Best 3D resolution in optical microscopy

Ashburn, VA, USA--"The best three-dimensional resolution ever seen with an optical microscope" is possible thanks to scientists at the Howard Hughes Medical Institute's Janelia Farm Research Campus. They adapted interferometry to make it compatible with the fluorescent molecules used to visualize proteins, producing a technique nearly 100 times more efficient than other 3D approaches in terms of light harvesting. The system enables pinpointing to within 10-20 nm.

Ashburn, VA, USA--Scientists at the Howard Hughes Medical Institute's Janelia Farm Research Campus say they have devised a new biological imaging tool capable of producing the best 3D resolution ever seen with an optical microscope. Here, the three-dimensional distribution of membrane proteins within a cell is evident: the red molecules are the deepest (farthest away) and purple are highest (closest).

The new tool adds a third dimension to a form of light microscopy that scientists at Janelia Farm have used for the last two years to create two-dimensional images that pinpoint the location of fluorescently labeled proteins with extremely high resolution. That approach is called photoactivated localization microscopy (PALM), and to push it to the next level the researchers borrowed interferometry--a strategy widely used in industry to measure miniscule distances, such as the height of components on a computer chip.

Combining interferometry with super-high resolution PALM produced iPALM—which enables researchers to see the three-dimensional architecture of cellular structures in extraordinary detail.

"This will be a good tool to really untangle things right down to the molecular structure level," said Harald Hess, who led the development of the new technology in the applied physics and instrumentation group at Janelia Farm. "iPALM needs only a modest amount of light to generate its sensitive measurements, and that's important for biological imaging," he says.

Hess and collaborators at the National Institutes of Health, Florida State University, and Janelia Farm, have already created detailed images of 3D structures previously not resolvable with light microscopy. Their "photo gallery" includes images of the microtubules that give cells structure; the two layers of a cell's outer membrane; and the focal adhesions that attach cells to their environment. Some of these images are included in a research article published in the February 2, 2009, issue of the Proceedings of the National Academy of Sciences describing the new technique.

Hess and Janelia Farm colleague Eric Betzig invented the PALM microscope in 2005. Scarcely three years later, it was one of a handful of new methods of "super-resolution" microscopy that were honored by Nature Methods in January 2009 as the "Method of the Year" for the previous year.

Conventional optical microscopes are limited by the wavelength of light. To achieve better resolution, PALM uses fluorescent labels that can be turned on and off with a pulse of light. Cells whose proteins are tagged with these labels are imaged repeatedly, with only a tiny subset of the fluorescent molecules turned on in each image. By compiling many thousands of these images, PALM creates a complete picture of the structure under study, pinpointing each fluorescently tagged protein.

More information:
The paper, Multilayer three-dimensional super resolution imaging of thick biological samples, in the Proceedings of the National Academy of Sciences.
Howard Hughes Medical Institute's Janelia Farm
Further iPALM technology and project details

Posted by Barbara G. Goode, barbarag@pennwell.com.

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