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Fluorescence Spectroscopy Reveals Cell's Components

Photonics Spectra
May 1998
Michael D. Wheeler

A team of researchers at the University of Illinois has employed fluorescence spectroscopy to find and analyze compounds crucial to cell metabolism.
Traditionally, scientists trying to detect compounds such as serotonin or tryptamine in animal cells have relied on crude methods involving special chemicals or enzymes. The problem: Much of it was guesswork.
In the last several years, scientists such as H.T. Chang and E.S. Yeung have refined these techniques with laser-induced native fluorescence. Building on some of the principles of this work, Jonathan V. Sweedler of the university's chemistry department and then graduate student Robert R. Fuller began work on a technique they hoped would provide a "snapshot" of a cell's metabolic process.
The process begins by placing a freshly isolated cell in a vial, where it undergoes homogenization and is drawn into a capillary. The researchers then jolt the cell with 20 kV of electricity.
The charge causes compounds in the cell to separate and to migrate toward one end of the capillary tube. Because each analyte is a different size, Sweedler and Fuller used size-to-mass separation parameters to determine the identity of each compound.

Laser fluorescence
The group also subjected the cell to a 257-nm frequency-doubled laser from Coherent Inc. of Palo Alto, Calif. This wavelength produces characteristic fluorescence emission spectra for many classes of biological compounds and identifies each component more completely than the separation technique alone. A charge-coupled device (CCD)/spectrograph collects the fluorescence emission from the analytes in less than 40 minutes.
The spectrograph acts as a diffraction grating, spatially distributing each wavelength. A CCD captures each wavelength so that researchers can monitor all wavelengths simultaneously and individually. This allows the researchers to record the entire fluorescence spectrum, not just a maximum wavelength. Achieving a complete spectral fingerprint is important in identifying the difference between serotonin and 5-hydroxytrypamine, which are hard to distinguish using only timing techniques.
Sweedler and Fuller hope this new information will lead to breakthroughs in treating disease.



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