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Sightings
Seeing Is Believing
Biophysicist Niels de Jonge longs to see what interests him, no matter how small. That includes individual proteins or receptors inside
mammalian cells. After all, who knows what surprises a good look at the real thing will yield? This drive, and an affection for tough problems, led the Oak Ridge National Laboratory and Vanderbilt University researcher to find a means to image whole cells in liquid with
a scanning transmission electron microscope. The technique improves the view inside a cell by a factor of more than 10 compared with
ultrahigh-resolution optical imaging with quick processing times. To do this, de Jonge needed to overcome a sizable obstacle: Scanning
transmission electron microscopy requires a high vacuum, a hostile environment for samples in liquid, including, of course, whole cells.
Associate Editor Catherine Clabby interviewed de Jonge about the innovation.
American Scientist: Tell us more about the limitations you
were trying to beat.
De Jonge: Since the origin in the 1930s of the electron micro-
scope, there’s always been a wish to image cells just like you
can do with light microscopy. It’s always been difficult due to
the liquid. There have been some solutions, for example with
the transmission electron microscope (TEM). But those only
work with thin samples and not whole cells. If you go thicker,
the contrast mechanism of the TEM prevents high resolution.
Other approaches used the scanning electron microscope
(SEM). There have been some good examples of a vacuum
chamber with water vapor and another with special capsules.
But SEM is a surface technique. You can look a little bit under
the skin, but not much farther than 50 nanometers.
My vision has been to image a whole eukaryotic cell
mostly in its native state. Those are rather thick: 5 to 10
micrometers. There has been huge progress with optical
microscopy where you can look at whole cells. But the
resolution is not so good. You can see regions where tagged
receptors are but you cannot always see individual proteins
or receptors. That’s really what you’re aiming for.
American Scientist: How did you overcome all that?
De Jonge: I came into contact with a very good scanning
transmission electron microscope (STEM) team at Oak Ridge
National Laboratory where I tried to apply STEM to this particular problem. Only a couple of groups use STEM in biology
studies. Traditionally TEM is used. The reason has been that
TEM is best for biological samples because it has good resolution with carbon. All biological materials have carbon. But we
want to look through thick samples. If the sample produces
too much signal, you can’t image it. I was looking for a technique with low contrast on carbon, which STEM has.
In the samples, you can use high-contrast labels—we’ve
used gold—to tag molecules. The idea is that you can see
very small labels of high atomic number inside the thicker
layer of material of lower atomic number. To protect the cells,
we used a special microfluidic device made from silicon
chips. They have very thin silicon nitride windows that are
electron transparent. You can make a sandwich with two of
those chips. Liquid can be enclosed between the windows,
separated from the vacuum, while the electron beams can go
through the whole thing. And we needed a holder for placing the liquid enclosure inside the electron microscope.
American Scientist: What improvements in resolution did
you achieve?
De Jonge: We have a resolution of 4 nanometers, which you
cannot achieve optically. And STEM is faster than the finestresolution optical imaging. If you go beyond a resolution of
50 nanometers with optical imaging, it’s very slow. It’s a very
complicated matter for data processing. STEM can take an
image in a matter of seconds. But there are limitations: The
electron beam creates radiation damage.
I believe if you combine optical imaging and STEM you
have a very viable imaging technology. You can look first in
optical and see what processes are happening and in which
regions certain receptors are located. Then you can zoom in
with the electron microscope on a whole cell in the sample.
You can actually see which proteins are interacting with
which receptors.
American Scientist: In a Proceedings of the National Academy of
Sciences article this year, you and collaborators Diana Peckys,
Gert-Jan Kremers and David Piston described how you imaged single, gold-tagged epidermal growth factor molecules
bound to receptors inside fixed African green monkey fibroblast cells. In this case, the resolution reached 4 nanometers.
In what other kinds of studies do you envision this new tool
would be useful?
De Jonge: It’s such a large field. What I am trying to do is find
collaborations with biomedical researchers. I want to learn
what kinds of problems they have and how I can help. We
continue to work, for example, with epidermal growth factor.
I’m also interested in the ways viruses interact with cells.
American Scientist: Will you attempt to keep improving this
technology?
De Jonge: I’m working on developing a new way to get 3-D
images from electron microscopes. The traditional technology
involves tilting the sample. With the fluid holder, I don’t want
to have to tilt anymore. Cells are 3-D structures. You want 3-D
images of them.
In Sightings, American Scientist publishes examples of innovative scientific imaging from diverse research fields.
238
American Scientist, Volume 97
© 2009 Sigma Xi, The Scientific Research Society. Reproduction
with permission only. Contact [email protected].
10 micrometers
a
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Above (a) is the view of tagged epidermal growth factor molecules in a monkey fibroblast cell achieved with confocal laser microscopy. Resolution (b) in this case improves with a new scanning transmission electron microscope technique. A microfluidic device (c) with electron-transparent windows that seals samples from a vacuum and a specially designed holder (d) made the new approach (e) possible.
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© 2009 Sigma Xi, The Scientific Research Society. Reproduction
with permission only. Contact [email protected].
2009 May–June
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