By Larry Bernard
Medical researchers who want to study the microscopic distributions of key pro teins, DNA, messenger signals, metabolic states and molecular mobility have a new tool that can show the activity and behavior of living cells under a variety of conditions.
Cornell researchers have developed new microscope technology using pulsed lasers and fluorescent markers to detect and image cellular activity with sensitivity to detect and recognize tens of individual molecules in focal volumes as small as 1/10th of a millionth of a millionth of a sugar cube. These advanced microscopes can reveal fun damental biological processes in living cells -- metabolism, wound healing, behavior of malignant cells and nerve communication -- opening a new world for investigators of biological systems.
Watt W. Webb, Cornell professor of applied physics, described the technology Feb. 9 at the annual AAAS meeting in a "Topical Lecture on Science Innovation" presenta tion titled "Non-Linear Laser Microscopy."
"We have the ability now to image dynamics of specific molecular distributions and signals in living cells with a sensitivity and diversity that heretofore was unattain able, without disruption of life processes," Webb said. "This gives us a valuable and remarkably benign new tool for a host of biomedical investigations. Because there is no excitation of the tissue outside the focal area, cells tolerate repeated images of pro tein auto-fluorescence."
The technology works like this: A scanned laser in the 700 to 900 nanometer range (deep red to infrared) fires very short pulses (10 -13 seconds, or 100 millionths of a billionth of a second duration) focused by the microscope so that two or three photons arrive at the same time (10 -16 seconds, or less than a millionth of a billionth of a second) at a molecule, and excite the fluorescence of the molecule relevant to biological activity. The sample emits the fluorescence photons, producing a three-dimensional image. Pho tons are collected and the resulting three-dimensionally digital image can be viewed and analyzed on a computer monitor.
"No one realized what a wide range of light wavelengths would excite fluorescent molecules by two-photon absorption because the physical measurements of the excitation were difficult. Now we have found new and easy ways of obtaining the molecular data we needed for non-linear microscopy," Webb said. Chris Xu, a graduate student in Webb's laboratory, solved this problem and perfected the method in collaboration with Winfried Denk of AT&T Bell Labs.
"You can excite the native auto-fluorescence of living tissue," said Webb, a Fellow of the AAAS and a member of the National Academy of Sciences and the National Academy of Engineering. Two-photon excitation of mitochondrial NADH molecules provides a measure of metabolic state of cells. Three-photon excitation with red laser light can be used to image the activity of key proteins, particularly those containing the amino acid tryptophan that ordinarily absorbs only deep ultraviolet light.
"We can map signal proteins through the ultraviolet fluorescence of tryptophan and detect secretory granules containing serotonin and other neurotransmitters to study their role in communication amongst cells," Webb said.
Webb and his colleagues also are adapting the technology to image fluorescent markers and signal indicators deep into tissues. Thick-tissue penetration has been re markably successful reaching the half-millimeter range. Two-photon excitation can image antibody labels through the depth of human skin, in order to examine effects of damage and aging, and chromosomes and mitochondria can be imaged simultaneously deep in living flower buds where pollen grains are formed in order to study conse quences of genetic mutations.
Webb, who invented the technology in 1989 with Denk and Jim Strickler, now at McKinsey Co., has been developing user -friendly instrumentation and methods as well as using it for biophysical investiga tions for the last five years with pre- and post-doctoral students. Cornell holds the patent on the technology, which is available for licensing. Webb also is director of Cornell's Developmental Resource for Bio physical Imaging and Opto-electronics, funded by the National Institutes of Health and the National Science Foundation.
He credits a long line of students for helping develop the technology he described: Ed Brown, Ingrid Brust-Mascher, Winfried Denk, Jeff Guild, Sudipta Maiti, Jerome Mertz, Jen nifer Nichols, Dave Piston, Jason Shear, Becky Williams, Chris Xu and Warren Zipfel. Webb gratefully acknowledges biological research collaborators Kathy Conley, Reiner Kohler and Maureen Hanson of Cornell's Department of Genetics and Development; Jim O'Malley and Mika Salpeter of Cornell's neurobiology program; Kevin Yuan of Unilever Research; Jon Lederer of the University of Maryland School of Medicine; Barry Masters of Uniform Services University of the Health Sciences; and Bob Summers of the State University of New York at Buffalo.
Other applications include:
· Imaging chromosomes in living tumor cells, in developing sea urchin embryos and in growing petunia buds. Cell divisions have been successfully followed through many generations, yielding insights into development control.
·The technology to study "sex in plants," by examining the cells where pollen grains form in the flower bud, in an effort to learn why certain mutations cause male sterility.
·The ability to "watch" the cellular ac tivity of heart muscle cells under stimulation gives researchers a new way to study heart disease.
·Applications to eye surgery in which optical inspection of corneal cells is re stricted, to evaluate damage and recovery.
A sample image is available at <http://www.news.cornell.edu/AAAS96 /cellphoto.html>.