The whitening and opacifying agent titanium dioxide (TiO2) is used worldwide in various foodstuffs, toothpastes and pharmaceutical tablets. Its use as a food additive (E171 in EU) has raised concerns for human health. Although the buccal mucosa is the first area exposed, oral transmucosal passage of TiO2 particles has not been documented. Here we analyzed E171 particle translocation in vivo through the pig buccal mucosa and in vitro on human buccal TR146 cells, and the effects on proliferating and differentiated TR146 cells. In the buccal floor of pigs, isolated TiO2 particles and small aggregates were observed 30 min after sublingual deposition, and were recovered in the submandibular lymph nodes at 4 h. In TR146 cells, kinetic analyses showed high absorption capacities of TiO2 particles. The cytotoxicity, genotoxicity and oxidative stress were investigated in TR146 cells exposed to E171 in comparison with two TiO2 size standards of 115 and 21 nm in diameter. All TiO2 samples were reported cytotoxic in proliferating cells but not following differentiation. Genotoxicity and slight oxidative stress were reported for the E171 and 115 nm TiO2 particles. These data highlight the buccal mucosa as an absorption route for the systemic passage of food-grade TiO2 particles. The greater toxicity on proliferating cells suggest potential impairement of oral epithelium renewal. In conclusion, this study emphasizes that buccal exposure should be considered during toxicokinetic studies and for risk assessment of TiO2 in human when used as food additive, including in toothpastes and pharmaceutical formulations.

Background Edible gold (Au) is commonly used as a food additive (E175 in EU) for confectionery and cake decorations, coatings and in beverages. Food-grade gold is most often composed of thin Au sheets or flakes exhibiting micro- and nanometric dimensions in their thickness. Concerns about the impact of mineral particles used as food additives on human health are increasing with respect to the particular physico-chemical properties of nanosized particles, which enable them to cross biological barriers and interact with various body cell compartments. In this study, male and female mice were exposed daily to E175 or an Au nanomaterial (Ref-Au) incorporated into food at relevant human dose for 90 days in order to determine the potential toxicity of edible gold. Results E175 or Ref-Au exposure in mice did not induce any histomorphological damage of the liver, spleen or intestine, nor any genotoxic effects in the colon and liver despite an apparent higher intestinal absorption level of Au particles in mice exposed to Ref-Au compared to the E175 food additive. No changes in the intestinal microbiota were reported after treatment with Ref-Au, regardless of sex. In contrast, after E175 exposure, an increase in the Firmicutes/Bacteroidetes ratio and in the abundance of Proteobacteria were observed in females, while a decrease in the production of short-chain fatty acids occurred in both sexes. Moreover, increased production of IL-6, TNFα and IL-1β was observed in the colon of female mice at the end of the 90-day exposure to E175, whereas, decreased IL-6, IL-1β, IL-17 and TGFβ levels were found in the male colon. Conclusions These results revealed that a 90-day exposure to E175 added to the diet alters the gut microbiota and intestinal immune response in a sex-dependent manner in mice. Within the dose range of human exposure to E175, these alterations remained low in both sexes and mostly appeared to be nontoxic. However, at the higher dose, the observed gut dysbiosis and the intestinal low-grade inflammation in female mice could favour the occurrence of metabolic disorders supporting the establishment of toxic reference values for the safe use of gold as food additive.

A dedicated setup was developed for simultaneous measurement of pressure and volume in a single eye of semi-hard cheese. A known level of gas pressure was applied to the cheese eye and the resulting eye inflation was monitored using Magnetic Resonance Imaging (MRI). Image analysis methods were developed to measure the eye volume, horizontal and vertical diameters of the eye and the deflected shape of the top surface of the cylinder of cheese under study. Two amounts of pressure were applied to attempt to reproduce a creep-recovery experiment in situ. In the last stage, lowering of pressure was applied in order to investigate time-independent elasticity. The core of the semi-hard cheese was found to show no relevant time-independent elasticity during processing in a 90 h experiment. A low amount of pressure (< 3.5 kPa) was able to inflate already existing eyes in semi-hard cheese within the linear domain.

We used triple-quadrupole and high-resolution inductively coupled plasma-mass spectrometry (ICP-MS) in single-particle mode to characterize the food additive E171 (titanium dioxide, TiO2) in chewing gum, chocolate candy, and cake decoration in the same sample extracts. Then, we spiked TiO2 particles (with similar characteristics as E171) to milk as an example of a calcium-rich matrix. The obtained particle size distributions with both techniques were highly similar in terms of shape and median and mean diameters. Median diameters were in the range of 123 to 209 nm and mean diameters from 146 to 223 nm. In addition, they were in agreement with results obtained by scanning electron microscopy and asymmetric flow field-flow fractionation coupled to multi-angle light scattering and ICP-MS. Repeatable determination of number-based particle size distributions was possible with both ICP-MS techniques even in a calcium-rich matrix showing that both instruments were similarly efficient in resolving the Ca interferences. The combination of spICP-MS with microscopy and TiO2 recovery allowed validating the methods and identifying the presence of aggregated/agglomerated particles in one sample. For the TiO2 powder and the two remaining food products, recoveries were higher than 60%. Both instruments are fit for purpose even if the analyses were performed with differences in detector mode, dwell times, and calculation tools. This shows that both techniques may be used as long as operating conditions are optimized and applicability range is defined.

The morphology of amorphous silica NPs in three dimensions (3D) of space is analysed using a single technique: the transmission electron microscope in Scanning Transmission Electron Microscopy with High Angle Annular Dark Field (HAADF-STEM) imaging mode. For this purpose, a method consisting in adjusting the HAADF-STEM signal by a function describing the thickness of material in a sphere has been developed to determine the height of the nano-object and tested on particles having quasi-spherical shape. This approach is suitable only in the case of a nanomaterial chemically homogeneous. A reference present in the image is required for calibrating the signal strength. This reference can be either a particle of known shape or a particle whose height was previously measured with another technique (i.e. Atomic Force Microscopy). Thus, the study of the small silica particles by HAADF-STEM highlighted their spheroidal shape but also their preferential orientation on the substrate.

Scanning Electron Microscopy (SEM) technique is widely used to characterize nanoparticle (NP) size. The landing energy (LE) of the primary electron beam is considered to be a key parameter related to the ability of electrons to penetrate the sample. However, few studies have been carried out so far on the influence of this parameter on the measurement of NP size by SEM. The increasing needs for reference materials consisting of size-controlled NP suspension for microscope calibration induce new issues. This paper focuses on the effect of electron landing energy on the measurement of the equivalent diameter of several NP populations by SEM. To evaluate the influence of LE, particles of different sizes and chemical compositions were analyzed. The results showed the variation of the measured diameter as a function of LE. SEM secondary electron (SE) yield modeling by the Monte Carlo method allowed us to relate this variation to the information volume in the material. Finally, the use of reference particles and transmission electron microscopy (TEM) allowed us to determine an optimal value of LE to be applied, depending on the chemical composition and particle size to limit the bias in the SEM measurement. We showed that this operating point can be simply determined without reference nanomaterials by scanning an LE range.

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Commercial products containing nanomaterials are already a part of our everyday life. The dimensional parameters play an important role for identifying nanoparticles. Various techniques are available for measuring the particles size and shape. However, electron microscopy-based techniques are often considered as the preferred methods for characterizing their dimensional properties. Samples preparations remain a key step but no standardized protocol is currently available. Scanning electron microscopy measurements require well-dispersed particle population corresponding to a statistically representative sample. This work presents a new approach, to prepare samples based on spincoating technique in order to measure in a reliable manner the constituent particle size. This method consists of three key processing steps: (i) extraction from complex media, (ii) redispersion by means of probe sonication and (iii) deposition by spincoating. The approach combines several factors such as pH, zeta potential, duration, concentration, sonication and spin coater parameters, that can influence agglomeration state of nanoparticles.

Particle size distribution (PSD) is an important factor determining the efficiency of industrial manufacturing processes for nanomaterials, assuring the reproducibility and safety of the final product. Among the instruments that have been developed to determine size and PSD of nanoparticle dispersions, the easiest to handle are based on indirect measurements; therefore, it is recommended to use at least two approaches to evaluate the PSD. This work evaluates the possibility of using direct size measurement methods based on the analysis of images of multimodal dispersion of nanomaterials by electron microscopy. Samples for measurement of the PSD were prepared by different deposition methods from a multimodal dispersion of poly(isobutylcyanoacrylate) nanoparticles. Grids prepared by flotation showed particle agglomeration and segregation between large and small particles and was found unsuitable for obtaining relevant measurement of the PSD. In contrast, spin-coating produced a homogenous and random deposition of well isolated particles on the substrate used to prepare the samples for electron microscopy. This deposition method was suitable for evaluating the PSD of this highly heterogenous dispersion. Deposition strategies are therefore essential to provide a statistically representative sample for PSD measurement of nanomaterial-based products using a direct measurement method.

Background Titanium dioxide (TiO 2 ) is broadly used in common consumer goods, including as a food additive (E171 in Europe) for colouring and opacifying properties. The E171 additive contains TiO 2 nanoparticles (NPs), part of them being absorbed in the intestine and accumulated in several systemic organs. Exposure to TiO 2 -NPs in rodents during pregnancy resulted in alteration of placental functions and a materno-foetal transfer of NPs, both with toxic effects on the foetus. However, no human data are available for pregnant women exposed to food-grade TiO 2 -NPs and their potential transfer to the foetus. In this study, human placentae collected at term from normal pregnancies and meconium (the first stool of newborns) from unpaired mothers/children were analysed using inductively coupled plasma mass spectrometry (ICP-MS) and scanning transmission electron microscopy (STEM) coupled to energy-dispersive X-ray (EDX) spectroscopy for their titanium (Ti) contents and for analysis of TiO 2 particle deposition, respectively. Using an ex vivo placenta perfusion model, we also assessed the transplacental passage of food-grade TiO 2 particles. Results By ICP-MS analysis, we evidenced the presence of Ti in all placentae (basal level ranging from 0.01 to 0.48 mg/kg of tissue) and in 50% of the meconium samples (0.02–1.50 mg/kg), suggesting a materno-foetal passage of Ti. STEM-EDX observation of the placental tissues confirmed the presence of TiO 2 -NPs in addition to iron (Fe), tin (Sn), aluminium (Al) and silicon (Si) as mixed or isolated particle deposits. TiO 2 particles, as well as Si, Al, Fe and zinc (Zn) particles were also recovered in the meconium. In placenta perfusion experiments, confocal imaging and SEM-EDX analysis of foetal exudate confirmed a low transfer of food-grade TiO 2 particles to the foetal side, which was barely quantifiable by ICP-MS. Diameter measurements showed that 70 to 100% of the TiO 2 particles recovered in the foetal exudate were nanosized. Conclusions Altogether, these results show a materno-foetal transfer of TiO 2 particles during pregnancy, with food-grade TiO 2 as a potential source for foetal exposure to NPs. These data emphasize the need for risk assessment of chronic exposure to TiO 2 -NPs during pregnancy.

Recent advances in deep generative models based on convolutional neural networks (CNNs) are used to demonstrate the potential of these approaches for the estimation of particle size distribution on images of aggregated TiO2 particles obtained by Scanning Electron Microscopy (SEM). This very promising framework shall permit effective automation of SEM measurements analysis. Indeed, common image processing softwares bring the end-users with segmentation algorithms as well as measuring tools to estimate individual particle diameters. In the case of aggregated nanoparticles, most particles suffer missing contents and are not considered in the computations. In this paper, we use a recently developed method called 'context encoder's to predict missing parts of the nanoparticles. The approach is tested against simulated and real dropped image regions.

Scanning Electron Microscopy (SEM) is considered as a reference technique for the determination of nanoparticle (NP) dimensional properties. Nevertheless, the image analysis is a critical step of SEM measuring process and the initial segmentation phase consisting in determining the contour of each nano-object to be measured must be correctly carried out in order to identify all pixels belonging to it. Several techniques can be applied to extract NP from SEM images and evaluate their diameter like thresholding or watershed. However, due to the lack of reference nanomaterials, few papers deals with the uncertainty associated with these segmentation methods. This article proposes a novel approach to extract the NP boundaries from SEM images using a remarkable point. The method is based on the observation that, by varying the electron beam size, the secondary electron profiles crosses each other at this point. First, a theoretical study has been performed using Monte Carlo simulation on silica NP to evaluate the robustness of the method compared with more conventional segmentation techniques (Active Contour or binarization at Full Width at Half-Maximum, FWHM). The simulation results show especially a systematic discrepancy between the NP real size and the measurements performed with both conventional methods. Moreover, generated errors are NP size-dependent. By contrast, it has been demonstrated that a very good agreement between measured and simulated diameters has been obtained with this new technique. As an example, this method of the remarkable point has been applied on SEM images of silica particles. The quality of the segmentation has been shown on silica reference nanoparticles by measuring the modal equivalent projected area diameter and comparing with calibration certificate. The results show that the NP contour can be very accurately delimited with using this point. The measurement uncertainty has been also reduced from 4.3 nm (k = 2) with conventional methods to 2.6 nm (k = 2) using the remarkable point.

At this time, there is no instrument capable of measuring a nano-object along the three spatial dimensions with a controlled uncertainty. The combination of several instruments is thus necessary to metrologically characterize the dimensional properties of a nano-object. This paper proposes a new approach of hybrid metrology taking advantage of the complementary nature of atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques for measuring the main characteristic parameters of nanoparticle (NP) dimensions in 3D. The NP area equivalent, the minimal and the maximal Feret diameters are determined by SEM and the NP height is measured by AFM. In this context, a kind of new NP repositioning system consisting of a lithographed silicon substrate has been specifically developed. This device makes it possible to combine AFM and SEM size measurements performed exactly on the same set of NPs. In order to establish the proof-of-concept of this approach and assess the performance of both instruments, measurements were carried out on several samples of spherical silica NP populations ranging from 5 to 110 nm. The spherical nature of silica NPs imposes naturally the equality between their height and their lateral diameters. However, discrepancies between AFM and SEM measurements have been observed, showing significant deviation from sphericity as a function of the nanoparticle size.