Laser Microscopy Technique Reduces Sample Damage

Daniel J. Dufresne

SAN JOSE, Calif. -- Breakthroughs in a relatively new laser-based microscopy technique have produced high-resolution images of living specimens with relatively little photodamage. The less harmful nature of the infrared light it uses makes the technique, called multiphoton excitation, more useful than earlier excitation techniques using confocal microscopy in many areas of bioresearch.
Cornell University physicist Watt Webb announced to a standing-room-only audience at the SPIE Biomedical Optics '97 conference last month that his team has successfully harnessed short-pulse infrared laser light to induce fluorescence in living cell specimens using a six-photon excitation technique. Webb is withholding further discussion of the six-photon excitation success until after his paper on the subject has been published, but his previous publications on two- and three-photon excitation offer a glimpse into the technique.
In the Jan. 24 issue of Science, Webb's team wrote that it achieved three-photon excitation by illuminating tissue samples with short, intense bursts of light from a Spectra-Physics Tsunami mode-locked Ti:sapphire laser. The pulses are so short that photon triplets (i.e., groups of three photons) strike the focal point over a brief enough time span to be considered simultaneous according to quantum theory. These triplets produce three times the energy (and frequency) of the photons that the laser emitted.
The group used three-photon fluorescence microscopy to quantify concentrations of the neurotransmitter serotonin in granules of live rat leukemia cells. Serotonin fluoresces without the aid of a dye when illuminated at about 200 nm, and photon triplets within pulses of the Tsunami's 700-nm light produced this autofluorescence.

A less destructive technique

Multiphoton excitation localizes light energy in the focal plane, so there is less photodamage than in conventional confocal fluorescence microscopy, which bombards specimens with high-intensity, short-wavelength light. The three-photon technique appears to be less destructive than two-photon excitation; no comparison was available yet for the six-photon technique.
Another benefit of multiphoton excitation is its use of infrared lasers for the initial pulses: Infrared light penetrates tissue better than other wavelengths can.
Science reported that Cornell has granted Bio-Rad Laboratories of Hercules, Calif., a license to develop a commercial multiphoton excitation instrument and reported that such instruments are estimated to cost several hundred thousand dollars with current laser technology.