The major obstacle to proving the kiss-and-run theory has been visually resolving synaptic vesicles. Light microscopes cannot distinguish objects that are closer than one half of the wavelength of light being used because diffraction blurs the elements into a single fuzzy image. So though the best confocal microscopes can discriminate objects as close as 200 nm, they are not capable of resolving single synaptic vesicles, which are only about 40-50 nm across. But now, microscopist Stefan Hell and neurobiologist Reinhard Jahn, both at the Max Planck Institute for Biophysical Chemistry in Gottingen, Germany, have teamed up to overcome that diffraction problem and track recycling vesicles in stimulated neurons.

Joint first authors Katrin Willig and Silvio Rizzoli and colleagues used a technique called stimulated emission depletion (STED) to break the diffraction barrier and improve resolution manyfold. Pioneered by Hell, STED uses a deceptively simple trick to improve the resolution of fluorescence microscopy. Superimposed on the regular excitation beam is a donut-shaped beam of light that de-excites fluorophores by driving electrons back toward the ground state from their excited states. In effect, STED quenches fluorescence and acts as an aperture to narrow fluorescence emission to a focal spot about 60 nm in diameter, effecting resolution to 45 nm.

A Microscope in Good Stead
STED microscopy improves resolution to about 45 nm. Compared to confocal microscopy (left), STED (right) can resolve numerous spots of synaptotagmin, a major synaptic vesicle protein, within synaptic boutons in primary hippocampal neurons (see insets). [Image courtesy of Reinhard Jahn and Stefan Hell, Max Planck Institute for Biophysical Chemistry, Gottingen, Germany.]

The authors used STED to study the behavior of synaptotagmin, a major synaptic vesicle protein, in hippocampal primary neurons. They used the microscope to visualize both internal and cell surface protein. They labeled the latter by adding fluorescent synaptotagmin antibodies to cultures on ice. Internal protein was labeled once the cultures were warmed to 37 degrees centigrade in the presence of calcium—this stimulated endocytosis of synaptotagmin-bound antibodies.

The authors found that both pools of protein appeared as discrete dots rather than diffuse patches, suggesting that even when fused to the cell membrane, synaptotagmin remains in small clusters. This in itself lends support to the kiss-and-run theory, but to capture it in action, the researchers examined how synaptotagmin behaves when synaptic vesicle recycling is ramped up. When they stimulated the neurons with potassium chloride, total internal and cell surface staining was dramatically increased. However, the pattern remained unchanged—synaptotagmin on the cell surface still appeared as discrete dots, demonstrating that at least this one vesicle component does not diffuse through the cell membrane once synaptic vesicles dock there.

“Their study provides some of the most compelling evidence to date that at least some membrane constituents remain grouped together after vesicles fuse with the plasma membrane (rather than diffusing freely within the membrane like a drop of water on water), which is consistent with the kiss-and-run theory,” writes Garth Simpson, Purdue University, West Lafayette, Indiana, in an accompanying Nature News & Views. Should the same be seen for other vesicle components, then support for the kiss-and-run theory should receive a huge boost.

In the meantime, it is worth noting that there is no theoretical limit to the resolution attainable by STED microscopy. By narrowing the aperture of the STED beam, the resolution can be improved further, potentially allowing measurements of single molecules. In fact, Hell’s group has recently achieved a focal spot of only 16 nm across (see Westphal and Hell, 2005).—Tom Fagan.

References:
Willig KI, Rizzoli SO, Westphal V, Jahn R, Hell SW. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature. April 13, 2006;440:935-939. Abstract

Simpson GJ. The diffraction barrier broken. Nature. April 13, 2006;440:879-880. Abstract