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【bio-news】高分辩率荧光显微镜：突破科研的极限
http://www.nature.com/nmeth/journal/v6/n1/full/nmeth.f.234.html
After a long period of measured development and a recent surge of technical advances driven by physicists, super-resolution fluorescence microscopy emerged in 2008 as a powerful tool for biologists. Kelly Rae Chi reports.
In the summer of 2005, cell biologist Jennifer Lippincott-Schwartz tore down the red lights in her darkroom at the National Institutes of Health in Bethesda, Maryland. Lippincott-Schwartz was making room to host then-unemployed physicists Eric Betzig and Harald Hess. They were working on photoactivation localization microscopy (PALM), a new technology they hoped could dramatically increase the resolving power of fluorescence imaging and be used to see nanometer-scale biology.
Betzig, Hess, and Lippincott-Schwartz's group worked in the tiny room through that winter, wearing their coats and hats in the unheated room and collecting data. Hess admits he and Betzig, now both at the Howard Hughes Medical Institute's Janelia Farm Research Campus in Ashburn, Virginia, didn't know much about biology. For 15 years, they had been thinking about high-resolution imaging. When they learned about photoactivatable green fluorescent protein—which was invented by Lippincott-Schwartz and George Patterson in 2002—they saw it as the missing link in their quest to improve imaging resolution.
"They were so excited," Lippincott-Schwartz remembers. "And I remember the first images. It was hard to know what we were looking at." It was not until she saw the fluorescence images laid over an electron micrograph that Lippincott-Schwartz believed that their method worked. "I thought, 'This is really right.' It was really, truly amazing."
Super-resolution fluorescence microscopy has, in the last few years, hit its stride and allowed researchers to see cellular processes unfolding at nanometer scales. The days of interpreting fuzzy blobs in the 200 to 750 nm size range are over. Though the ideas behind super-resolution microscopy were born and raised in academic institutions starting in the late 1980s, the last few years have seen an explosion of technology, along with the start of commercialization. And now, with dozens of labs setting up their own instruments and tweaking their samples, the most exciting moment, especially for biologists like Lippincott-Schwartz, is seeing it work.
Stefan Hell breaks the barrier
Since 1873, when Ernst Abbe first proposed the rule of diffraction-limited imaging, researchers had believed that the ability to resolve two nearby points was fundamentally limited by the wavelength of light. More than a century later, Stefan Hell, now director of the Max Planck Institute of Biophysical Chemistry in Göttingen, Germany, was the first to show, theoretically and experimentally, that one can use a light microscope to resolve objects on the nanometer scale, below the diffraction limit.
As a graduate student in the mid-1980s, working at the University of Heidelberg in Germany with a low-temperature solid state physicist as his adviser, Hell first realized that it would be possible to improve resolution by having not just a single lens focus the light onto a point but by having two large-aperture lenses jointly doing so. Working on his own at home, his fellowship having expired after he completed his PhD thesis in 1990, he devised the 4Pi microscope based on this idea. "I thought, 'How would you realize the idea [of high resolution] using two opposing lenses?'" he recalls. "I conceived that, and I laid it out on paper."
Hell needed a place to show that the principles worked. So, his idea in hand, he went to the European Molecular Biology Laboratory (EMBL) in Heidelberg, where he began work in 1991, supported by a postdoctoral fellowship from the German Science Foundation.
In those early days, many researchers, including prominent physicists, thought that Hell wouldn't get very far in improving resolution. With what little financial independence he had, Hell was taking a risk. But he was tied to the idea of breaking the diffraction barrier. "I stayed in science just because I wanted to improve the spatial resolution," he says.
Sunney Xie, now a professor of chemistry at Harvard University in Cambridge, Massachusetts, met Hell in the 1990s and saw some of his early talks on high-resolution 4Pi microscopy. "He was just so original," Xie says. "He was not afraid to challenge conventional wisdom when he set out to do what he believed in."
Hell's risk-taking paid off. In 1992, he showed for the first time that the 4Pi microscope could improve resolution to three to seven times that of a conventional microscope. But although it improved resolution along the z axis, it still did not overcome the limiting role of diffraction.
At his next postdoctoral stint, at the University of Turku in Finland, Hell had an idea one Saturday morning while sitting on his bed in a student dorm and reading a book on the quantum theory of light. He thought that, with the right lasers, he could fluorescently activate a spot and then shrink that spot by depleting the emission in a doughnut-shaped area surrounding it. He would later call the imaging method STED, or stimulated emission depletion. "I instantly went into the lab to make those assessments," he recalls. "That was one of the most exciting moments in my career."
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作者:admin@医学,生命科学 2011-02-25 17:12
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