Advantages of Two-Photon Imaging
Dr. James P. Smith
BUFFALO, N.Y. -- During the past decade, photon scanning tunneling microscopy has achieved resolution better than 100 nm, overcoming the optical diffraction limits. Recently, scientists at the State University of New York at Buffalo developed a two-photon system that has better signal-to-noise ratio and optical contrast than the one-photon microscopes.
Photon scanning tunneling microscopy is an optical fluorescence method in which a laser excites one side of a thin sample while a fiber probe scans the opposite surface and collects the emitted photons. The aluminum-coated fiber probe has an apex diameter of about 200 nm. As with other scanning probe microscope systems, it is rastered using a piezo-tube scanner while its tip remains a few nanometers above the sample surface.
A Hamamatsu R943-02 photomultiplier detects the photons that are collected by the probe. The photons are processed to create an image or sent to a Kaiser Optical Systems spectrograph equipped with a Princeton Instruments CCD camera. The excitation source is an 800-nm Ti:sapphire laser with an average of 12 mW. The laser beam passes through a prism, reflecting from the surface below the sample. The beam focuses on the sample, which is mounted with an index-matching oil on the prism.
Researchers at the State University of New York at Buffalo are using an experimental setup such as this for two-photon fluorescence imaging and spectroscopy. The system can be used to spatially and spectrally probe emitting regions on the nanometer scale.
According to team leader Paras N. Prasad, two-photon imaging has many advantages over one-photon imaging. One is that you can get better spatial resolution because of the quadratic density dependence of a two-photon process. A single-photon process can collect information only very close to the surface. "We can look through the layers of paint, for example, all the way to the substrate. We can see corrosion, delamination and other effects," he said.
Some materials can absorb two photons to create an excited state, and a photon emitted after the absorption of two near-infrared photons can be in the visible or ultraviolet range. Most substances do not fluoresce when exposed to near-infrared radiation, so they do not contribute to the background in a two-photon setup.
"One-photon excitation also results in significant spreading of excitation, and this limits resolution," Prasad said. Two-photon scanning tunneling microscopy also has lower sample damage from near-IR radiation, he added.
Prasad's group, whose results were published in the Jan. 3 issue of Applied Physics Letters, is concentrating on the development of other two-photon-absorbing materials, including multibranched large molecular compounds, dyes, erbium-doped nanoparticles and semiconductor quantum dots. Prasad also plans to develop a kit that can convert a conventional optical microscope into a two-photon laser scanning microscope at an affordable price.
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