Berkeley Lab scientists demonstrate the promise of synchrotron infrared spectroscopy of living cells for medical applications

APRIL 30, 2012
Berkeley Lab scientists observed phosphorylation in living PC12 cells stimulated by nerve growth factor as they differentiated and sent out neuron-like neurites. The researchers imaged individual cells and simultaneously obtained absorption spectra using synchrotron radiation from the Advanced Light Source. Cells not stimulated with nerve growth factor did not differentiate and showed different infrared absorption spectra.

Berkeley Lab scientists observed phosphorylation in living PC12 cells stimulated by nerve growth factor as they differentiated and sent out neuron-like neurites. The researchers imaged individual cells and simultaneously obtained absorption spectra using synchrotron radiation from the Advanced Light Source. Cells not stimulated with nerve growth factor did not differentiate and showed different infrared absorption spectra.

Knowing how a living cell works means knowing how the chemistry inside the cell changes as the functions of the cell change. Protein phosphorylation, for example, controls everything from cell proliferation to differentiation to metabolism to signaling, and even programmed cell death (apoptosis), in cells from bacteria to humans. It’s a chemical process that has long been intensively studied, not least in hopes of treating or eliminating a wide range of diseases. But until now the close-up view – watching phosphorylation work at the molecular level as individual cells change over time – has been impossible without damaging the cells or interfering with the very processes that are being examined.

“To look into phosphorylation, researchers have labeled specific phosphorylated proteins with antibodies that carry fluorescent dyes,” says Hoi-Ying Holman of the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab). “That gives you a great image, but you have to know exactly what to label before you can even begin.”

Holman and her coworkers worked with colleagues from the San Diego and Berkeley campuses of the University of California to develop a new technique for monitoring protein phosphorylation inside single living cells, tracking them over a week’s time as they underwent a series of major changes.

“Now we can follow cellular chemical changes without preconceived notions of what they might be,” says Holman, a pioneer in infrared (IR) studies of living cells who is director of the Berkeley Synchrotron Infrared Structural Biology program at Berkeley Lab’s Advanced Light Source (ALS) and head of the Chemical Ecology Research group in the Earth Sciences Division . “We’ve monitored unlabeled living cells by studying the nonperturbing absorption of a wide spectrum of bright synchrotron infrared radiation from the ALS.”

The researchers report their results in the American Chemical Society journal Analytical Chemistry.

Cellular Analysis: A powerful source of infrared light reveals phosphorylation within individual cells
Micrograph of differentiating cell.
Inside Story
Researchers can now track protein phosphorylation inside single cells like these, as they differentiate to become nerve cells.
Credit: Anal. Chem.

A bright light may help illuminate cellular secrets such as how they grow, differentiate, or respond to drug therapy. Researchers have focused infrared radiation from a synchrotron light source on single cells and measured protein modifications as the cells developed into nerve cells (Anal. Chem., DOI:10.1021/ac300308x).

Phosphorylation—the attachment of a phosphate group to a protein—plays a key role in cells’ metabolism, proliferation, and differentiation. But the existing techniques for studying this process in single cells, including fluorescence-based ones, require researchers to put chemical tags on proteins of interest in advance. As a result, says Hoi-Ying Holman of Lawrence Berkeley National Laboratory, researchers can use the techniques only when they know what proteins they’re looking for.

IR spectroscopy can measure phosphorylation without labeling, but most instruments don’t use light bright enough to detect the weak signals from single cells. So Holman used a synchrotron, a source of intense IR radiation that generates data with a signal-to-noise ratio 100 to 1000 times as high as that of standard IR spectrometers.

To demonstrate the technique, Holman’s team used the synchrotron light to watch what happens inside individual rat cells called PC12 as they differentiated into nerve cells. Previous studies have shown that protein phosphorylation increases during this transition.

In the experiment, the researchers treated a culture of PC12 cells with a signaling protein called nerve growth factor, which triggers differentiation. Then, going one cell at a time, they focused a beam of light on a cell and collected data at several time points during the following hour and up to seven days later. Using data they collected previously from PC12 cells with artificially high levels of protein phosphorylation, the scientists could pinpoint the phosphorylation signal among the signals of other molecules in the cell.

Holman was surprised at how quickly phosphorylation levels changed in each of the cells. Levels spiked in just five minute, she says, then came “in waves,” as the cells then experienced a decrease in phosphorylation, followed by a second rise. “It’s like you are watching a movie,” she says. The researchers observed that the timing correlates with the stage of differentiation, supporting phosphorylation’s role as a driving force in nerve cell maturation.

Studying phosphorylation in real time may be just the beginning. Since IR can track many types of molecules, Holman hopes to monitor other chemical transformations, such as methylation or changes in carbohydrate composition. She thinks it could prove useful to study cell signaling or the effects of radiation on cancer cells.

Likewise, Gregory Enns of Stanford University sees almost unlimited possibilities for the approach. “This is just great stuff,” he says. “The ability to interrogate a single cell is a wonderful tool with widespread implications.” Enns hopes to use this technique to test candidate drugs by comparing healthy cells to cells from people with diseases both before and after treatment.

C&E News April 11, 2012.

An introduction to IR spectroscopy from the Royal Society of Chemistry. Includes vibrational modes of molecules, how an FTIR spectrometer works, what the ATR method is, some spectral interpretation and spectral library matches.

Help figure out what your IR spectrum means with the Infrared Spectrum Song!

Feng WangTwo Berkeley Lab researchers received this year’s Presidential Early Career Award for Scientists and Engineers (PECASE): Christian Bauer (not shown, Physics Division) and Feng Wang of the Materials Sciences Division. Wang is an ALS user on Beamline 1.4 (see ALS Science Highlight Bilayer Graphene Gets a Bandgap and this month's Berkeley Lab News Center article A Whole New Light on Graphene Metamaterials). Wang is cited for “pioneering research on ultrafast optical characterization of carbon nanostructures that has advanced the fundamental understanding of the electronic structure of graphene and is expected to enable the development of advanced-energy-relevant technologies.” PECASE awards are the U.S. government’s highest honors to outstanding scientists and engineers, early in their independent research careers.

Berkeley Lab scientists demonstrate a tunable graphene device, the first tool in a kit for putting terahertz light to work

IMAGE: The graphene microribbon array can be tuned in three ways. Varying the width of the ribbons changes plasmon resonant frequency and absorbs corresponding frequencies of terahertz light. Plasmon response is much stronger when there is a dense concentration of charge carriers (electrons or holes), controlled by varying the top gate voltage. Finally, light polarized perpendicularly to the ribbons is strongly absorbed at the plasmon resonant frequency, while parallel polarization shows no such response.

Long-wavelength terahertz light is invisible – it's at the farthest end of the far infrared – but it's useful for everything from detecting explosives at the airport to designing drugs to diagnosing skin cancer. Now, for the first time, scientists at the U.S. Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California at Berkeley have demonstrated a microscale device made of graphene – the remarkable form of carbon that's only one atom thick – whose strong response to light at terahertz frequencies can be tuned with exquisite precision.

"The heart of our device is an array made of graphene ribbons only millionths of a meter wide," says Feng Wang of Berkeley Lab's Materials Sciences Division, who is also an assistant professor of physics at UC Berkeley, and who led the research team. "By varying the width of the ribbons and the concentration of charge carriers in them, we can control the collective oscillations of electrons in the microribbons."

The name for such collective oscillations of electrons is "plasmons," a word that sounds abstruse but describes effects as familiar as the glowing colors in stained-glass windows.

"Plasmons in high-frequency visible light happen in three-dimensional metal nanostructures," Wang says. The colors of medieval stained glass, for example, result from oscillating collections of electrons on the surfaces of nanoparticles of gold, copper, and other metals, and depend on their size and shape. "But graphene is only one atom thick, and its electrons move in only two dimensions. In 2D systems, plasmons occur at much lower frequencies."


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