Strategy for Ensuring the Metrological Traceability of Nanoparticle Size Measurements by SEM.

The concept of measurement traceability is crucial for ensuring the data reliability and the comparability of measurement results provided by different instruments and operators. In the field of nanoparticle metrology, determining the size of nanoparticles using electron microscopy-based techniques remains a real challenge. In laboratory settings, the establishment of traceability regarding the instrument calibration procedures, the assessment of uncertainties associated with instruments/operators/samples/environments, as well as the complexities related to electron-sample interactions, are often neglected. In this article, we describe the calibration procedure set up at the LNE (Laboratoire National de métrologie et d'Essais) and propose an evaluation method for determining the uncertainties in measuring nanoparticle size by SEM (Scanning Electron Microscopy). This study investigates the impact of the energy of the primary electrons (PEs) generated by the electron beam and accelerating voltage on the reliability of size measurements. The convolution between the signals coming from a nanoparticle and the substrate on which the particle is deposited induces edge effects that can have a negative impact on the measurement results. Finally, a diagram describing the various stages involved in establishing traceability for SEM measurements of nanoparticle size is proposed to facilitate the work of future operators.

Metrological Protocols for Reaching Reliable and SI-Traceable Size Results for Multi-Modal and Complexly Shaped Reference Nanoparticles.

The study described in this paper was conducted in the framework of the European nPSize project (EMPIR program) with the main objective of proposing new reference certified nanomaterials for the market in order to improve the reliability and traceability of nanoparticle size measurements. For this purpose, bimodal populations as well as complexly shaped nanoparticles (bipyramids, cubes, and rods) were synthesized. An inter-laboratory comparison was organized for comparing the size measurements of the selected nanoparticle samples performed with electron microscopy (TEM, SEM, and TSEM), scanning probe microscopy (AFM), or small-angle X-ray scattering (SAXS). The results demonstrate good consistency of the measured size by the different techniques in cases where special care was taken for sample preparation, instrument calibration, and the clear definition of the measurand. For each characterization method, the calibration process is described and a semi-quantitative table grouping the main error sources is proposed for estimating the uncertainties associated with the measurements. Regarding microscopy-based techniques applied to complexly shaped nanoparticles, data dispersion can be observed when the size measurements are affected by the orientation of the nanoparticles on the substrate. For the most complex materials, hybrid approaches combining several complementary techniques were tested, with the outcome being that the reliability of the size results was improved.

Correlative Analysis of the Dimensional Properties of Bipyramidal Titania Nanoparticles by Complementing Electron Microscopy with Other Methods.

In this paper, the accurate determination of the size and size distribution of bipyramidal anatase nanoparticles (NPs) after deposition as single particles on a silicon substrate by correlative Scanning Electron Microscopy (SEM) with Atomic Force Microscopy (AFM) analysis is described as a new measurement procedure for metrological purposes. The knowledge of the exact orientation of the NPs is a crucial step in extracting the real 3D dimensions of the particles. Two approaches are proposed to determine the geometrical orientation of individual nano-bipyramides: (i) AFM profiling along the long bipyramid axis and (ii) stage tilting followed by SEM imaging. Furthermore, a recently developed method, Transmission Kikuchi Diffraction (TKD), which needs preparation of the crystalline NPs on electron-transparent substrates such as TEM grids, has been tested with respect to its capability of identifying the geometrical orientation of the individual NPs. With the NPs prepared homogeneously on a TEM grid, the transmission mode in a SEM, i.e., STEM-in-SEM (or T-SEM), can be also applied to extract accurate projection dimensions of the nanoparticles from the same sample area as that analysed by SEM, TKD and possibly AFM. Finally, Small Angle X-ray Scattering (SAXS) can be used as an ensemble technique able to measure the NPs in liquid suspension and, with ab-initio knowledge of the NP shape from the descriptive imaging techniques, to provide traceable NP size distribution and particle concentration.

Influence of electron landing energy on the measurement of the dimensional properties of nanoparticle populations imaged by SEM.

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.

Deep Learning Based Instance Segmentation of Titanium Dioxide Particles in the Form of Agglomerates in Scanning Electron Microscopy.

The size characterization of particles present in the form of agglomerates in images measured by scanning electron microscopy (SEM) requires a powerful image segmentation tool in order to properly define the boundaries of each particle. In this work, we propose to use an algorithm from the deep statistical learning community, the Mask-RCNN, coupled with transfer learning to overcome the problem of generalization of the commonly used image processing methods such as watershed or active contour. Indeed, the adjustment of the parameters of these algorithms is almost systematically necessary and slows down the automation of the processing chain. The Mask-RCNN is adapted here to the case study and we present results obtained on titanium dioxide samples (non-spherical particles) with a level of performance evaluated by different metrics such as the DICE coefficient, which reaches an average value of 0.95 on the test images.

A new method for measuring nanoparticle diameter from a set of SEM images using a remarkable point.

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.

Development of a new hybrid approach combining AFM and SEM for the nanoparticle dimensional metrology.

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.