BALTIMORE -- Medical researchers who want to study the microscopic distributions of key proteins, 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 University 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 fundamental 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 today (Feb. 9) at the annual meeting of the American Association for the Advancement of Science (AAAS) in a Topical Lecture on Science Innovation 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 unattainable, 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 protein autofluorescence."

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. Photons 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 remarkably 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 consequences 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 investigations 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 Biophysical 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, Jennifer 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 of two-photon laser scanning microscopy 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. Webb's collaborators have imaged cell division of a sea urchin embryo from its first formation until the new animal swims away.
• 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 activity 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 restricted, to evaluate damage and recovery.

"Current developments in this area include high sensitivity real-time imaging," Webb said. "The forthcoming small, low- cost femtosecond lasers should make the technology more economical. The technology is ready for general use and we want to license an industrial manufacturer capable of making the instrumentation available. New applications include 3-D resolved second messenger signal propagation in cells, imaging of biochemical constituents of structure in thick tissue and monitoring cellular metabolism and genetic change in response to cancer therapy."

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Photo caption: A dividing rat basophil leukemia cell in early anaphase (center), imaged by a two-photon laser scanning microscope invented at Cornell. The cell structures are labeled with different colored dyes, excited by two photon excitation: blue is plasma membrane; white is DNA; red is mitochondria. The image was reconstructed using IBM's Data Explorer software on the IBM SP2 parallel processor supercomputer at the Cornell Theory Center. EDITORS: After Feb. 12, Watt W. Webb can be reached at (607) 255-3331.