Q&A: What is multiphoton microscopy?

NASA Tech Briefs, Jul 2004 by Segelken, Roger

Multiphoton microscopy, or two-photon laser microscopy, produces high-resolution, three-dimensional images of tissues (i.e. pre-cancerous cells) with minimal damage to living cells. The technique can be conducted in still-living tissue inside (in vivo) or outside (ex vivo) of the body in order to depict details of cells and cellular processes across the third and fourth (time) dimensions. Originally applied in biological research, multiphoton microscopy is making its way in to medical imaging.

The procedure begins when extremely short, intense pulses of laser light are directed at cells below the surface. The rapid-fire nature of multiphoton microscopy increases the probability that two or three photons will interact with individual biological molecules at the same time, combining their energies. The cumulative effect is the equivalent of delivering one photon with twice the energy (half the wavelength, in the case of two-photon excitation) or three times the energy (one-third the wavelength in three-photon excitation) to illuminate the smallest details. As a scanning laser microscope moves the focused beam of pulsed photons across a sample at a precise depth (plane of focus), cells above or below the plane are not affected. When repeated scans at different focal planes are "stacked" by computer processing, a detailed three-dimensional picture emerges.

The technique was co-invented by Watt W. Webb, a biophysicist who is director of the National Institutes of Health-funded Developmental Resource for Biophysical Imaging and Opto-electronics (DRBIO) at Cornell, and the S.B. Eckert Professor in Engineering. Co-inventor Winfied Denk is a director of Germany's MaxPlanck-Institute for Medical Research in Biomedical Optics.

Technology-transfer and collaborative-research agreements were recently signed by the Cornell Center for Technology, Enterprise, and Commercialization (CCTEC), and Carl Zeissjena GmbH (a maker of microscopy instrumentation) in order to help make the technique, which is protected by patents dating back to july 1991, widely available for routine use.

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This information was contributed by Roger Segelken of Cornell University. For more information, please contact Mr. Segelhm at (607) 255-9736 or hrs2@corndl.edu. Visit the Cornell Center for Technology, Enterprise, & Commercialization (CCTEC) online at www.cctec.cornell.edu. Additional information is available from the Developmental Resource for Biophysical Imaging and Opto-ekctronics (DRBIO) at wwiv. drbio. Cornell, edu.