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Dye Improves Insulin Studies

Photonics Spectra
May 2001
Brent D. Johnson, Senior News Editor

Insulin therapy helps to control diabetes, but a better understanding of the disease could spare diabetics a lot of pain.

Robert Kennedy, a professor of chemistry at the University of Florida in Gainesville, has investigated insulin-secreting cells using Zinquin, a fluorescent zinc dye. In normal pancreatic beta cells, when glucose enters a cell it causes the secretion of an insulin-zinc complex. He put the zinc dye in anionic form around the beta cells to image and measure the release of insulin.

appsDye1.gif
Robert Kennedy bathed pancreatic beta cells in FluoZin-3, and continuously scanned them (one image per second) with a blue laser line to generate fluorescent images. Image a is before stimulation; b and c are during K+ stimulation; and d is after stimulation. The increase in intensity in the extracellular region is clear more than 10 µm from the cell because of the probe's sensitivity.

Histidines on insulin bind with zinc.

Scientists have theorized that one of the evolutionary mechanisms that brought this about was the tendency of the insulin-zinc complex to precipitate into crystal form, which allows greater storage space. Whatever its cause, Kennedy hopes to exploit the characteristic to determine what might prevent beta cells from secreting insulin; this could lead to new therapies for diabetes.

Kyle Gee, a researcher at Molecular Probes Inc., learned about Kennedy's work in the December issue of Biophotonics International. He thought Kennedy might benefit from samples of a new fluorescent zinc dye, FluoZin-3, that he had been developing.

Kennedy tried the experimental dye and found that it dramatically improved his results. Using his Nikon RCM 8000 fast-scanning confocal microscope, he obtained fluorescence images much better than those achieved with the older dye, he said.

Signal-to-noise results are also better for several reasons. The new dye is much less cell-permeant, so background fluorescence is less than 1 percent of that in previous results because of lower interference from intracellular dye fluorescence. FluoZin-3 uses a 488-nm laser wavelength for excitation, compared with the 350-nm wavelength for Zinquin. The 488-nm excitation not only reduces background, but also causes much less damage to the cell. And the fluorescence increases more than 100-fold over baseline during insulin/zinc secretion.

Kennedy said FluoZin-3 works well for imaging extracellular secretions of zinc with insulin. His next test will be to see whether the dye can measure intracellular zinc as well.


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