Comparing video-rate STED nanoscopy and confocal microscopy of living neurons

We compare the performance of video-rate Stimulated Emission Depletion (STED) and confocal microscopy in imaging the interior of living neurons. A lateral resolution of 65 nm is observed in STED movies of 28 frames per second, which is 4-fold higher in spatial resolution than in their confocal counterparts. S FED microscopy, but not confocal microscopy, allows discrimination of single features at high spatial densities. Specific patterns of movement within the confined space of the axon are revealed in STED microscopy, while confocal imaging is limited to reporting gross motion. Further progress is to be expected, as we demonstrate that the use of continuous wave (CW) beams for excitation and STED is viable for video-rate STED recording of living neurons. Tentatively providing a larger photon flux, CW beams should facilitate extending fast STED imaging towards imaging fainter living samples. [GRAPHICS] Synaptic vesicles within an axon. Single frames of movies recorded at 28 frames per second. Confocal microscopy (left) can only reproduce the axon, whereas STED microscopy (right) can resolve moving objects in the neuron; '+' indicates that the data are linearly deconvolved.