Two-photon excitation is gaining rapidly in interest and significance in spectroscopy and microscopy. Here we introduce a new approach that suggests versatile optical labels suitable for both one-and two-photon excitation and also two-photon-excited ultrasensitive, nondestructive chemical probing. The underlying spectroscopic effect is the incoherent inelastic scattering of two photons on the vibrational quantum states called hyper-Raman scattering (HRS). The rather weak effect can be strengthened greatly if HRS takes place in the local optical fields of gold and silver nanostructures. This so-called surface-enhanced HRS (SEHRS) is the two-photon analogue to surface-enhanced Raman scattering (SERS). SEHRS provides structurally sensitive vibrational information complementary to those obtained by SERS. SEHRS combines the advantages of two-photon spectroscopy with the structural information of vibrational spectroscopy and the high-sensitivity and nanometer-scale local confinement of plasmonics-based spectroscopy. We infer effective two-photon cross-sections for SEHRS on the order of 10 ؊46 to 10 ؊45 cm 4 ⅐s, similar to or higher than the best ''action'' cross-sections (product of the two-photon absorption cross-section and fluorescence quantum yield) for two-photon fluorescence, and we demonstrate HRS on biological structures such as single cells after incubation with gold nanoparticles.local optical fields ͉ plasmonics ͉ Raman spectroscopy ͉ two-photon spectroscopy ͉ biological structures T he probing of electronic or vibrational states in molecules by two photons provides several advantages over one-photon excitation, including the application of light of a longer wavelength and the limitation of the excitation volume in a sample (1, 2). These specific characteristics of two-photon excitation are of particular interest for biological applications of spectroscopy and microscopy. The development of optical probes that are suitable for two-photon excitation is therefore an important task in advancing optical techniques for biological applications. In addition, in biospectroscopy, another challenge is to obtain molecular structural information on biological samples and the development of methods to achieve this at high sensitivity, high lateral resolution in situ or in vivo, and without the need for purification.In general, by the inelastic Raman scattering process, molecular vibrations and thereby molecular structure, composition, and interactions, can be detected by the observation of scattered light with a shifted wavelength compared with the wavelength of an excitation source. The Raman shift corresponds to the energy of the molecular vibration with which an excitation photon interacts. The fingerprint-like Raman spectral information has been used to study biological samples for decades and has gained popularity in particular in combination with microscopy, where it is used for multiparameter imaging of cells and tissues based on their intrinsic molecular composition (3-5). Hyper-Raman scattering (HRS), illustrat...