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 Feb. 9 at the annual meeting of the American Association for the Advancement of Science (AAAS) in Baltimore 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."

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 3D digital image can be viewed and analyzed on a computer monitor.

"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," said Webb, who invented the technology in 1989 with Winfried Denk of AT&T Bell Labs and Jim Strickler, now at McKinsey Co.

Other uses: to examine effects of damage and aging on skin; and chromosomes and mitochondria can be imaged simultaneously deep in living flower buds where pollen grains are formed to study consequences of genetic mutations. Webb has been developing user-friendly instrumentation and methods 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.