Using picosecond excitation at 1064 nm, surface-enhanced hyper-Raman scattering (SEHRS) spectra of the nucleobases adenine, guanine, cytosine, thymine, and uracil with two different types of silver nanoparticles were obtained. Comparing the SEHRS spectra with SERS data from the identical samples excited at 532 nm and with known infrared spectra, the major bands in the spectra are assigned. Due to the different selection rules for the one- and two-photon excited Raman scattering, we observe strong variation in relative signal strengths of many molecular vibrations obtained in SEHRS and SERS spectra. The two-photon excited spectra of the nucleobases are found to be very sensitive with respect to molecule–nanoparticle interactions. Using both the SEHRS and SERS data, a comprehensive vibrational characterization of the interaction of nucleobases with silver nanostructures can be achieved.

Surface-enhanced hyper-Raman scattering (SEHRS) and surface-enhanced Raman scattering (SERS) of para-mercaptobenzoic acid (pMBA) were studied with an excitation wavelength of 1064 nm, using different silver nanostructures as substrates for both SEHRS and SERS. The spectra acquired for different pH values between pH 2 and pH 12 were compared with SERS data obtained from the identical samples at 532 nm excitation. Comparison of the ratios of the enhancement factors from SEHRS and SERS experiments with those from calculations using plasmonic absorbance spectra suggests that the difference between total surface-enhancement factors of SEHRS and SERS for pMBA is mainly explained by a difference between the electromagnetic contributions for linear and non-linear SERS. SERS and SEHRS spectra obtained at near-infrared (NIR) excitation indicate an overall reduction of enhancement by a factor of 2-3 at very low and very high pH, compared to neutral pH. Our data provide evidence that different molecular vibrations and/or different adsorption species are probed in SERS and SEHRS, and that SEHRS is very sensitive to slight changes in the pMBA-nanostructure interactions. We conclude that the combination of SEHRS and SERS using NIR excitation is more powerful for micro-environmental pH sensing than one-photon spectra excited in the visible range alone.

We observed strong surface-enhanced Raman scattering on discontinuous nanostructured aluminum films using 785 nm excitation even though dielectric constants of this metal suggest plasmon supported spectroscopy in the ultraviolet range. The excitation of SERS correlates with plasmon resonances in the 1.3-2.5 eV range identified in electron energy loss spectra.

We investigate distributions of crystal violet and malachite green on plasmonic surfaces by principal component analysis (PCA) imaging of surface-enhanced hyper-Raman scattering (SEHRS) data.

scattering, that is, spontaneous, two-photon excited Raman scattering, of organic molecules becomes strong when it occurs as surface-enhanced hyper Raman scattering (SEHRS), in the proximity of plasmonic nanostructures. Its advantages over one-photon excited surface-enhanced Raman scattering (SERS) include complementary vibrational information resulting from different selection rules, probing of very small focal volumes, and beneficial excitation with long wavelengths. Here, imaging of macrophage cells by SEHRS is demonstrated, using SEHRS labels consisting of silver nanoparticles and two different molecules, 2-naphthalenethiol and para-mercaptobenzoic acid, that are excited offresonance. The vibrational signatures of the molecules are discriminated using hyperspectral analysis and provide information about the subcellular localization of the SEHRS probes. The SEHRS based hyperspectral imaging approach presented here uses principal component analysis (PCA) to localize the reporter molecules inside the cells and is augmented by hierarchical cluster analysis (HCA). The high sensitivity of SEHRS spectra with respect to small environmental changes can be utilized for mapping of physiological parameters in the endosomal system of the cells. This is illustrated by discussing the spatial distribution of endosomes of varying pH inside the cytosol.