NONLINEAR MICROSCOPY: STED microscopy continues to push the bounds of resolution

A group of researchers at the Max Planck Institute for Biophysical Chemistry (Gottingen, Germany) has beaten the diffraction limit by a factor of 40 in a world-record demonstration of the power of stimulated-emission-depletion (STED) microscopy.

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A group of researchers at the Max Planck Institute for Biophysical Chemistry (Gottingen, Germany) has beaten the diffraction limit by a factor of 40 in a world-record demonstration of the power of stimulated-emission-depletion (STED) microscopy.1

The group has studied individual color centers in a diamond crystal–in this case, nitrogen vacancies. Such color centers, besides being interesting physical systems in their own right, could have future applications ranging from biolabeling to quantum cryptography.

Since the 1990s, STED has cheated classical optics by allowing resolution beyond the diffraction limit. A number of research groups around the world have picked up on STED as a research tool, and there is even a commercially available microscope based on STED. But the group that invented the technique, headed up by MPI’s Stefan Hell, still does it best.

STED spectroscopy works by collectively exciting a fluorescent system with a pump beam, and then using a slightly redshifted “STED beam” to selectively drive down the excitation in an annulus around a particular point. The tiny area left excited then fluoresces and can be resolved with far greater precision than other types of spectroscopy permit, with the resolution now a variable controlled by the experimenter.

“It’s not limited by the wavelength anymore; it scales with the square root of the intensity,” says Eva Rittweger, lead author of the work. “If you crank up the intensity, then your resolution goes up.”

Pinpointing color centers

The team cranked up the intensity on a very particular system, however. “Color centers are very interesting fluorescent systems for doing STED because they show ideal behavior, almost textbook-like,” Rittweger says. “Also, they’re very photostable; you can have observation times up to hours, and that was something that was considered impossible before.”

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A STED microscope easily resolves individual nitrogen vacancies in a diamond crystal (center), while a conventional confocal microscope shows nothing of interest (left). The positions of the vacancies can then be found to a 0.15 nm precision (right). (Courtesy of the Max Planck Institute for Biophysical Chemistry)
Click here to enlarge image

The result is images of individual nitrogen vacancies with a resolution down to just 5.8 nm, with 1.5 Å precision. While that ability bodes well for future fundamental studies on such topics as crystal growth, it also has implications for a number of potential practical uses of color centers.

There is particular interest from the spintronics community because the spin state of the vacancies can be probed by looking at their fluorescence. Quantum-optics researchers want to make use of them as single-photon sources. Ongoing efforts to grow diamond nanoparticles could see them used in conjunction with STED microscopy as a biolabeling substrate to provide unprecedented resolution for microscopy of cells.

Looking further into the future, crystals that are densely packed with individually addressable color centers could provide dense, light-based data storage.

For now, there is a lot to be learned from the hundreds of different color-center types reported in diamond, and the group is working to refine the experimental setup even further. The ultimate resolution and sensitivity of the technique depend on a perfectly doughnut-shaped STED beam, so the researchers know there is a gain to be had by working on the “hole” of the doughnut.

“It’s very important that zero is really zero–and in reality it’s not,” Rittweger says. Simply using a shorter-wavelength STED beam would result in another gain.

Keeping at it, the researchers note, opens up the fascinating prospect of sensing directly the size of the electron cloud and its immediate environment–truly uncharted dimensions.

As Rittweger puts it, “All of this work is still at the beginning.”

–Jason Palmer

REFERENCE

  1. Rittweger et. al., Nature Photonics (2009), DOI: 10.1038/nphoton.2009.2

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