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Fluorescently labeled nanoparticles are widely used to investigate nanoparticle cell interactions by fluorescence microscopy. Owing to limited lateral and axial resolution, nanostructures (

For about a decade, superresolution fluorescence microscopy has been advancing steadily, maturing from the proof-of-principle stage to routine application. Of the various techniques, STED (stimulated emission depletion) microscopy was the first to break the diffraction barrier. Today, it is a prominent and versatile form of superresolution light microscopy. STED microscopy has shed a sharper light on numerous topics in cell biology, but also in material sciences. Both disciplines extend into the nanometer range, making detailed studies of structural and functional relationships difficult or even impossible to achieve using diffraction-limited microscopy. With recent advancements like spectral multiplexing or live-cell imaging, STED microscopy makes nanoscale materials and components of the cell accessible for fluorescence-based investigations. With multicolor superresolution imaging, even the interactions between biological and engineered nanostructures can be studied in detail. This review gives an introduction into the working principle of STED microscopy, provides a detailed overview of recent advancements and new techniques implemented for use with STED microscopy and shows how these have been applied in the life sciences and nanotechnologies.

The widespread use of engineered nanomaterials increases the exposure of the materials to humans. Therefore, it is necessary to know how these materials interact with cells. One approach is to trace particles by fluorescent labeling. The aim of the present work was to study the behavior of silica particles in A549 cells. For the first time, we applied stimulated emission depletion (STED) microscopy for this approach. Therefore, SiO2 particles conjugated with Atto647N were prepared by L-arginine-catalyzed hydrolysis of tetraethoxysilane. The Atto647N labeled SiO2 particles exhibit a mean size of 128 ± 7 nm and a zeta-potential of −11 mV in cell culture medium. STED microscopy enables subdiffraction resolution imaging of single Atto647N labeled SiO2 particles not only in pure solution but also in a cellular environment. To visualize Atto647N labeled SiO2 particles inside A549 cells, the membrane was labeled and image stacks, that give three-dimensional information, were taken after 5, 24, and 48 h exposure of particles to cells. During this incubation period, an increase in particle uptake was observed and STED micrographs allowed us to evaluate the agglomeration of Atto647N labeled SiO2 particles inside A549 cells. Our results show that STED microscopy is a powerful technique to study particles in a cellular environment.