to BioTechniques free email alert service to receive content updates.
100,000 Doughnuts Improve STED Imaging

07/08/2013
Megan Scudellari

A new technique for super-resolution imaging allows scientists to view living cells across a large field of view quickly and with low light. How could you use this method? Find out...


For more than a century physicists believed that the resolution of a light microscope was limited to only 200 to 500 nanometers—far too large of an area to visualize a single human cell in detail. Physicists called this limit the diffraction barrier, and many believed it to be carved in stone.

Human PtK2 cells expressing a fluorescent dye. Total image acquisition time was ~2 seconds. Scale bars, 10 μm. Image size = 133 x 100 µm. Source: Andriy Chmyrov, Stefan W. Hell, Max Planck Institute for Biophysical Chemistry, Göttingen




But in 1994, Stefan Hell and colleagues smashed the diffraction barrier to bits by developing a fluorescence imaging method called stimulated emission depletion (STED), which is capable of resolving details down to 35 nm (1). Over the following decade, Hell, now at the Max Planck Institute for Biophysical Chemistry in Germany, continued to pioneer the field of nanoscopy, showing that STED can produce 3-D, multi-color images and to visualize live cells.

Today, Hell has done it again. In a new Nature Methods paper, Hell describes a novel nanoscopy method that uses light at much lower intensities than STED and produces a much larger field of view at super-resolution (2). The new technique should make super-resolution imaging of live cells more accessible to scientists and laboratories.

“People thought in the past it was not possible to reconcile imaging large fields of view with high speed and low light levels,” said Hell. “This is a clear step toward showing that there is physics in this world that allows us to reconcile [those two things.]”

With the STED technique, Hell was able to image an area he fondly refers to as a doughnut because of its shape. After staining cells with fluorescent dye, he shone a light on the area, causing the cells to fluoresce. But when multiple cells fluoresce next to each other, it’s difficult to view them or their features. So Hell countered that original fluorescence with a second, donut-shaped pulse of light that quenched the excited dye molecules everywhere it touched—except in the center hole of the donut, where a single spot continued fluorescing. That spot had a resolution of an impressive 35 nm.

But STED requires strong pulses of light, which can damage cells and can only be used to image one very small area at a time, requiring scientists to do many long laborious scans under the microscope to view a large area. “In order to see larger fields of view in a very short period of time, we had to parallelize the imaging process—to get information from a large field of view at the same time,” said Hell.

To do that, Hell designed reversible saturable optical fluorescence transitions, or RESOLFT. With this new technique, it is possible to create doughnuts by simply crossing two unique patterns of low intensity light. And because the light intensity is so low, one is able to view more than 100,000 doughnuts at the same time, creating a large field of view with the same high resolution as STED.

Hell’s new paper describes how to set up the RESOLFT imaging platform, but to do so requires expertise in optics. For those without such expertise, Hell has a spinoff company working to commercialize the method for all laboratories.

References

1. Hell, S.W., et al. 1994. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780-782.

2. Chmyrov, A., et al. 2013. Nanoscopy with more than 100,000 ‘doughnuts’. Nat Methods, doi:10.1038/nmeth.2556.

Keywords:  microscopy