If you’re not watching recent work in biology, you might have thought that light microscopy hit its limits years ago. After all, it’s been around a long time. But to the contrary, microscopic imaging technology just keeps getting better and better. Here you can look with unprecedented clarity at just one of the many dynamic processes going on within a living cell. Specifically, this video shows actin fibers (orange-red), which are key components of the cell’s cytoskeleton, slowly pulling clathrin-coated pits (green), which are basket-like structures containing molecular cargo, away from the cell’s external membrane and deeper within the cell.
This remarkable live-action view was produced using one of two new forms of extended-resolution, structured illumination microscopy (SIM). SIM is faster than other forms of super-resolution fluorescence microscopy. It’s also less damaging to cells, making it the go-to method for live-cell imaging. The downside has been SIM’s limited resolution—just twice that of conventional light microscopes. However, Nobel Prize-winner Eric Betzig and postdoc Dong Li of Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, along with colleagues including Jordan Beach and John Hammer at NIH’s National Heart, Lung, and Blood Institute, recently came up with two different solutions to enhance SIM’s spatial resolution.
Betzig was one of three scientists awarded the 2014 Nobel Prize in Chemistry for the development of another innovative microscopic imaging technology, known as super-resolved fluorescence microscopy. That technology is credited with producing the first super-resolution images of intact cells. (As an interesting aside, Betzig’s microscope was first developed at NIH’s Eunice Kennedy Shriver National Institute of Child Health and Human Development, at a time when Betzig had been working out of a cottage in rural Michigan!)
Betzig and his colleagues recognized, however, that their Nobel Prize-winning approach had certain limitations when it came to imaging events as they unfold in real-time inside living cells. The high level of light required by super-resolved fluorescence microscopy damages the cells and causes the markers that scientists use to track molecules to fade quickly. To get around this obstacle, the researchers turned their energies toward optimizing the potential of SIM technology, which despite its lower resolution does not inflict as much damage on the samples being studied.
Those efforts proved successful, resulting in two new SIM imaging technologies described in a recent issue of the journal Science . In one approach, the researchers used a commercially available microscope lens with an ultrahigh numerical aperture. This improved the resolution in SIM from 100 nanometers to 84 nanometers. In the second, they used a clever trick, applying striped patterns of light at two wavelengths to activate different subsets of fluorescently labeled proteins in a sample. Those two patterns offer more information and a greater level of detail without undue damage to the cells. It also improved the resolution of SIM to 45 nanometers, revealing structural details at the level of large macromolecular complexes.
The researchers say they are now eager to work with biologists to continue to explore potential applications of the new SIM techniques for understanding cell processes with important implications for health research. It will be interesting to see what they and others will uncover next using these innovative technologies.
 Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Li D, Shao L, Chen BC, Zhang X, Zhang M, Moses B, Milkie DE, Beach JR, Hammer JA 3rd, Pasham M, Kirchhausen T, Baird MA, Davidson MW, Xu P, Betzig E. Science. 2015 Aug 28;349(6251).
Betzig Lab (Janelia Research Campus/Howard Hughes Medical Institute, Ashburn VA)
John Hammer (National Heart, Lung, and Blood Institute/NIH)
Size of the Nanoscale (National Nanotechnology Initiative)
NIH Support: National Institute of General Medical Sciences; National Heart, Lung, and Blood Institute