For biological imaging, two-photon processes can image deeper into tissue and can do so with less damage. This is because the processes use longer wavelengths, and the interaction naturally confines the imaging to a smaller volume. However, two-photon processes do not provide molecular structure information.Now researchers at the Wellman Center for Photomedicine at Harvard Medical School in Boston and at the Federal Institute for Materials Research and Testing in Berlin have demonstrated two-photon vibrational spectroscopy, a method that combines the benefits of a two-photon process with molecular information. As the name implies, surface-enhanced hyper-Raman scattering, upon which the new technique is based, is related to standard Raman scattering. When photons collide with molecules, a small number are inelastically — or Raman — scattered, and energy is transferred between the photon and molecule. The resulting frequency shift relative to the incoming light provides information about the vibrational states of the molecule and serves as a chemical fingerprint.In hyper-Raman scattering, as in other two-photon processes, two photons interact, resulting in a shift with double the energy — or half the wavelength — of the incoming photons. The signal is weak, but when the scattering takes place near metallic nanostructures, the local optical fields increase the signal by tens of orders of magnitude.Most common fluorescence dyes have a two-photon cross section — a measure of the size of the interaction — in the range of one to 300 Goeppert-Mayer (GM). With surface-enhanced hyper-Raman scattering, the researchers measured cross sections of 10,000 to 100,000 GM, which is comparable to the best results to date for two-photon fluorescence of quantum dots.As described in the Nov. 14 issue of PNAS, the researchers developed a surface-enhanced hyper-Raman scattering label, which can also be used for single-photon surface-enhanced Raman scattering. It consisted of a reporter molecule attached to a gold or silver nanoparticle linked to a unit that showed an affinity for the desired target. Researchers developed an optical label suitable for one- and two-photon excitation based on surface-enhanced Raman scattering (SERS) and surface-enhanced hyper-Raman scattering (SEHRS). The example shows SEHRS (a) and SERS (b) spectra of a solution of the dye Rose Bengal on silver nanoaggregates. The schematic of the one- and two-photon excited inelastic scattering processes is shown in the energy level diagram. The concept of the label is on the bottom (c). Metallic nanoparticle aggregates (d) boost the signal from the reporter, while the targeting unit ensures that this comes only from the intended target. Images reprinted with permission of PNAS.Testing the techniqueWhen they began, the researchers expected surface-enhanced hyper-Raman scattering to be evident when using dyes such as Rose Bengal. However, when they tried the technique with the common biomolecule adenine, they realized that it could be used for a two-photon vibrational probe of biomolecules and that the probe had a large cross section, according to Katrin Kneipp of Harvard Medical School.The researchers used aggregates made from silver and gold nanoparticles that were 20 to 50 nm in size. They used a 1064-nm picosecond laser from High Q Laser Production GmbH of Hohenems, Austria, for excitation, and a cooled CCD and a single-stage spectrograph from Horiba Jobin Yvon detected the signal after it passed through notch filters at 532 and 1064 nm to reject scattered light. These measurements showed a series of peaks in the signal that corresponded to different molecular vibrational states, similar to standard Raman shift readings.Surface-enhanced hyper-Raman scattering (SEHRS) was used to collect intrinsic biochemical information from nanometer-scale volumes in individual cultured cells using two-photon excitation. Spectra were measured from dry cells after a 4-h uptake of gold nanoparticles (a). The spectra were measured at randomly chosen locations in the cell where gold nanoparticles must have been present for surface-enhanced hyper-Raman scattering to occur. Assignment of the vibrational bands to major contributing molecules and molecular groups present in the nanometer-proximity of the gold nanoparticles is indicated below the spectra. Micrographs show fixed cells after 4 h incubation with the gold nanoparticles (right) and corresponding controls (left) (b). Scale bars: 20 μm.(Lip. = lipids, Prot. = proteins, stretch = stretching vibration, def. = deformation, rock = rocking vibration).The researchers collected data for crystal violet and Rose Bengal dyes and for adenine using light focused to a 100-μm spot size. The samples were measured with gold nanoparticles in solutions and with a final molecular concentration of 10–7 to 10–8 M. Their findings contained an unexpected, but positive, result. “The surprising outcome of these studies was that the cross section of this process, surface-enhanced hyper-Raman scattering, was unexpectedly high,” Kneipp said. “It’s even better or the same order as two-photon-excited fluorescence.” Studying cellsThe scientists also performed surface-enhanced hyper-Raman scattering studies in cells, imaging them after a four-hour uptake of gold nanoparticles. For these experiments, they focused the excitation laser to a 1-μm spot size and used a microspectroscopic setup. They detected the presence of proteins, lipids, DNA and other molecules. An important point in probing small biological structures, noted Kneipp, was that the surface-enhanced hyper-Raman scattering effect was confined to a few nanometers, producing information from a volume far smaller than is possible with techniques constrained by the diffraction limit of half a wavelength of light.As for the future, she said that surface-enhanced hyper-Raman scattering enabled the creation of highly efficient two-photon labels that could be applied to a dye or biological molecule. The technique could find an application in vibrational studies of biological tissues, much as the researchers demonstrated with living cells.There are other molecular fingerprinting methods, such as standard Raman scattering or infrared-absorption spectroscopy. Surface-enhanced hyper-Raman scattering, according to Kneipp, can provide the same information as the other two, but to acquire structure information that is as complete as possible, one should apply both methods.