Johns Hopkins Gazette: January 22, 1996

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

     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

     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

     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

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