HOLD FOR RELEASE: THURSDAY, JAN.
23, 1997, 4 P.M. EST
Contact: Larry Bernard
Office: (607) 255-3651
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http://www.news.cornell.edu
Laser Microscope Images Serotonin in Live Cells
ITHACA, N.Y. -- Cornell University researchers, using non-linear
laser-microscope technology developed at Cornell, have produced images
displaying the neurotransmitter serotonin in live cells in real time, and
they have for the first time measured the concentration of serotonin in
secretory granules.
The microscope, which uses pulsed lasers for excitation, can record
ultraviolet (UV) fluorescence images of live cells without using UV
illumination to detect and image cellular activity.
"This technique caught serotonin granules in the act of releasing the
substance, without damaging the cell or changing the process and without
requiring any external fluorescent marker," said Watt W. Webb, Cornell
professor of applied and engineering physics, who led the work. "It is a
new way of doing three-dimensional UV microscopy in functioning cells and
the best way we know of for doing UV microscopy in thick tissues," Webb
said.
The studies were reported in a paper, "Measuring serotonin distribution in
live cells with three-photon excitation," published in the journal Science
(Jan. 24, 1997) by Webb and Sudipta Maiti, a postdoctoral researcher; Jason
B. Shear, former National Science Foundation post doctoral fellow now at
the University of Texas at Austin; Rebecca M. Williams, physics graduate
student; and Warren R. Zipfel, a research associate, all at Cornell.
Serotonin is a neurotransmitter that is becoming increasingly important as
medical science learns of its role in a host of human disorders. It has
been implicated in central nervous system disorders such as anxiety,
depression, obsessive-compulsive disorder, schizophrenia, stroke, obesity,
pain, hypertension, vascular disorders, migraine and even nausea.
Serotonin is synthesized in brain neurons and is released upon a nerve
impulse, where it interacts with receptors. The antidepressant Prozac
(fluoxetine) is thought to treat depression by inhibiting re-uptake of
serotonin into cells from which secretion occurs.
The Cornell technology may be useful in gaining an understanding of a broad
spectrum of physiological and psychological effects of this -- and other --
neurotransmitters. The technology could be useful, then, for researchers
in designing more effective drugs for a host of disorders.
Previous efforts to image neurotransmitter secretory granules have not
directly detected the neurotransmitter content or allowed visualization of
secretory processes. But the Cornell technology, called non-linear laser
scanning microscopy, can detect and image the serotonin and measure its
concentration and the total neurotransmitter content of individual granules
in intact cells.
Here is how it works: A laser in the 700 to 750 nanometer wavelength
(infrared) fires photons bunched in very short pulses (each 10-13 seconds
or a 10 millionth of a millionth of a second), which are focused by the
microscope so that there is a high probability that three photons arrive at
the same time (in about 10-16 seconds) at the same molecule near the focus.
Molecules such as serotonin and tryptophan, which can normally be excited
only with deep ultraviolet (~250nm) illumination, are now excited by
simultaneously absorbing three infrared photons and, subsequently,
fluorescence in the UV. These photons are collected as the laser is
scanned through the specimen, and the resulting 3-D image can be viewed and
analyzed on a computer monitor. For these studies, the researchers used
basal leukemia cells from rats.
"This three-photon excitation produces very high energy corresponding to
shorter absorption wavelengths than was possible before, without killing
the cells," Webb said. "All you could see before this technique was
diffuse brightness increase attributable to serotonin. But now we can see
individual granules and we have a way to measure the serotonin in active
cells."
Maiti, a postdoctoral associate, said that serotonin absorbs wavelengths
below 250 nanometers, a wavelength that is far shorter than the human eye
can see or than can penetrate tissue without damage. So quantitative
measurement of serotonin in live cells had not been possible. "There are
molecules that act like pumps. They concentrate serotonin within a cell.
We found very high concentrations, about 50 millimolar, in the secretory
granules," he said.
Williams is following the release of serotonin from a cell as it occurs
after allergic stimulation. "The larger granules secrete faster. Once the
serotonin is secreted it is available in the tissue to stimulate swelling
and fluid release," she said. The researchers used a pollen-like antigen
to add to the solution that turned the signal on in the cell. As that was
happening, the laser was illuminating the process to visualize the
granules.
Webb said he hopes to use the technology to look for serotonin secretions
deep inside the brain. The technology could be used for studies of the
nervous system, not just the immune system or allergic response, but to a
broad range of areas.
Webb invented the technology for scanning laser microscopy in 1989 with
Winfried Denk, now at AT&T Bell Labs, and Jim Strickler, now at McKinsey
Co., and has been using it since for biophysical investigations with pre-
and post-doctoral students. Cornell holds a patent on the technology,
which recently has been licensed to Bio-Rad Laboratories of California.
Webb directs Cornell's Development Resource for Biophysical Imaging and
Optoelectronics, funded by the National Institutes of Health and National
Science Foundation. For 3-D image reconstruction, the researchers used the
IBM SP2 supercomputer at the Cornell Theory Center, part of the NIH
Parallel Processing Resource for Biomedical Scientists.
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EDITORS: A microscope photo is available at
http://www.news.cornell.edu/science/Jan97/serotonin2.lb.html
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