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ANALYTICAL CURRENTS STED microscopy illuminates vesicle fate (a)
(b)
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REINHARD JAHN
The question of what happens to synaptic vesicles after they fuse into the plasma membrane of neurons is difficult to answer. Synaptic vesicles, ~40-nm-diam balls that contain neurotransmitters, can’t be resolved by conventional fluorescence microscopy, which is restricted by a diffraction limit of ~200 nm. But Reinhard Jahn and colleagues at the Max Planck Institute for Biophysical Chemistry (Germany) have now overcome this barrier with stimulated emission depletion (STED) microscopy. The technique provides a novel way to probe the fate of synaptic vesicles. STED microscopy is a form of farfield fluorescence microscopy that uses regular lenses to overcome the diffraction limit. Two beams of light overlap at the focal region. The first beam excites molecules just as it would in conventional fluorescence microscopy. The second beam suppresses the molecules’ fluorescence except in a narrow, ~50nm-diam region in the middle of the beam. As the ~50-nm region is scanned across a sample, any fluorescent molecule present within that region is detected.
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A comparison between (a) confocal and (b) STED microscopic images highlights the greater resolution of the latter technique. Scale bar = 500 nm.
Jahn and colleagues used STED microscopy to determine whether proteins on the synaptic vesicle membrane remained clustered together after exocytosis or whether they spread out over the plasma membrane. The investigators targeted a monoclonal antibody against synaptotagmin, a protein in the vesicle membrane, and used a fluorescently labeled secondary antibody for visualization. They investigated both resting and stimulated neurons and demonstrated that the synaptotagmin proteins stayed
close together after the vesicle fused to the neuronal plasma membrane. The findings indicated that the recycling of vesicles for subsequent rounds of exocytosis was helped by the clustering of the proteins in the plasma membrane. Jahn and colleagues point out that the ~50-nm resolution achieved in their experiments doesn’t represent the limits of STED microscopy. The investigators say they’ve carried out single-molecule experiments with a focal region of 16 nm. (Nature 2006, 440, 935–939)
Imaging of the oxidant peroxynitrite in living cells In humans, production of peroxynitrite may
colleagues at the University of Hong Kong
vidual oxidative species. Neuronal cells
be involved in acute and chronic inflamma-
developed a fluorescent probe, HKGreen-1,
that were incubated with HKGreen-1 gen-
tion, ischemic stroke, diabetes, sepsis, ath-
which has a ketone unit linked to a dichlo-
erated a fluorescence signal when they
erosclerosis, and other diseases. Because
rofluorescein moiety. Upon reaction with
were treated with a peroxynitrite donor.
this highly reactive oxidant has a very short
peroxynitrite in vitro, the probe fluoresced
The response was specific: When the cells
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