The subcellular visualisation of nanomaterials is crucial for a wide range of studies in nanomedicine and nanotoxicology. Although light microscopy usually requires less demanding sample preparation, compared to electron microscopy, it suffers from occlusion and resolution when observing nanoparticles. A main difference in the sample preparation is the reduction of cell’s thickness. Here we propose an improved light microscopy setting in which cells are spread on nanostructured patterns as to minimise their thickness, and at the same time minimise the overlap of nanoparticles themselves. Nanostructured substrates were prepared by depositing functionalised gold-RGD nanodots. We optimise the experimental conditions as to minimise cell’s thickness, which literally flattens the cell for further imaging procedures. The improved conditions are attained when cells reach their maximum spreading, and it is found when the dot-dot distance is 58nm. A threshold mechanism in cell adhesion is explained. When cells are maximally flat, confocal microscopy can easily detect the subcellular location of individual carbon nanotubes. This is a novel imaging concept with many potential applications in nanosciences, especially when a fast, reliable and inexpensive visualisation of nanoparticles is required.

Several studies have tackled the evaluation of the biocompatibility of nanomaterials in cell and tissue. Nonetheless to date, a quantitative technique for the assessment of the total intracellular nanocarrier dose administered has not been introduced. In this paper we develop two rapid and sensitive assays for the measurement of internalized nanomaterials in macrophages to be applied as vehicular carriers for drug delivery and contrast imaging applications. Five commercially available polystyrene particles with different diameters (from 20 to 1000 nm) were used and imaged by using a 3dimensional confocal imaging techniques. The two proposed assays are: “volumetric” assay which evaluate the spherical volume of internalized particles and 2) “max-flat” assay which evaluate the total differential area between cells and internalized particles. These two assays were then compared to a reference method. Among these three assays, the “max-flat” assay was found to be the most reliable and accurate to quantify and investigate the total content of internalized nanomaterials. The “max-flat” assay also allowed for a 3dimensional subcellular investigation of the adaptation and localization mechanisms between cytoskeleton and internalized materials, which may help to further increase the selectivity and delivery of active and passive biopolymers. Therefore, we believe that the here presented assays could become a useful tool to address many biomaterials questions, especially where the key issue is the quantitative assessment of dose effect issues related to the sizedependence response of nanomaterials.