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Published Online February 21, 2008
Science DOI: 10.1126/science.1154228

Reports

Submitted on December 17, 2007
Accepted on February 8, 2008

Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement

Volker Westphal 1{dagger}, Silvio O. Rizzoli 2{dagger}, Marcel A. Lauterbach 1, Dirk Kamin 3, Reinhard Jahn 4, Stefan W. Hell 1*

1 Department of NanoBiophotonics, Max-Planck-Institute for Biophysical Chemistry, Göttingen 37077, Germany.
2 Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen 37077, Germany.; STED Microscopy of Synaptic Function, European Neuroscience Institute, Göttingen, 37077, Germany.
3 STED Microscopy of Synaptic Function, European Neuroscience Institute, Göttingen, 37077, Germany.
4 Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen 37077, Germany.

* To whom correspondence should be addressed.
Stefan W. Hell , E-mail: shell{at}gwdg.de

{dagger}These authors contributed equally to this work.

We present video-rate (28 frames/s) far-field optical imaging with a focal spot size of 62 nm in living cells. Fluorescently labeled synaptic vesicles inside the axons of cultured neurons were recorded with STED (stimulated emission depletion) microscopy in a 2.5 x 1.8 µm² field of view. By reducing the cross-section area of the focal spot about 18-fold below the diffraction limit (260 nm), STED allowed us to map and describe the vesicle mobility within the highly confined space of synaptic boutons. While restricted within boutons, the vesicle movement was substantially faster in non-bouton areas, in line with the observation that a sizeable 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.




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