Figure 5

Multistage tissue sampling design for EFTEM analysis of nanoparticles, adapted from [21]. Stage 1 - Lung slices: exhaustive cutting of agar embedded lung lobes (with random start) into equally thick slices [38], followed by systematic sampling of slices, e.g. every second (with random start) and Epon embedding. Stage 2 - Tissue blocks and ultrathin sections: systematic sampling (with random start) of tissue blocks from lung slices using a point counting test system and cutting of ultrathin (≤ 50 nm) sections, which are placed on 600-mesh hexagonal copper grids and stained with lead citrate and uranyl acetate. Stage 3 - Quadrats on ultrathin sections: generation of a virtual field, completely contained within the ultrathin section, at 80× magnification. Field subdivision into a predetermined number of uniform quadrats and systematic subsampling of quadrats thereof (marked in grey). Stage 4 - Fields for nanoparticle analysis: Subsampling of a group of seven adjacent fields, delimited by the hexagonal TEM grid bars, within each quadrat at 6300× magnification, using a point counting test system. Tissue analysis within these hexagonal fields for (i) the presence of particles with matching nanoparticle characteristics and (ii) particle localization within the compartments of interest. Stages 3 and 4 can equally be applied on ultrathin sections of cell pellets. Alternative to stages 3 and 4 - Fields for nanoparticle analysis are sampled by picking a random hexagonal field on the ultrathin section as starting point at 80× magnification. From there on systematic tissue analysis in horizontal and vertical direction, using the automated goniometer of the microscope.