In an example of an extreme movie close-up, researchers have used a subwavelength-resolution technique to track synaptic vesicle movement at video rates. Scientists from Max Planck Institute for Biophysical Chemistry and from European Neuroscience Institute, both in Göttingen, Germany, followed the movement of vesicles — small, hollow vessels that transport biochemicals — as they traveled in boutons, the knoblike enlargements at the ends of nerve cells that form synapses. Researchers used a technique called stimulated emission depletion microscopy to create a nanoscale-resolution movie of a live cell. On the left is a confocal microscopy image of dyed synaptic vesicles in an axon of a living cultured hippocampal neuron. On the right is the same image from a video-rate stimulated emission depletion movie. The still frame had an exposure time of 35 ms. Arrows indicate vesicle movement in the five subsequent frames. Courtesy of Stefan W. Hell, Max Planck Institute for Biophysical Chemistry.The first real-time imaging with nanoscale resolution of living cells revealed pockets, or hot spots, that constrained vesicle synaptic travel. It is a finding that Max Planck Institute director Stefan W. Hell noted was somewhat surprising. “The pronounced appearance of hot spots was not expected. It is a genuine observation that will stimulate further biological investigation about the biochemical nature and physiological role of these spots.”To get these results, the researchers had to develop an optical imaging technology for live cells with a resolution comparable to that produced by electron microscopy. The latter method, however, can be used only on dead cells, and optical microscopy classically is limited by diffraction to a resolution of half the illumination wavelength.The researchers overcame the resolution barrier through a twist on standard fluorescence imaging known as stimulated emission depletion. In this technique, excitation light is focused onto a sample while a second doughnut-shaped spot of light of lower photon energy is overlapped with the first.Thanks to stimulated emission, the second light quenches molecules excited by the first. Thus, the periphery is rendered dark, and only a much smaller spot in the sample is fluorescent. Scanning the spot through the specimen allows subdiffraction imaging.The size of the spot is a function of the intensity of the two beams. That allowed the researchers to use the technique for video imaging. They tuned the spot size down to the point where just enough photons were collected in each frame so that features of interest could be safely differentiated from the background.The investigators performed imaging using a lab-built confocal system, with the scanning done in one direction by moving the sample. For the other direction, they used a galvo scanner made by Electro-Optical Products Corp. of Glendale, N.Y., to sweep the beam back and forth at a rate of 16 kHz. They generated the excitation beam using a 635-nm laser from PicoQuant GmbH of Berlin, while the stimulated emission beam at 750 nm was produced by a Newport Corp. Ti:sapphire laser. The researchers employed a PerkinElmer photodetector to measure the signal.With this setup, they followed the movement of ~40-nm-diameter vesicles in 1-μm-diameter presynaptic nerve bundles, having first labeled the vesicles with the dye Atto647N. They tracked the vesicles over a field of view measuring 2.5 × 1.8 μm at 28 fps with a resolution of 62 nm. They found that the vesicles spent most of their time barely moving but would occasionally experience bursts where they would move at ∼2 nm/ms.The group plans to upgrade its subwavelength-imaging abilities to allow increased frame rates or smaller resolutions. Either would require the production of more photons, which the group plans to try by using a CW laser. Other improvements must come from the work of chemists and biologists.“In the future, there might be fluorophores that are brighter than the one we have used,” Hell said.Science, online edition, Feb. 21, 2008, doi: 10.1126/science,1154228.