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  • Subdiffraction-Limit Microscopy Exposes Vesicle Dynamics

Photonics Spectra
Jul 2006
Daniel S. Burgess

Using a variant of fluorescence microscopy that enables subdiffraction-limit resolutions, scientists at Max Planck Institut für Biophysikalische Chemie in Göttingen, Germany, have imaged synaptic vesicle recycling in cultured neurons. The demonstration shows that at least some constituents of these structures — which ferry the neurotransmitters that enable neurons to communicate among themselves and to convey information to other cells and tissues in the body — remain intact on the cell membrane for later recycling, rather than being absorbed and broken down. It further exhibits the suitability of the microscopy technique for the investigation of biomolecular phenomena.

Silvio O. Rizzoli, a postdoctoral fellow at the institute, compared the vesicles in a synapse with small soap bubbles floating inside a larger one. To release their contents to the external world, the smaller bubbles must fuse with the surface membrane of the larger one.

An open question of interest to neurobiologists involves how a synaptic vesicle forms again after it has fused with a cell membrane. Rizzoli explained that there are two main hypotheses to explain this process. In one, the vesicle collapses and fuses with the membrane, and protein complexes in the neuron later extract, sort and reassemble the components. In another, known as “kiss and run,” the vesicle fuses only briefly with the cell membrane, expelling its contents through a pore that opens and closes again quickly enough that the vesicle does not collapse.

The problem for experimentalists seeking to investigate vesicle recycling using traditional fluorescence microscopy is that these structures are densely packed and are on the order of 40 nm in diameter. The hurdle is Abbe’s law, which dictates that the resolution of a light microscope can be no better than approximately one-half the wavelength of the light illuminating the sample.

A little more than a decade ago, however, Stefan W. Hell and his colleagues at the institute developed a corrective process for fluorescence microscopy called stimulated emission depletion. Although Abbe’s law still prevents them from focusing the excitation light to an actual spot smaller than about half the wavelength, they found that they can reduce its effective focal area with the addition of another beam of light that quenches the fluorescence response. By phase-modulating this de-excitation beam so that it forms a doughnut-shaped spot on the sample, they ensure that only the part of the sample in the “hole” — which theoretically can be arbitrarily smaller than the diffraction limit — will respond to the excitation.

Stimulated emission depletion reduces the effective focal area of the excitation spot in fluorescence microscopy to much less than the diffraction limit by selectively quenching the fluorescence response of the sample. The technique has been used to investigate synaptic vesicle recycling in neurons by imaging tagged synaptotagmin I, a protein on vesicle membranes.

In their work imaging synaptic vesicles, the researchers employed a homebuilt microscope with a Leica Microsystems GmbH 1.4-NA objective that incorporated a PicoQuant GmbH laser diode for 470-nm excitation and an optical parametric oscillator pumped with a Spectra-Physics Ti:sapphire laser for 615-nm de-excitation. A Hamamatsu Photonics KK spatial light modulator shaped the de-excitation beam into a doughnut with a 66-nm-diameter hole, enabling them to resolve pointlike objects separated by 45 nm. Antibodies tagged with the rhodamine dye Atto 532 were used to visualize synaptotagmin I, a protein on vesicle membranes, and the resulting fluorescence was collected using a PerkinElmer Inc. avalanche photodiode in photon-counting mode.

The results indicate that synaptotagmin I stays aggregated in clusters on the cell membrane after fusion, rather than mixing with it. Rizzoli said the team expects that studies of other vesicle proteins will reveal similar clustering.

He noted that the current work does not argue in favor of or against either of the hypotheses describing vesicle recycling, although the observation that the vesicle stays intact after full fusion has been viewed as a hallmark of kiss-and-run. He also cautioned that the findings at this point do not support particular explanations of neurochemical disorders over others.

The question remains, however, whether disruptions in the vesicle recycling process play a role in conditions involving neurotransmitter defects, and stimulated emission depletion potentially offers a means of exploring the issue. Rizzoli said that he and his colleagues will continue with their investigations of synaptic vesicle recycling, in both in vivo and in vitro conditions.

Hell’s group will continue to refine the microscopy technique, Rizzoli said, and it has achieved focal spot widths of 16 nm. He predicted that stimulated emission depletion will have a significant impact in the biological sciences, particularly if it can be used with multiple fluorophores to track the interaction of various proteins and organelles.

Nature, April 13, 2006, pp. 935-939.

fluorescence microscopy
Observation of samples using excitation produced fluorescence. A sample is placed within the excitation laser and the plane of observation is scanned. Emitted photons from the sample are filtered by a long pass dichroic optic and are detected and recorded for digital image reproduction. 
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