A revolution in light microscopy is letting scientists zoom in on structures never before visualized with visible light. New “super resolution” microscopy techniques under development in several labs allow scientists to view structures that were once too small to be seen under a light microscope, due to the inherent resolution limit imposed by the wavelength of light.
Scientists at Howard Hughes Medical Institute’s Janelia Farm Research Campus recently announced the creation of a technique called interferometric photoactivated localization microscopy (iPALM), which allows them to create three-dimensional pictures of structures inside cells at the highest resolution yet seen with an optical microscope.
The technique, details of which were published recently in Proceedings of the National Academy of Sciences, 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 small 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 two-dimensional picture.
To add a third dimension to PALM, the researchers turned to interferometry, a technique that is widely used 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, says Harald Hess, the Janelia Farm scientist who led the study. Click here to see images of cell structures created using iPALM.
John Sedat, professor of biochemistry and biophysics at the University of California, San Francisco, says that the paper is a “tour de force” in pushing the resolution of light microscopes. But 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, Sedat says, is to bring together advances in spatial resolution with real-time imaging of live cells.
Gleb Shtengel, one of the leaders of the new technique, says that although the time required to combine multiple pictures makes it difficult to capture fast-paced events with iPALM, “we are planning to expand it to live cell images of slower-moving events.”
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