FIB-SEM imaging of carbon nanotubes in mouse lung tissue

Ultrastructural characterisation is important for understanding carbon nanotube (CNT) toxicity and how the CNTs interact with cells and tissues. The standard method for this involves using transmission electron microscopy (TEM). However, in particular, the sample preparation, using a microtome to cut thin sample sections for TEM, can be challenging for investigation of regions with agglomerations of large and stiff CNTs because the CNTs cut with difficulty. As a consequence, the sectioning diamond knife may be damaged and the uncut CNTs are left protruding from the embedded block surface excluding them from TEM analysis. To provide an alternative to ultramicrotomy and subsequent TEM imaging, we studied focused ion beam scanning electron microscopy (FIB-SEM) of CNTs in the lungs of mice, and we evaluated the applicability of the method compared to TEM. FIB-SEM can provide serial section volume imaging not easily obtained with TEM, but it is time-consuming to locate CNTs in the tissue. We demonstrate that protruding CNTs after ultramicrotomy can be used to locate the region of interest, and we present FIB-SEM images of CNTs in lung tissue. FIB-SEM imaging was applied to lung tissue from mice which had been intratracheally instilled with two different multiwalled CNTs; one being short and thin, and the other longer and thicker. FIB-SEM was found to be most suitable for detection of the large CNTs (Ø ca. 70 nm), and to be well suited for studying CNT agglomerates in biological samples which is challenging using standard TEM techniques. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-013-7566-x) contains supplementary material, which is available to authorized users

Time-Dependent Subcellular Distribution and Effects of Carbon Nanotubes in Lungs of Mice

BACKGROUND AND METHODS:Pulmonary deposited carbon nanotubes (CNTs) are cleared very slowly from the lung, but there is limited information on how CNTs interact with the lung tissue over time. To address this, three different multiwalled CNTs were intratracheally instilled into female C57BL/6 mice: one short (850 nm) and tangled, and two longer (4 μm and 5.7 μm) and thicker. We assessed the cellular interaction with these CNTs using transmission electron microscopy (TEM) 1, 3 and 28 days after instillation. RESULTS:TEM analysis revealed that the three CNTs followed the same overall progression pattern over time. Initially, CNTs were taken up either by a diffusion mechanism or via endocytosis. Then CNTs were agglomerated in vesicles in macrophages. Lastly, at 28 days post-exposure, evidence suggesting CNT escape from vesicle enclosures were found. The longer and thicker CNTs more often perturbed and escaped vesicular enclosures in macrophages compared to the smaller CNTs. Bronchoalveolar lavage (BAL) showed that the CNT exposure induced both an eosinophil influx and also eosinophilic crystalline pneumonia. CONCLUSION:Two very different types of multiwalled CNTs had very similar pattern of cellular interactions in lung tissue, with the longer and thicker CNTs resulting in more severe effects in terms of eosinophil influx and incidence of eosinophilic crystalline pneumonia (ECP)

Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA damage. These results are important guidelines to the design and interpretation of experiments involving nanowire-based transfection and electrical characterization of living cells

Nanotoxicology: 2D analysis in a 3D matrix

Engineered nanoparticles are the fastest growing nanotechnology product, and of increasing environmental concern. Examples include antimicrobial silver nanoparticles, iron oxide particles used in bulk scale for environmental remediation, and micro- and nano-plastics. The latter are added to a broad range of cosmetic products. Larger plastic pieces pollute the environment by breaking down naturally to form smaller particles. These particles may attract and concentrate hydrophobic persistent organic pollutants on their surface, which might later be released once inside a biological matrix, as has been shown indirectly in e.g. birds [1]. All these particles are found in nature and humans are exposed to them daily (e.g. in beer [2]). People who regularly consume cultured bivalves may ingest up to 11.000 micro plastic particles per year by this route alone, even ignoring particles smaller than 5 µm [3]. Classically, nanoparticle uptake studies have been performed by acid digestion and chemical analysis [4]. But how do these particles interact with organisms and their tissue? Are they taken up in the tissue [5], or are they passively passing through the digestion system? Such questions are interesting in particular with regard to cytotoxic particles, such as silver, or very small particles that might enter the nucleus and interfere with DNA replication. Particles with the capability to carry toxic compounds, such as plastic, also have the potential for causing disruption of normal cellular function. In order to further our understanding of how such particles interact with tissue at the cellular level, nanoscale analysis is required, either to verify the nature of the particle (STEM EDX point analysis of single particles, Figure 1 a-c) or to trace substances released from their surface (NanoSIMS). However, before such analysis, a first challenge is to locate the particles in the affected cells. This has proven a major obstacle because of the relatively large size of the organism investigated. Indeed, screening a large volume of the organism under investigation to locate nanoparticles before EDX and NanoSIMS analysis is thus often a large part of the work. We work with model organisms such as microalgae, daphnia and earthworms that are likely to come into contact with, and accumulate nanoparticles. During the past few years we have employed various methods to localize different particles inside the organisms, e.g. serial block-face imaging, FIB-SEM imaging, freeze-dried cryo-sections and serial sections (Figure 1 d). Correlation between screening- and analysis techniques is key to locate, and be able to investigate the effects of nanoparticles in a biological matrix. At the moment we are exploring methods to screen large amounts of serial sections made by a custom-made knife, and methods for transferring interesting sections to carriers compatible with either STEM EDX or NanoSIMS. References [1] K. Tanaka et al., Marine Poll Bull 69 (1-2) (2013) 219. [2] G. Liebezeit and E. Liebezeit, Food Addit. Contam. Part a Chem. Anal. Control Exposure Risk Assess (2014). [3] L. van Cauwenberghe, and C. R. Janssen. Environ Pollut 193 (2014) 65. [4] L. M. Skjolding et al., Ecotoxicology 23 (2014) 1172. [5] P. Rosenkrantz et al., Environ Toxicol Chem 28 (2009) 2142. [6] We would like to thank Irina Kolotueva for invaluable technical advice. This research was supported by The Society of Electron Microscope Technology, and the European Research Council (Grant no. 281579). FIG. 1. (a-c) HAADF STEM images of D. magna gut epithelia exposed to 10 nm gold nanoparticles (Au NP) (0.4 mg Au/L) for 24h. Corresponding EDX spectra are superimposed. For clarity the C-peaks are capped and only up to 2.5 keV is depicted. (a) Au NP at microvilli, scale bar = 100 nm, (b) Au NP at base of microvilli, scale bar = 100 nm, (c) Os-rich particles in gut cell lipid droplet, scale bar = 50 nm, (d) Serial sections of L. variegatus exposed to iron particles on Si-wafer