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A Better View of the Molecule


A Better View of the Molecule
New method of microscopy using fluorescence photoactivation could help address biological questions

Assistant Professor of Physics and Astronomy Sam Hess, left, and Assistant Professor of Chemical and Biological Engineering Mike Mason
Assistant Professor of Physics and Astronomy Sam Hess, left, and Assistant Professor of Chemical and Biological Engineering Mike Mason

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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 400700 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

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