SPECTROMICROSCOPY: Bright infrared beam follows changes in living human cells

May 1, 2000
An infrared (IR) spectromicroscopy technique that enables the observation of subtle chemical and molecular changes in individual human cells nondestructively without killing the cells or using intrusive probes was described in late March at the annual meeting of the American Physical Society (APS; Minneapolis, MN) by researchers from Lawrence Berkeley National Laboratory

An infrared (IR) spectromicroscopy technique that enables the observation of subtle chemical and molecular changes in individual human cells nondestructively without killing the cells or using intrusive probes was described in late March at the annual meeting of the American Physical Society (APS; Minneapolis, MN) by researchers from Lawrence Berkeley National Laboratory (LBNL; Berkeley, CA). Although new to applications in living human cells, the technique, Synchrotron Radiation-Based Fourier Transform Infrared (SR-FTIR) spectromicroscopy, has been previously used for studies in environmental, forensic, and materials sciences.

"Traditional methods of biomedical research either require killing cells, averaging results from many cells, or introducing dyes or tagged proteins or other agents that can affect cell chemistrymethods that usually involve tedious sample preparation and long delays between experiment and result," said Hoi-Ying Holman of LBNL's Earth Sciences Division, the principal investigator in developing the new technique. "Now we can study individual cells in real time without introducing extraneous factors." Holman described the technique a week after the APS meeting at the annual meeting of the American Chemical Society (San Francisco, CA).

The technique relies on the ability to focus the infrared synchrotron beam to a spot less than 10 µm in diameter that also happens to be a little smaller than a typical mammalian cell (see figure). "Because every specific molecule absorbs a specific set of infrared wavelengths, the beam reflected from an individual cell yields a unique spectrum that can distinguish among different cell lines, different phases in the cell cycle, and the different chemical reactions and physical changes within cells," according to Michael Martin, a member of the Advanced Light Source research team at LBNL. The beam cank also be positioned with an accuracy of 1 µm, and a rapid pulse-repetition rate (2 ns between pulses) enables observation of rapid cell changes.

In recent work, Holman and her collaborators have concentrated on the response of cultured human cells, including lines originated from lung and liver tissue, to low doses of environmental agents. "We studied changes in cells caused by oxidizing agents in dilute amounts typical of environmental exposure," she said. "Hydrogen peroxide is a strong oxidizer, and bleomycin is an antibiotic that is a weaker oxidizer but still damages DNA."

Hydrogen peroxide causes predominantly single-strand breaks in DNA, while bleomycin induces a large number of double-strand breaks. Under SR-FTIR spectroscopy, damage from each chemical showed up as distinctly different spectral changes. In addition, the cell-wide damage caused by x-rays in lung cells produced a different spectroscopic signature compared to unexposed cells.

"We also used the new technique to detect changes caused by dioxin in liver tumor cells," Holman said. The dioxin molecule binds to a specific receptor, which then binds to a site on the cell's DNA, regulating a gene that expresses one of the cytochromesproteins that catalyze the breakdown of aromatic carcinogens and other organic molecules.

"Increasing the dose of dioxin caused marked changes in the SR-FTIR spectrum, but increasing a control compound that doesn't bind to that receptor didn't show these spectral changes," she said, indicating that dioxin's biological influence is related to its interaction with the binding site.

By mapping biological and chemical reactions as they occur in individual living cells over a period of hours or days—in response to dilute, environmentally relevant concentrations of chemical substances and radiation—the new technique enables researchers to perform basic studies at the subcellular level.

About the Author

Hassaun A. Jones-Bey | Senior Editor and Freelance Writer

Hassaun A. Jones-Bey was a senior editor and then freelance writer for Laser Focus World.

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