IR Fourier Analysis Reveals Individual Cell Mechanisms
BERKELEY, Calif., May 7, 2012 — To understand how living cells work, it is important to understand the chemistry inside the cell. One such process is protein phosphorylation, which controls cell proliferation, differentiation, metabolism, signaling as well as apoptosis. Before now, it was impossible to observe the changes as a single cell underwent phosphorylation. However, a technique has been developed to observe such chemical mechanisms up close.
A research collaboration between the universities of Berkeley and San Diego used the Berkeley synchrotron IR beam line 1.4.3 to perform Fourier transform infrared analysis (FTIA) spectromicroscopy on a collection of mammal cells to monitor the protein phosphorylation of single living cells.
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.(Images: Berkeley Lab)
Previously, researchers labeled certain phosphorylated proteins with a fluorescent marker so that they could track the proteins. “That gives you a great image," said Hoi-Ying Holman of the US Department of Energy’s Lawrence Berkeley National Laboratory and director of the Berkeley Synchrotron Infrared Structural Biology Program. "But you have to know exactly what to label before you can even begin.”
The phosphorylation process adds one or more phosphate groups to three amino acid residues in proteins. This activates the protein, while removing a phosphate group deactivates it. The researchers' goal is to isolate what is happening in the process, and at what timescale, without killing the cells. Unlike x-rays, infrared light has an energy high enough to image the cells, but not high enough to damage or kill them. Various states of the cell absorb various wavelengths of the infrared light, and the Fourier transform algorithm can record all of the signals simultaneously, showing exactly where the cell is changing.
PC12 cells treated with nerve growth factor underwent a series of changes resulting from phosphorylation. On Day 3, the cells sent out neurites, resembling the growth of nerve cells. Spectromicroscopy at beamline 1.4.3 of the Advanced Light Source tracked specific local chemical changes in the living cells.
The current experiment uses a line of cultured cells, called PC12. Lian Cheng, a postgraduate fellow at Berkeley Lab, introduced nerve growth factor to the PC12 cells to induce them to differentiate. The cells were left to culture on gold-coated slides in chambers maintained at human body temperature. FTIR spectra were collected before and after the nerve growth was introduced. After that, spectra were taken at short intervals.
Berkeley Lab's Zhao Hao had predicted what the results should be based on quantum chemistry simulations. The researchers compared their results with these predictions and confirmed the monitoring of phosphorylation phases, their timing and their target proteins.
“This experiment was a proof of the concept,” Cheng said. “We demonstrated the dynamics of protein phosphorylation in controlling differentiation in this biological system using synchrotron infrared spectromicroscopy, and we pointed the way to answering the many questions a biologist has to ask about measuring the coordination of specific processes in real time.”
In the top panel, various modes of imaging of the same cell show the differences between visible light microscopy and fluorescence imaging, and in the lower panel, the images resulting from Fourier transform infrared spectromicroscopy. Infrared absorption at various frequencies pinpoints different cell components at specific locations in the living cell.
Although unable to follow individual cells, the investigators did observe the differentiation in the PC12 cells in real time without labeling or damaging the cells.
Other related experiments have involved the design of equipment to maintain mammalian cells in a thin culture, automatically adjusting the temperature, humidity and nutrition levels. Another is monitoring cell development in human cells, bacteria and plants using hypermicrospectroscopy to choose the optimal frequency window for the sample.
“Many researchers from the medical communities are interested in using the technology, and we are particularly interested in collaborating with university centers and private firms that are seeking a broad view of how promising drugs act within specific cells,” Holman said.
The results were reported in Analytical Chemistry.
The research was performed at Berkeley Lab's Advanced Light Source, which is supported by the Energy Department's Office of Science.
For more information, visit: www.lbl.gov
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