Biosingularity

Super-Resolution Microscopy Takes on a Third Dimension

Posted on: February 11, 2009

The shapes of some of the tiniest cellular structures are coming into sharper focus at the Howard Hughes Medical Institute’s Janelia Farm Research Campus, where scientists have developed a new imaging technology that produces the best three-dimensional resolution ever seen with an optical microscope.

With this new tool, scientists can pinpoint fluorescent labels in their images to within 10-20 nanometers – about ten times the size of an average protein – in all three dimensions. The researchers say they now have an extremely powerful technology that will help reveal how biomolecules organize themselves into the structures and signaling complexes that drive cellular functions.

The three-dimensional distribution of membrane proteins within a cell revealed through iPALM imaging. The vertical position of fluorescently labeled farnesyl proteins has been color coded, with red molecules being the deepest and purple the highest.

The three-dimensional distribution of membrane proteins within a cell revealed through iPALM imaging. The vertical position of fluorescently labeled farnesyl proteins has been color coded, with red molecules being the deepest and purple the highest.

Their new method adds a third dimension to a cutting-edge 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. To push this form of microscopy to the next level — three-dimensional imaging — the researchers borrowed a strategy widely used in industry to measure vanishingly small distances, such as the subtle variations in height on the surface of a computer chip.

Janelia Farm scientist Harald Hess and his colleagues adapted that technique, known as interferometry, to make it compatible with the fluorescent molecules often used by biologists to visualize proteins. When interferometry is combined with the super-high resolution photoactivated localization microscopy (PALM), researchers can 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 Hess, who led the development of the new technology in the applied physics and instrumentation group at Janelia Farm.

Hess and collaborators at the National Institutes of Health, Florida State University, and Janelia Farm, who call their new tool interferometric photoactivated localization microscopy (iPALM), have already created detailed images of three-dimensional 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.

>>>> Rest of the article and Multimedia images in HHMI site

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