Technique Allows 'Filming' of Cellular Processes

With a new technique that images molecular mechanisms in living cells, researchers at Lawrence Berkeley National Laboratory have enabled scientists to broaden their understanding of both the way drugs work and the progress of diseases in cells derived from humans.

Previously, scientists had to either label cells with specific markers or kill cells before being able to measure their overall activity. The new technique, called Synchrotron Radiation-Based Fourier Transform Infrared Spectromicroscopy, permits researchers to watch and measure changes in a single cell over time, like a "movie."

Using the new technique, scientists can grow cells and then impose whatever conditions that they are interested in studying on them. Then, researchers expose the treated cells to the synchrotron infrared light.

By looking at spectral changes, the scientists can determine the effect of a variety of factors - including drugs, environmental contaminants and radiation - on individual cells.

"In this case, we do not introduce extraneous factors; the cells are still alive; we just monitor what is happening," said Hoi-Ying Holman, the researcher who developed the new technique. "We do not need to do any other treatments or sample preparation. Usually, people would label cells with markers and observe their changes when excited with light, but most of the time they have to extract the content of the cells or lyse the cell with chemicals. The sample treatment procedure is generally lengthy, labor intensive and the cells are dead."

Because drugs affect cells in different ways, researchers could use the new technique to determine the specific mechanisms that two similar drugs go through as a way to screen for efficacy and overall cellular activity.

The scientists looked at the initial effects of dioxin - a powerful toxin that has a single target within a cell - and demonstrated that the chemical binds to a specific site on the cell's DNA and affects the regulation of a cancer-fighting gene.

The researchers found that increasing the amount of dioxin in the cell caused marked changes in the spectrum not present in the control samples, indicating that the influence that dioxin has on a cell is related to the specific way it interacts with its binding site.

The researchers were also able to show the different spectra during the different stages of the cell cycle, and were able to clearly demonstrate changes between the pre-DNA synthesis stage, the DNA replication stage and the cell division phase.

In the future, scientists are planning to use the new technique to study the effects of radiation and drug therapies for malignant brain tumors and damage due to oxidative stress diseases including atherosclerosis, diabetes and rheumatoid arthritis.

Scientists can monitor the molecular happenings within the cell by looking at the infrared spectrum of individual cells. Analyzing these spectra permit researchers to identify whether chemical bonds are being made, weakened or are remaining unchanged.

"The technique measures the absorption of infrared light at specific wavenumbers, which correspond to vibrations of chemical bonds in molecules," Holman said. "The measurement is sensitive to the presence of chemical functional groups in a molecule within a biological sample. With appropriate interpretations of measured infrared spectra, you can study chemical and structural changes in a biological sample as the changes are occurring over time."

Monitoring the chemical activity within the cell requires the use of infrared synchrotron light, which is more focused and has a higher signal-to-noise ratio than the conventional light sources used in infrared microscopes.

The synchrotron light source permits scientists to increase the sensitivity of their experiment, said Michael Martin, one of the researchers who was involved in developing the machinery used in the study.

"We measure all wavelengths in the middle of the infrared spectrum," Martin said. "That way, we can measure everything at once."

Using infrared light is useful for two reasons - it does not damage the cell, so the low-energy light can be used to study living cells. In addition, almost every chemical bond has a signature wavelength in the infrared region that can be monitored by studying spectral data.

After obtaining the spectral data, scientists can work backward to figure out which molecules in the cells are changing.

"Because almost every biomolecule has a little absorption in this region, the high brightness of the synchrotron light allows us to have the extra ability to recognize what type of molecule is changing," Martin said. "In dying cells, we can see shifts in the regions absorbed by proteins, indicating that the proteins are starting to change structurally. There are specific infrared absorption regions for proteins, DNA, RNA, lipids, hydrocarbons and phosphates."

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