We present video-rate (28 frames per second) far-field optical imaging with a focal spot size of 62 nanometers in living cells. Fluorescently labeled synaptic vesicles inside the axons of cultured neurons were recorded with stimulated emission depletion (STED) microscopy in a 2.5-micrometer by 1.8-micrometer field of view. By reducing the cross-sectional area of the focal spot by about a factor of 18 below the diffraction limit (260 nanometers), STED allowed us to map and describe the vesicle mobility within the highly confined space of synaptic boutons. Although restricted within boutons, the vesicle movement was substantially faster in nonbouton areas, consistent with the observation that a sizable vesicle pool continuously transits through the axons. Our study demonstrates the emerging ability of optical microscopy to investigate intracellular physiological processes on the nanoscale in real time.

In the axons of cultured hippocampal neurons, actin forms various structures, including bundles, patches (involved in the preservation of neuronal polarity), and a recently reported periodic ring-like structure. Nevertheless, the overlaying organization of actin in neurons and in the axon initial segment (AIS) is still unclear, due mainly to a lack of adequate imaging methods. By harnessing live-cell stimulated emission depletion (STED) nanoscopy and the fluorescent probe SiR-Actin, we show that the periodic subcortical actin structure is in fact present in both axons and dendrites. The periodic cytoskeleton organization is also found in the peripheral nervous system, specifically at the nodes of Ranvier. The actin patches in the AIS co-localize with pre-synaptic markers. Cytosolic actin organization strongly depends on the developmental stage and subcellular localization. Altogether, the results of this study reveal unique neuronal cytoskeletal features.

A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500–630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye–ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure–property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one‐ and two‐color images of living cells with an optical resolution of 40–60 nm.

The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.