When researchers at the University of California teamed a near-infrared laser with a near-field scanning optical microscope, they created a high-resolution imaging technique with chemical specificity. The new system combines an atomic force microscope's high surface resolution with the ability to produce optical images that overcome the diffraction limit of light. By tuning the laser to a wavelength three times longer than a given chemical's absorption band, the researchers excited the target in much the same way as if they had used light at the actual absorption wavelength, but in a three-photon process. "Three photons of equal energy are mixed together in the sample to produce a single photon of three times the energy, which is detected," said Richard D. Schaller, a member of the research group led by Richard J. Saykally. To test whether the technique probes the bulk properties of a sample or only the surface, the scientists tested red blood cells. They used a near-field scanning optical microscope for imaging and an optical parametric amplifier, pumped by a femtosecond-pulse, 800-nm Ti:sapphire laser, as the excitation source. "Oxyhemoglobin has an absorption band centered around 420 nm," Schaller said. "So when we tune our laser to 1260 nm, we observe a stronger signal from the red blood cell than from the surrounding glass [slide]." This indicated that the third-harmonic technique probes the bulk properties, he explained, because the oxyhemoglobin is in the bulk of the blood cells. End of staining Schaller said that the researchers have not yet conducted experiments to determine the sensitivity of the technique, which they reported in the Nov. 1, 2000, issue of Analytical Chemistry. They are exploring the chemical physics of the method and have used it to image several different samples, including purple bacteria and spinach chloroplasts. The imaging technique may prove useful in biological science as a way to heighten optical contrast without using fluorescent markers that may affect the behavior of the sample. The longer wavelengths also are less likely than two-photon fluorescence to damage fragile biological samples. "It can make biological staining unnecessary," Schaller said.