On Research: New Microscope Brings Tiniest Worlds Into Sharper Focus Emil Venere ------------------------------------ Homewood News and Information It's as though biologists have put on their glasses for the first time--a powerful new tool is letting scientists see details never before visible with light microscopes. The device is called a near-field scanning microscope, and Hopkins is one of only a handful of universities to begin using the instrument for biological research. "We are seeing things that nobody's ever seen before, and for a biologist, that's exciting," said biology professor Michael Edidin. Unlike other advanced microscopes, the new device operates with visible light, enabling biologists to view living specimens that have been labeled with fluorescent dye to make them stand out. "Any method that's been used for the last 150 years of light microscopy can be used here, with 5 to 10 times better resolution," he said. The instrument was developed at AT&T Bell Labs. But the prototype could not be used to look at wet samples, meaning biologists could not view living specimens. Hopkins physicist Jeeseong Hwang, however, working closely with physicist Eric Betzig at Bell Labs, managed to use the instrument so that carefully dried cells could be viewed. "That taste was enough to say OK, I want one too, and we built our own at Hopkins," Edidin said. The instrument cost about $100,000 to build and has been funded largely through the National Institutes of Health, with additional money from the Biology Department. The new instrument has been operating here since mid-October. The innovation has already yielded important findings. For example, with ordinary microscopes scientists used to see a vague, featureless landscape on the surfaces of cell membranes. Suddenly, this previously nondescript world is revealing surprising structure--no small development, since these cell membrane features are vital for relaying cell-to-cell signals that dictate key biological functions. While using the microscope to study cells that trigger the production of antibodies in humans, Edidin and scientists in his lab were startled by the clarity of the images. They were looking at the cell membranes surrounding human leukocyte antigens-- proteins that, when not matched properly, cause the body to reject any kind of tissue grafts, including bone marrow and organ transplants. What they learned will help biologists make a more accurate model of how the membranes work, which is essential to reducing tissue rejection in transplants. If the HLA in the transplanted tissue does not match the recipient's HLA type, it will trigger rejection. Understanding more about the membranes surrounding HLA molecules also will help biologists learn how receptors on cell surfaces are triggered. Scientists had thought that the molecules making up the membranes were evenly distributed, floating independently on the cell surfaces. Research with the near-field scanning microscope, however, found something quite different: they are not evenly distributed at all but appear to be interconnected patches on the cell membranes. Although biologists had theorized about the possible existence of this structure, it had never before been seen, Edidin noted. "There is much more detail that says that these membranes are more complicated than we thought," he said. Until the near-field scanning microscope was developed, biologists were unable to use visible light to see objects smaller than half of a wavelength of light. Other advanced high-resolution microscopes, such as electron microscopes and scanning tunneling microscopes, use electron beams and electric current, respectively. While they provide high-resolution images, they cannot be used in fluorescence microscopy, which greatly enhances structural details. The Hopkins scientists marked molecules with a fluorescent dye and then viewed them with the new microscope. Researchers were able to see features as small as 30 nanometers, or billionths of an inch--that's 10 times better than the finest resolution achieved with conventional light microscopes. Edidin explained the advance this way: The laws of physics state that objects smaller than about half the diameter of a wavelength of light cannot be resolved using traditional visible light microscopes. If you punched a hole of that diameter through an opaque screen and then shined a light through the hole, no light would be seen coming through the screen unless you could get extremely close to the hole. That's exactly how the new device gets around the previous visible-light limitation. The microscope's tip, an ultra-thin optical fiber, is moved as close as 10 billionths of an inch to the subject being viewed. That's only about 30 times the diameter of an atom. Scientists achieve this remarkable closeness by causing the tip to vibrate and then measuring changes in vibration as the tip nears the specimen being viewed, explained Hwang, a postdoctoral fellow. The tip slows down, ever so slightly, as it encounters forces that surround molecules, indicating its proximity to the specimen, he said. If the process is hard to describe, the result isn't. "The main thing is you get this gorgeous resolution," Edidin said.
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