Darkfield and confocal laser scanning microscopy both allow for a simultaneous observation of live cells and single nanoparticles. Accordingly, a characterization of nanoparticle uptake and intracellular mobility appears possible within living cells. Single particle tracking allows to measure the size of a diffusing particle close to a cell. However, within the more complex system of a cell’s cytoplasm normal, confined or anomalous diffusion together with directed motion may occur. In this work we present a method to automatically classify and segment single trajectories into their respective motion types. Single trajectories were found to contain more than one motion type. We have trained a random forest with 9 different features. The average error over all motion types for synthetic trajectories was 7.2%. The software was successfully applied to trajectories of positive controls for normal- and constrained diffusion. Trajectories captured by nanoparticle tracking analysis served as positive control for normal diffusion. Nanoparticles inserted into a diblock copolymer membrane was used to generate constrained diffusion. Finally we segmented trajectories of diffusing (nano-)particles in V79 cells captured with both darkfield- and confocal laser scanning microscopy. The software called “TraJClassifier” is freely available as ImageJ/Fiji plugin via https://git.io/v6uz2.

Dark field microscopy is a widely unknown method to measure the particle size distribution of diffusing nanoparticles by particle tracking. Here we demonstrate that by using the surface plasmonic resonance of Au nanoparticles, size differences of ca. 20 nm can be identified within the particle size distribution. For that purpose, we developed a software tool which helps to analyze color videos of diffusing nanoparticles retrieved from CCD or CMOS cameras. Polystyrene beads with a diameter of 100 and 200 nm were used to compare the results to those obtained with a well-established laser-based particle tracking system. The methodology will be discussed in the light of recent developments in the emerging field of optical nanoparticle tracking.Electronic supplementary materialThe online version of this article (doi:10.1007/s11051-014-2419-x) contains supplementary material, which is available to authorized users.

An approach for the size measurement of particulate (nano)materials by transmission electron microscopy was evaluated. The approach combines standard operating procedures for specimen preparation, imaging, and image analysis, and it was evaluated on a series of certified reference materials and representative test materials with varying physical properties, including particle size, shape, and agglomeration state. The measurement of the median value of the minimal external particle diameter distribution was intra-laboratory validated. The validation study included an assessment of the limit of detection, working range, selectivity, precision, trueness, robustness, and ruggedness. An uncertainty that was associated to intermediate precision in the range of 1–7% and an expanded measurement uncertainty in the range of 7–20% were obtained, depending on the material and image analysis mode. No bias was observed when assessing the trueness of the approach on the certified reference materials ERM-FD100 and ERM-FD304. The image analysis method was validated in an inter-laboratory study by 19 laboratories, which resulted in a within-laboratory precision in the range of 2–8% and a between-laboratory precision of between 2% and 14%. The automation and standardization of the proposed approach significantly improves labour and cost efficiency for the accurate and precise size measurement of the particulate materials. The approach is shown to be implementable in many other electron microscopy laboratories.

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