Contact: Aimee Dolloff, (207) 581-3777; Samuel T. Hess, (207) 581-1022
Scientists at the University of Maine have developed a new way of looking at the molecular organization of cells by creating a microscope system they call FPALM (Fluorescence Photoactivation Localization Microscopy).
They already have used FPALM to image living cells with membranes that contain a protein that enables infection by the influenza virus. They also have used the system to image a variety of other biological and some non-biological systems.
“In principle, FPALM can be used to image any sample that can be labeled with an appropriate fluorescent marker,” UMaine physics graduate student Travis Gould said.
Influenza uses a protein, hemagglutinin (HA) to infect healthy cells. In the first step of infection, HA enables the virus to attach to the membrane of a healthy cell.
It is believed that how the individual HA molecules are arranged in the membranes is crucial for infection to occur. Unfortunately, due to the limited resolution of conventional microscopes, it hasn’t been possible to create images of such molecules on a small enough scale to test the biological models that predict how they may be organized.
“This problem was actually the motivation for inventing FPALM in the first place,” Gould said. “In our work on this question we were able to image living cells and disprove several of the existing models of membrane organization.”
The recent extension of FPALM to include three-dimensional imaging and provide information about the orientation of single molecules will greatly increase the ability of FPALM to address important biological questions.”
The system breaks a fundamental limit on the resolution of lens-based microscopes, known as the diffraction barrier, that has existed for more than 100 years.
UMaine graduate students Gould and Mudalige Gunewardene, research scientist Manasa Gudheti, and professors Julie Gosse and Samuel T. Hess of UMaine, along with colleagues at the Albert Einstein College of Medicine in New York and the National Institute of Child Health and Human Development in Maryland, recently had their findings published in the “Nature Methods” science journal.
When detecting light through a lens-based imaging system, in this case a microscope, structures smaller than about half the wavelength of the detected light can’t be resolved because of diffraction. Diffraction is the phenomenon that occurs when a wave, such as light, encounters an obstacle, such as a lens.
A normal microscope looks at all of the molecules at once, which can make the individual molecules difficult to see. It’s like trying to pinpoint individual drops of water in a stream.
The FPALM system uses photoactivatable dyes to identify individual molecules and separate them.
“Surprisingly, it is relatively easy and inexpensive to adapt conventional, commercially available microscopes for FPALM imaging,” Travis Gould says. “In fact, in labs where conventional fluorescence imaging is already being performed, the only additional equipment required would be a camera sensitive enough to detect the light emitted by a single molecule and potentially an additional laser.”
While it does take longer to produce an image using FPALM, the system provides about 10 times better resolution. Its resolution capabilities exceed those of the most powerful confocal light microscopes currently available.