A laser scanning microscope collects information from a thin focal plane, disregarding out-of-focus information. It has become the standard imaging method to characterise cellular morphology and structures, both in static and living samples. Laser scanning microscopy at high resolution combined with digital image restoration is also a powerful tool to analyse intracellular localisation of fluorescently labelled nanoparticles (NPs), such as their colocalisation within membrane-bound compartments (e.g. endosomes or lysosomes), cellular organelles (e.g. mitochondria or nucleus), or, alternatively, as free NPs in the cytosol, thus providing essential information about cellular uptake mechanisms and trafficking of NPs.

Laser scanning microscopy (LSM), also known as confocal LSM, is a valuable tool for obtaining high spatial resolution and three-dimensional (3D) reconstruction of specific fluorescently labelled structures at the light microscopic level [1]. During the past few years it has emerged as the standard imaging method to characterise cellular morphology and structures in static and living samples. LSM combined with digital image restoration provides a powerful instrument to analyse cellular architecture, expression of specific proteins, and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models [2].
The immense potential of nano-sized particles (NPs; 1-100nm) for diagnostic and therapeutic applications stands in sharp contrast to a growing number of critical reports regarding their potential toxicity [3]. In order to understand how NPs interact with cellular systems, potentially causing adverse effects, their detection and localisation within cells is of central importance. Once intracellular NPs are identified, their distribution in different cellular compartments, such as endosomes, lysosomes, mitochondria, the nucleus or cytosol, may also provide some indications as to any potential toxicity.

Due to the small size of NPs, their intracellular identification is demanding and time consuming. For the detection of both fluorescent and/or electron dense NPs, light and transmission electron microscopy methods can be applied [4].

Transmission electron microscopy with a resolution range from Ångström to nm is the method of choice for detection of electron dense NPs. But, in addition to complex and long sample section preparation, stereological approaches are required to understand the 3D distribution of NPs within a defined reference volume [5].

During the last years many new NPs labelled with fluorophores or with fluorescence specificity have been introduced, such as semiconductor nanocrystals (i.e. quantum dots) that are robust and bright light emitters [6]. Another very promising technique is the design of the so-called core-shell NPs with fluorophores embedded in their shell [7]. Such fluorescently labelled NPs can be visualised by LSM combined with digital image restoration to analyse their intracellular localisation. However, it is important to understand that in LSM the resolution is limited to 200 nm and 500-900 nm in the lateral and axial dimensions, respectively, though de-convolution algorithms may increase the resolution 2-3 fold [8]. Nevertheless, considering the possibility to visualise the uptake of fluorescently labelled NPs into living cells or to perform colocalisation studies with fluorescently labelled organelles in 3D objects (fixed or living cells) [9], LSM provides an excellent tool to gain new insights into NP-cell interactions and NP uptake mechanisms.

SPIONS Preparation and Visualisation Method

We have used super-paramagnetic iron oxide NPs (SPIONS) coated with poly(vinylalcohol) and vinyl alcohol/vinyl amine co-polymer. Essentially, the particles consist of an iron oxide core coated with a hydrophilic polymer shell to which the fluorescence dye Oregon green has been covalently coupled (fig. 1A Inset) [7]. Since one of our research focuses is to understand how SPIONS affect the human immune system, we have investigated particle interactions with dendritic cells, known to be the one of the key immune competent cells in the human body. Human monocyte-derived dendritic cells (MDDC) were cultured as described [10]. It is possible to visualise SPIONS by cryo-transmission electron microscopy (fig. 1A), however, since SPIONS do not have such an electron dense core as, for instance, gold NPs [5], determining their localisation inside cells without any time consuming and technically difficult methods, such as electron energy loss spectroscopy to identify a certain element [4], is difficult to assess. Therefore, LSM was performed on MDDC to characterise uptake, intracellular localisation and the association of SPIONS with the endosomal compartment. For this approach MDDC were treated with SPIONS at different concentrations (10 µg/mL and 20 µg/mL) during 4 hours and subsequently incubated with Transferrin Alexa 633 (100µg/mL; endosomes) for 20 minutes. Thereafter, cells were fixed and the cytoskeleton stained with Phalloidin-Rhodamine and the cell nuclei with DAPI as described [10].