Fiber Optic Array Provides Real-Time Gel Analysis
LUND, Sweden -- Capillary electrophoresis has become a useful analytical tool, with laser-induced fluorescence imaging offering improved detection.
Researchers from Lund University and the Lund Institute of Technology have taken it a step further, developing an optical fiber array system that allows real-time imaging of electrophoretic separations.
In standard laser-induced fluorescence imaging, both the laser and the photomultiplier tube are positioned near the end of the capillary to excite and detect fluorescence. Alternatively, researchers have experimented with imaging the length of the capillary with a camera lens system and a charge-coupled device (CCD) camera. This enables real-time detection and gives the researchers more insight into how the separations develop.
The new technique, which was detailed in the Oct. 1, 1999, issue of Analytical Chemistry, replaces the camera lenses with an array of fiber optics perpendicular to the capillary, making the setup more compact and efficient. Because the fibers can be placed closer to the capillary, the signal-to-noise ratio is improved by a factor of 10.
The setup uses either a 405-nm krypton-ion laser from Coherent Inc. of Santa Clara, Calif., or a 488-nm argon-ion laser from Spectra-Physics Inc. of Mountain View, Calif., to excite fluorescence from below the capillary. The optical fibers are positioned along the capillary. At the detection end, the fibers form a grid, which delivers the signal through a lens into a CCD camera.
A fiber optic array allows researchers to image capillary electrophoresis in real time. Courtesy of Staffan Nilsson, Lund University.
The ends of the 125-µm fibers (from Polymicro Technologies of Phoenix) are slightly rounded so that they act as tiny lenses, increasing the efficiency of the fibers themselves. The system's resolution "is quite sufficient in terms of electrophoretic
efficiency," said research leader Staffan Nilsson of Lund University's Center for Chemistry and Chemical Engineering. "Going to a smaller diameter would increase the resolution but at the expense of a lower light collection."
The system has commercial potential in capillary isoelectric focusing, Nilsson said, in which a sample must be presented to the end-point detector by force. "The focusing takes two minutes, the presentation of the result for the end-point detector takes an additional 15 to 30 minutes, where at least 30 percent of the performance is lost [to bad resolution]," he said. "Why wait for the results when you can follow the whole process in real time?"
Theoretically, the fiber optic system should improve the lens-based system by as much as a factor of 33. With this in mind, the researchers are working to create a fiber array that is wider, has a higher resolution and is more efficient at collecting light from the sample.
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