Scientists at Howard Hughes Medical Institute's Janelia Farm Research Campus in Ashburn, Va., have developed interferometric photoactivated localization microscopy (iPALM), which allows them to create 3D images of structures inside cells at high resolution with an optical microscope, according to research published online before print Feb. 6 in the Proceedings of the National Academy of Sciences.
The technique adds a third dimension to a previous approach, called PALM, which uses fluorescent molecules that can be switched on and off to resolve the details of small structures under a light microscope. With PALM, only a fraction of the fluorescent molecules inside a cell are switched on at any given time, transforming a haze of light into a relatively sparse set of bright spots that can be resolved individually and that reveal the position of proteins tagged with fluorescent molecules. By stitching many images together, researchers create a complete 2D image, according to the researchers.
To add a third dimension to PALM, the researchers turned to interferometry--a widely used technique for measuring angles and distances on the microscopic scale. Light from fluorescent molecules in the sample is captured from above and below, and the two light beams are sent to a beam splitter that directs them to three different cameras. The amount of light that reaches each camera can be used to calculate the vertical position of each fluorescent molecule within the sample.
"In the end, we're able to get the position in all three directions of a molecule in less than 20 nanometers, or about 10 times the size of an average protein," said Harald Hess, lead study author.
John Sedat, professor of biochemistry and biophysics at the University of California, San Francisco, said that the paper is a "tour de force" in pushing the resolution of light microscopes. He adds that one of the trade-offs of using such high-spatial resolution for biological imaging is that it currently requires cells to be killed and chemically fixed, so it can't capture events in real time. The challenge for the field is to bring together advances in spatial resolution with real-time imaging of live cells, he said.