Science has limits. Defined by theories, laws and formulaic
equations, these limits define the boundaries within which scientific
discoveries are made.
In most cases, having boundaries that are clearly defined by
justifiable rules is a good thing. In science, however, the existing
limits often interfere with the overall quest: the search for a cure,
the advancement of technology, the depth of our understanding of
ourselves and our universe. For scientists, the goal is often to break
through those limits to reveal the discoveries on the other side.
For University of Maine Assistant Professor of Physics and
Astronomy Sam Hess, a breakthrough idea that would redefine the limits
of scientific microscopy arrived late at night, accompanied by a heavy
backbeat and a lot of yelling.
A raucous party at his neighbors' apartment had made it impossible
for Hess to sleep. Instead of simply pulling a pillow over his head
and contemplating moving, Hess considered the limits of physics. In
particular, he mulled over the refraction barrier, which has long set
the magnification limit of light microscopy as the size of the
wavelength of visible light — roughly 400–700 nanometers.
"It was 2 in the morning. I was tossing and turning, thinking about
this idea, and at the time it seemed like it could work," says Hess,
laughing about the circumstances that surrounded his discovery. "The
party continued, and I continued to think about the idea and I just
couldn't prove it wrong, so I decided to go downstairs and write it
down. I figured I would have a good laugh when I read it in the
morning."
As it turned out, Hess couldn't find any problems with his approach
in the morning either, and neither could his colleagues. With a nod of
encouragement from some of his most trusted peers, he decided to take
the next step, enlisting the help of fellow UMaine professor and
friend Mike Mason.
"We had talked about different ways to approach this problem a
number of times. I had done some work on a similar technique as a
postdoc, and Sam knew a lot about photoactive dyes," says Mason,
assistant professor of chemical and biological engineering. "When he
came to me with the idea, we knew we had to get working on it."
After a review of the literature yielded no mention of the new
approach by other researchers, Hess and Mason discussed the new
technique at length, outlining the steps necessary to build and test a
prototype. The new microscope system, dubbed FPALM (Fluorescence
Photoactivation Localization Microscopy), combines existing
technologies to build an image based on the florescence of individual
molecules. The device's magnification capabilities exceed those of the
most powerful confocal light microscopes available.
"A normal microscope looks at all of the molecules at once, which
can make the individual molecules difficult to see, like drops in a
stream of water," says Hess. "The separation between individual
objects needs to be larger than the microscope's resolution, otherwise
the light coming back through the microscope is blurred and the
objects are indistinct. This new technique allows us to find out where
the molecules are and separate them as individual entities. The key is
in the use of photoactive dyes."
The device uses lasers to excite dye molecules on the surface of
the subject being observed. The laser causes a portion of the
molecules to fluoresce, and the light given off creates an image that
is captured digitally. The process is repeated as new sets of
molecules are excited, and the individual images, each reminiscent of
a starry sky at night, are layered with the help of a computer to
create a composite image. The resolution of the new image is at least
eight times better than any traditional light microscope available
today.
Mason, whose research utilizes single molecule imagery to probe
local processes in biology and materials science, brought in a highly
sensitive camera to aid in detection of the faint single molecules.
The prototype yielded its first preliminary data in October 2005.
The first few tests had barely ended before disaster struck the
project. A steam pipe in Hess' lab burst, ruining some of the highly
sensitive equipment and setting the project back several months. To
make matters worse, Hess learned that another research team was close
to publishing its findings on a similar microscopy technique.
"The pipe was repaired, the lab was cleaned up, we got another
camera and we began taking more measurements," says Hess. "We heard
that another group had submitted its paper already, so we really had
to push. It was stressful, but it was also very motivating. We spent a
lot of late nights, but we were able to get our paper published in
Biophysical Journal within a few days of when the other group's work
was published."
In the final stages of the peer review process, Hess and Mason were
asked to calibrate their device to show that the new microscope did,
indeed, break the refraction barrier. They collaborated with a team of
researchers at UMaine's Laboratory for Surface Science and Technology
— George Bernhardt, Scott Collins and Patrick Spinney — who were able
to make a calibration sample and image it by atomic force microscopy
in just three days.
"The difficulty in calibrating our microscope was in finding an
accurate standard to measure against. If you have a microscope that
can look at things that are smaller than any other microscope can see,
how do you verify what is being seen?" says Hess.
Their published paper earned important recognition on Science
magazine's Top Ten Discoveries of 2006 list.
"I can really see this being used in a lot of labs. The entire
device costs about one-fifth of the cost of a confocal microscope, and
its resolution is 10 times better. Theoretically, the technique could
eventually yield images that have resolutions that are 100 times
better or more," says Hess.
"There's a lot more to do in this area, and I'm so motivated to
keep it going, I can't sit still."
by David Munson
July-August, 2007
Click Here
for more stories from the current issue of UMaine Today Magazine.
