well as the ability to determine their localization in different cellular compartments. However, only few particle types, such as gold NPs, show unique characteristics like particle shape and electron density that can be easily recognized within cellular compartments. To confirm the presence of NPs and their localization inside cells, additional elemental analysis of the NP compositions is therefore often required . This can be performed on TEM level by energy filtered TEM, since each chemical element shows a characteristic electron energy loss spectrum.
In this study, elemental analysis was performed on intracellular quantum dots (QDs). Semi-conductor QD nanocrystals  have gained increased attention in recent years due to their novel fluorescent characteristics and subsequently, their potential advantages as diagnostic and therapeutic tools [3, 4, 5]. Therefore, intensive research has focused upon understanding the potential toxic effects of QDs, prior to their use within such medical applications . This is predominantly due to QDs consisting of a heavy-metal core material, such as cadmium-telluride (CdTe) or cadmium-selenide (CdSe), which is covered by a zinc sulfide (ZnS) shell. Although not fully understood, it is these constituents which have subsequently been suggested as driving QD associated toxicity. The QDs used in this study were coated with NH2 polyethylene glycol (PEG) and have previously been shown to cause no cytotoxicity  or pro-inflammatory cytokine stimulation in J774.A1 cells after 2 h . However, the NH2 PEG QDs do induce an increased intracellular Ca2+ concentration after 30 min and a decreased glutathione level after 2 h exposure with 40 nM QD in this macrophage cell-line . In addition, it has also been shown that the specific intracellular localization (such as within the nucleus, cytosol, mitochondria or vesicles) significantly determines QD toxicity [8, 9].
Since QDs are highly fluorescent, research using laser scanning microscopy (LSM) has been used to identify QD intracellular localization via a series of fluorescent markers for intracellular organelles, such as the cytosol, nucleus or intracellular vesicles [9, 10]. Despite the advantages of LSM techniques, light microscopic resolution is limited for the size scale of NPs. TEM, however, provides an adequate resolution at a single particle level and, theoretically, due to the heavy-metal core of QDs, TEM is a viable option for determining their intracellular localization. However, the relatively weak electron density of QDs compared to TEM sample staining agents, such as osmium, uranyl acetate and lead citrate, as well as their small size (~5 nm) similar to one of cytoplasmic protein complexes, makes it extremely difficult to detect QDs inside cells. Therefore, electron spectroscopic imaging (ESI)  was performed to confirm the intracellular QDs.
To investigate intracellular particle localization, J774.A1 murine 'macrophage-like' cells were cultured in a 24-well plate, at a density of 2.5 × 105 cells/mL as previously described , and further exposed to 40 nM QDs for 2 h in an environment of 37°C, 5% CO2. Investigation of the intracellular localization of the QDs was performed initially via LSM (Zeiss 510 Meta; Axiovert 200 M, Lasers: HeNe 633 nm, and Ar 488 nm), which confirmed that QDs had entered the macrophages . The cells were then fixed with 1 M glutaraldehyde in 0.1 M Na-cacodylate diluted in PBS at pH 7.3, for 3 h at 4°C. The samples were then embedded for TEM by post-fixation in 1% osmium tetroxide in 0.1 M Na-cacodylate buffer for 45 min, washing with 0.1 M Na-cacodylate buffer at 3 and 10 min changes, dehydration in graded concentrations of acetone (50%, 70%, 90% and 100%) and embedded in Araldite resin. The embedded samples were then cut to 60 nm thick ultrathin sections, mounted onto square 400 mesh copper grids (Agar Scientific, Essex, England) and stained with uranyl acetate and lead citrate. The QDs intracellular localization was subsequently investigated using ESI as described before.
Results and Discussion
The results of this study emphasize the need for better characterization of intracellular NPs, as not all detected electron dense or irregular, nano-sized, intracellular structures represent NPs. Only a limited number of NP types show very unique characteristics, including particle shape and electron density, which can be easily and exclusively recognized within cells. Despite this fact, several studies investigating intracellular localization by TEM have not performed any form of elemental analysis to confirm the presence of intracellular NPs [14, 15, 16]. In each example, additional elemental analysis such as ESI or Energy Dispersive X-ray Spectroscopy (EDXS) would be indispensable to the conclusions made by these studies. In light of this fact, statements made within the literature concerning the intracellular localization of NPs without adequate analysis should therefore be taken with caution. Obtaining reliable information pertaining to the intracellular localization of NPs is of increasing importance due to the need to understand NP-cell interactions. As the intracellular localization of NPs has been shown to be related to their toxicity , information regarding the precise intracellular localization of NPs is not only imperative in order to understand the potential adverse effects of exposure to NPs, but also to realize the proposed advantages that are posed by nanotechnology.
Competing financial interests
The authors declare that they have no competing interests.
The authors would like to acknowledge Stephen Mitchell (Royal (Dick) Veterinarian College of the University of Edinburgh, UK) as well as Andrea Stokes and Mohammed Ouanella (Institute of Anatomy, University of Bern, Switzerland) for their technical assistance in preparing the electron microscopy samples and Kirsten Dobson for proof reading the manuscript. We thank Dr. Alfred Bretscher for the funding of the Tecnai F20 TEM.
This study was supported by grants of the Animal Free Research Foundation, the Doerenkamp-Zbinden Foundation and the Swiss National Science Foundation (3100A0_118420).
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