Molecular Movies

By Kirsten Allen

In 1962, an efficient light-emitting green fluorescent protein was derived from the jellyfish Aequorea victoria, floating near the Pacific Northwest coast. Its breakthrough in bioimaging and biomedicine came about 30 years later, when scientists discovered its genetic encodability and began implementing these proteins into cells to assist in the visualization of all kinds of biological processes. Dr. Chong Fang, an assistant professor of chemistry at Oregon State University, along with the help of his postdoctoral fellow Dr. Weimin Liu, graduate student Breland Oscar, and Canadian collaborators Professor Robert Campbell and Dr. Yongxin Zhao from University of Alberta Canada, has taken the phenomenon one step further. Fang has created a breakthrough optical engineering feat which, when triggered by a sophisticated sequence of short pulse lasers, allows a CCD camera to capture stop-motion picture images of proton transfer, with implications for myriad biological functions from nerve impulses to cancer metastasis.

The key point to understand is the speed at which this process occurs. The measurements are created by use of short pulse lasers, the photons of which excite and illuminate the bioluminescent proteins with precise timing. This all takes place in the matter of femtoseconds, or one millionth of one billionth of a second. To put it another way, a femtosecond compared to one second is about the same as one second compared to 32 million years. 

“After the femtosecond actinic pump pulse initiates photochemistry, the picosecond Raman pump and femtosecond Raman probe pulse pair interrogates the molecular system to record the stimulated Raman signal. This signal carries the information about the conformational dynamics of the molecule during chemical reactions, and we can analyze the data to retrieve the molecular ‘movie’ with unprecedented spatial and temporal resolutions,” says Fang. 

Mind boggling? A little. But because this new technology is able to track proton transfer associated with calcium ion binding at a fluorescent protein biosensor, Fang and his team can measure the fastest and most basic aspects of all living systems and virtually see all levels of bioprocesses. Using a newly developed apparatus capable of generating different laser pulses, each pulse with a different color, wavelength, and power, various laser beams reflect off mirrors, gratings, and lenses, which guide three laser beams through a forest of optics to energize the proton and initiate a chemical reaction, while the camera stands ready to capture the movement and record the images on a computer. Each laser pulse can be prepared with desirable tunability to excite the different samples efficiently, resulting in high signal-to-noise ratios of a typically weak Raman signal so a clear portrait of the atomic motions can be captured.

Proton movement is integral to everything from respiration to cell metabolism, and even plant photosynthesis. It also powers the fluorescent protein biosensor that can image calcium ions in living systems. Fang and his team are at a crossroads of understanding how a fundamental biological process works and how to begin implementing the movie technology and apply their findings to answer more pressing questions. What Fang is focused on is making better tools to help scientists observe all level of chemistry in action. “This type of new molecular ‘movies’ will enable the rational design of biomolecular machineries, including but not limited to fluorescent protein biosensors, to achieve transformative advances in materials science and life sciences,” Fang said. In other words, scientists can now observe the molecular processes as they progress rather than only being able to observe the beginning state and the end state. Of course, this breakthrough is already being implemented in the medical field as protein engineers are racing to develop better biosensors, and Fang foresees the potential, upon an increase in funding and, hopefully, receipt of a federal grant or two, to have the ability to further improve those biosensors with prescribed functions, and send them to locations throughout the body to better image calcium ions, and perhaps begin to battle various diseases. “Knowing what happens in between is a powerful approach to build a mechanistic understanding of how things work at the atomic level and on their intrinsic timescales,” Fang adds.

At this point, Dr. Fang is leading the way in this breakthrough. “We have versatile lasers and a skilled team, and can use technical advances to expose atomic motions in all kinds of chemical reactions as they occur. There is no other technique at this point that can capture these vivid molecular movies at the speed of which we are,” he says. Fang and his team have recently published their work with in-depth discussions and illustrations in Proceedings of the National Academy of Sciences.