We report two-photon excited nonresonant surface-enhanced hyper Raman scattering (SEHRS) spectra of tricyclic antidepressant (TCA) molecules during their interaction with biocompatible gold nanostructures and with silver nanostructures. The SEHRS spectra of amitriptyline, desipramine, and imipramine are compared with surface-enhanced Raman scattering (SERS) spectra on both kinds of nanoparticles, obtained with excitation at 532 and 785 nm. The SEHRS spectra of the TCA molecules show several intense contributions by infrared-active vibrations. Combining SEHRS with SERS therefore enables a comprehensive vibrational characterization of the interaction of the molecules with the nanostructures. SEHRS and SERS data indicate that the molecules interact with the silver nanostructures mainly via their ring moiety. In contrast, in the interaction with gold, the methylaminopropyl side chain plays a very important role, along with parts of the ring system. It is possible to obtain the spectra of the molecules with near-infrared excitation and with gold nanoparticles in cell culture media. The spectral signatures of the drug molecules collected at low pH values characteristic of late endosomal stages or of acidified tissues are very stable and show only small changes in the interaction of the TCA with the gold nanoparticles. The results will help to develop tools for the characterization of new nanoparticle-based drug delivery platforms in real biological environments.

The mechanism of the plasmon-catalyzed reaction of p-aminothiophenol (PATP) to 4,4′-dimercaptoazobenzene (DMAB) on the surface of metal nanoparticles has been discussed using data from surface-enhanced Raman scattering of DMAB. Oxides and hydroxides formed in a plasmon-catalyzed process were proposed to play a central role in the reaction. Here, we report DMAB formation on gold nanoparticles occurring in the presence of the metal cations Ag+, Au3+, Pt4+, and Hg2+. The experiments were carried out under conditions where formation of gold oxide or hydroxide from the nanoparticles can be excluded and at high pH where the formation of the corresponding oxidic species from the metal ions is favored. On the basis of our results, we conclude that, under these conditions, the selective oxidation of PATP to DMAB takes place via formation of a metal oxide from the ionic species in a plasmon-catalyzed process. By evidencing the necessity of the presence of the metal cations, the reported results underpin the importance of metal oxides in the reaction.Keywords: 4,4′-dimercaptoazobenzene; metal ions; p-aminothiophenol; plasmonic catalysis; surface-enhanced Raman scattering;

Hyper Raman 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 off-resonance. 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.

Surface enhanced hyper Raman scattering (SEHRS) is the spontaneous, two-photon excited Raman scattering that occurs for molecules residing in high local optical fields of plasmonic nanostructures. Being regarded as a non-linear analogue of surface enhanced Raman scattering (SERS), SEHRS shares most of its properties, but also has additional characteristics. They include complementary spectroscopic information resulting from different selection rules and a stronger enhancement due to the non-linearity in excitation. In practical spectroscopy, this can translate to advantages, which include a high selectivity when probing molecule–surface interactions, the possibility of probing molecules at low concentrations due to the strong enhancement, and the advantages that come with excitation in the near-infrared. In this review, we give examples of the wealth of vibrational spectroscopic information that can be obtained by SEHRS and discuss work that has contributed to understanding the effect and that therefore provides directions for SEHRS spectroscopy. Future applications could range from biophotonics to materials research.

Gold nanostructures that serve as probes for nanospectroscopic analysis of eukaryotic cell cultures can be obtained by the in situ reduction of tetrachloroauric acid (HAuCl4). To understand the formation process of such intracellularly grown particles depending on the incubation medium, the reaction was carried out with 3T3 fibroblast cells in three different incubation media, phosphate buffer, Dulbecco's Modified Eagle Medium (DMEM), and standard cell culture medium (DMEM with fetal calf serum). The size, the optical properties, the biomolecular corona, and the localization of the gold nanoparticles formed in situ vary for the different conditions. The combination of surface-enhanced Raman scattering (SERS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) microscopic mapping and transmission electron microscopy (TEM) provides complementary perspectives on plasmonic nanoparticles and non-plasmonic gold compounds inside the cells. While for the incubation with HAuCl4 in PBS, gold particles provide optical signals from the nucleus, the incubation in standard cell culture medium leads to scavenging of the toxic molecules and the formation of spots of high gold concentration in the cytoplasm without formation of SERS-active particles inside the cells. The biomolecular corona of nanoparticles formed in situ after incubation in buffer and DMEM differs, suggesting that different intracellular molecular species serve for reduction and stabilization. Comparison with data obtained from ready-made gold nanoparticles suggests complementary application of in situ and ex situ generated nanostructures for optical probing.

Surface-enhanced Raman scattering (SERS) probes and labels exploit surface-enhanced Raman signatures of specifically chosen reporter molecules and intrinsic biomolecules in the plasmonic near field of gold and silver nanostructures. They offer several advantages over other optical labels and sensors with respect to sensitivity and selectivity, mobility, and biocompatibility. Here, the multifunctionality and versatility of SERS labels that come from the vibrational spectroscopic information and their ability to act as nanoprobes of a biological environment are discussed. Surface-enhanced Raman scattering probes have the potential to become the next-generation sensor technology for monitoring cells and tissues. They improve our understanding of cellular function and will play a major role in future theranostic applications.

In this work, we report nonresonant surface-enhanced hyper-Raman (SEHRS) spectra of the amino acids tryptophan, histidine, phenylalanine, and tyrosine using silver nanoparticles. The spectra are obtained at an excitation wavelength of 1064 nm and compared to the corresponding surface-enhanced Raman scattering (SERS) spectra measured at 532 nm excitation. The majority of the bands in the SEHRS spectra are assigned. Important hallmarks of the spectra include strongly diminished or absent bands from the ring breathing modes. SEHRS and SERS spectra obtained from histidine and tyrosine indicate changes at slightly varied amino acid concentration. Small changes in the SEHRS spectra were more pronounced than variation in the corresponding SERS data, supporting the high sensitivity of the SEHRS spectra with respect to structural changes due to small variations in surface environment. The possibility to measure nonresonant SEHRS spectra of amino acids in solution and the complementary information obtained from the spectra demonstrates the potential of this method for future investigations of proteins and more complicated biological structures and their interaction with nanostructures.

Multifunctional composite nanoprobes consisting of iron oxide nanoparticles linked to silver and gold nanoparticles, Ag–Magnetite and Au–Magnetite, respectively, were introduced by endocytic uptake into cultured fibroblast cells. The cells containing the non-toxic nanoprobes were shown to be displaceable in an external magnetic field and can be manipulated in microfluidic channels. The distribution of the composite nanostructures that are contained in the endosomal system is discussed on the basis of surface-enhanced Raman scattering (SERS) mapping, quantitative laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping, and cryo soft X-ray tomography (cryo soft-XRT). Cryo soft-XRT of intact, vitrified cells reveals that the composite nanoprobes form intra-endosomal aggregates. The nanoprobes provide SERS signals from the biomolecular composition of their surface in the endosomal environment. The SERS data indicate the high stability of the nanoprobes and of their plasmonic properties in the harsh environment of endosomes and lysosomes. The spectra point at the molecular composition at the surface of the Ag–Magnetite and Au–Magnetite nanostructures that is very similar to that of other composite structures, but different from the composition of pure silver and gold SERS nanoprobes used for intracellular investigations. As shown by the LA-ICP-MS data, the uptake efficiency of the magnetite composites is approximately two to three times higher than that of the pure gold and silver nanoparticles.

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. As a two-photon excited Raman scattering process, SEHRS provides chemical structure information based on molecular vibrations, but follows different selection rules than the normal, one-photon excited surface-enhanced Raman scattering (SERS). Therefore, simultaneous hyperspectral mapping using SEHRS excited at 1064 nm and SERS excited at 532 nm improves spatially resolved multivariate discrimination based on complementary vibrational information. The possibility to map distributions of the structurally similar dyes crystal violet and malachite green demonstrates the potential of this approach for multiplex imaging of complex systems.

Porous MgF2-over-nanoparticles (MON) surfaces are fabricated from immobilized gold nanoparticles of different sizes on a glass surface by coating them with a magnesium fluoride layer. High mechanical stability of the resulting plasmonic surface is obtained, and optical spectroscopy across a very wide optical window is enabled. The nanoscopic characterization by scanning force microscopy and electron microscopy shows a uniform assembly of the gold nanoparticles in monolayers and a complete coating by magnesium fluoride. Surface-enhanced Raman scattering (SERS) experiments provide evidence that organic analyte molecules have free access to the gold surface, and interact with the immobilized nanoparticles in a very similar fashion as with uncoated surfaces. As the spectroscopic results indicate, the coating leads to properties that are favourable for plasmonic enhancement of optical processes excited in the visible and near-infrared. As demonstrated by experiments using SERS, as well as by finite difference time domain (3D-FDTD) simulations, enhancement factors are obtained that allow for analytical applications with optical excitations ranging from the visible to the near infrared.

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.

A thiol-modified carotene, 7′-apo-7′-(4-mercaptomethylphenyl)-β-carotene, was used to obtain nonresonant surface-enhanced Raman scattering (SERS) spectra of carotene at an excitation wavelength of 1064 nm, which were compared with resonant SERS spectra at an excitation wavelength of 532 nm. These spectra and surface-enhanced hyper-Raman scattering (SEHRS) spectra of the functionalized carotene were compared with the spectra of nonmodified β-carotene. Using SERS, normal Raman, and SEHRS spectra, all obtained for the resonant case, the interaction of the carotene molecules with silver nanoparticles, as well as the influence of the resonance enhancement and the SERS enhancement on the spectra, were investigated. The interaction with the silver surface occurs for both functionalized and nonfunctionalized β-carotene, but only the stronger functionalization-induced interaction enables the acquisition of nonresonant SERS spectra of β-carotene at low concentrations. The resonant SEHRS and SERS spectra are very similar. Nevertheless, the SEHRS spectra contain additional bands of infrared-active modes of carotene. Increased contributions from bands that experience low resonance enhancement point to a strong interaction between silver nanoparticles and electronic levels of the molecules, thereby giving rise to a decrease in the resonance enhancement in SERS and SEHRS.

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 correlate the localization of silver nanoparticles inside cells with respect to the cellular architecture with the molecular information in the vicinity of the particle surface by combining nanoscale 3D cryo-soft X-ray tomography (cryo-SXT) with surface-enhanced Raman scattering (SERS). The interaction of the silver nanoparticle surface with small molecules and biopolymers was monitored by SERS in vitro over time in living cells. The spectra indicate a stable, time-independent surface composition of silver nanoparticles, despite the changing environment in the endosomal structure. Cryo-SXT reveals a characteristic ring-shaped organization of the silver nanoparticles in endosomes of different cell types. The ring-like structures inside the endosomes suggest a strong association among silver particles and with membrane structures. The comparison of the data with those obtained with gold nanoparticles suggests that the interactions between the nanoparticles and with the endosomal component are influenced by the molecular composition of the corona.

The interaction of nanoparticles with hemoglobin (Hb), a major constituent of red blood cells, is important in nanotoxicity research. We report SERS spectra of Hb using gold and silver nanoparticles at very small nanoparticle:Hb molecule ratios, that is, under conditions relevant for SERS-based nanotoxicity experiments with red blood cells at high sensitivity. We show that the structural information obtained from the experiment is highly dependent on the type of SERS substrate and the conditions under which the interaction of nanoparticles with Hb molecules takes place. In experiments with isolated red blood cells, we demonstrate that the dependence of the spectra on the type of nanoparticle used as the SERS substrate extends to whole red blood cells and red blood cell components. Regarding the applicability of SERS to red blood cells in vivo, evidence is provided that the molecular information contained in the spectra is highly dependent on the material and size of the nanoparticles. The results indicate specific interactions of gold and silver nanoparticles with Hb and the red blood cell membrane, and reflect the hemolytic activity of silver nanoparticles. The results of this study help improve our understanding of the interactions of silver and gold nanoparticles with red blood cells.

The interaction of nanomaterials with biomolecules, cells, and organisms plays an important role in cell biology, toxicology, and nanotechnology. Spontaneous Raman scattering can be used to probe biomolecules, cells, whole animals, and nanomaterials alike, opening interesting avenues to study the interaction of nanoparticles with complex biological systems. In this review we discuss work in biomedical Raman spectroscopy that has either been concerned directly with nanostructures and biosystems, or that indicates important directions for successful future studies on processes associated with nano-bio-interactions.

Gold and silver nanoparticles can be immobilized on glass slides using aminosilane linkers. Here, we demonstrate that particle monolayer surfaces can also be generated by simultaneous immobilization of both gold and silver nanoparticles with the same organosilane linker. These new surfaces display surface-enhanced Raman scattering (SERS) enhancement typical for gold or silver monolayers, depending on the ratio of the two types of nanoparticles and, at the same time, have the capability to probe complex analytes composed from various molecules which adsorb at only one of the metals. The reported results from scanning electron microscopy, scanning force microscopy, and UV/vis absorbance for surfaces containing one or two types of nanoparticles indicate that an enhancement level above 104 is related to nanoaggregates that form in the 2D plane. High and stable enhancement factors over a wide range of analyte concentrations along with high homogeneity of the enhancement at the microscopic scale make the plasmonic nanoparticle mix-and-match surfaces ideal substrates for use in microscopic SERS sensing.

Silver nanoparticles were generated based on citrate reduction in the ultrastructure of the sporopollenin biopolymer of Ambrosia artemisiifolia (ragweed) and Secale cereale (rye). The nanoparticles enable the acquisition of SERS spectra and thereby a vibrational characterization of the local molecular structure of sporopollenin.

Surface-enhanced Raman scattering (SERS) labels and probes consisting of gold and silver nanoaggregates and attached reporter molecules can be identified by the Raman signature of the reporter molecule. At the same time, SERS hybrid probes deliver sensitive molecular structural information on their nanoenvironment. Here we demonstrate full exploitation of the multifunctional and multiplexing capabilities inherent to such nanoprobes by applying cluster methods and principal components approaches for discrimination beyond the visual inspection of individual spectra that has been practiced so far. The reported results indicate that fast, multivariate evaluation of whole sets of multiple probes is feasible. Spectra of five different reporters were shown to be separable by hierarchical clustering and by principal components analysis (PCA). In a duplex imaging approach in live cells, hierarchical cluster analysis, K-means clustering, and PCA were used for imaging the positions of different types of SERS probes along with the spectral information from cellular constituents. Parallel to cellular imaging experiments, cytotoxicity of the SERS hybrid probes containing aromatic thiols as reporters is assessed. The reported results suggest multiplexing applications of the nontoxic SERS nanoprobes in high density sensing and imaging in complex biological structures.Keywords: 2-naphthalenethiol; 3T3 cells; cytotoxicity; hierarchical cluster analysis; imaging; nanosensor; para-aminobenzenethiol; principal component analysis; surface-enhanced Raman scattering

This review introduces multifunctional optical nanosensors based on surface-enhanced Raman scattering (SERS) and demonstrates their application in live cells. The novel nanosensors have the potential to improve our understanding of cellular processes on the molecular level. The hybrid sensor consists of gold or silver nanoparticles with an attached reporter species. The sensor can be detected and imaged based on the SERS signature of the reporter. This results in several advantages, such as high spectral specificity, multiplex capabilities, improved contrast, and photostability. SERS sensors not only highlight cellular structures, based on enhanced Raman spectra of intrinsic cellular molecules measured in the local optical fields of the gold nanoparticles, they also provide molecular structural information on their cellular environment. Moreover, the SERS signature of the reporter can deliver information on the local pH value inside a cell at subendosomal resolution. SERS sensors are suitable for one- and two-photon excitation.From the Clinical EditorThis review introduces multifunctional optical nanosensors based on surface enhanced Raman scattering (SERS) and demonstrates their application in live cells. These hybrid sensors consist of gold or silver nanoparticles with an attached reporter species. The sensor can be detected and imaged based on the SERS signature of the reporter. SERS sensors highlight cellular structures and provide molecular structural information on their cellular environment. They can also deliver information on the intracellular pH-value at subendosomal resolution.

Raman signatures of the carotenoid component are studied in individual pollen grains from different species of trees. The information is obtained as differences in the strong pre-resonant Raman spectra measured before and after photodepletion of the carotenoid molecules. The results provide the first in situ evidence of interspecies differences in pollen carotenoid content, structure, and/or assembly between plant species without prior purification. The analysis of carotenoids in situ is confirmed by high-performance thin-layer chromatography (HPTLC)-supported resonance Raman data measured directly on the HPTLC plates after separation of carotenoids in pollen extracts. Utilization of the in situ, extraction-free procedure in carotenoid analysis will improve sensitivity and structural selectivity and provides insight into carotenoid structure and composition in single pollen grains.

We report on the in situ characterization of tree pollen molecular composition based on Raman spectroscopy. Different from purification-based analysis, the nondestructive approach allows (i) to analyze various classes of molecules simultaneously at microscopic resolution and (ii) to acquire fingerprint-like chemical information that was used for the classification of pollen from different species. Hierarchical cluster analysis of spectra from fresh pollen samples of 15 species partly related at the genus level and family level indicates separation of species based on the complete Raman spectral signature and yields classification in accord with biological systematics. The results have implications for the further elucidation of pollen biochemistry and also for the development of chemistry-based online pollen identification methods.

Throughout the history of preparation of biological samples for microscopy the choice of the mounting medium was sometimes dictated merely by availability of the used media. Thus, a plethora of resins and other organic polymers as well as complex mixtures are found to serve as mounting agents in microscope slide collections of museums of natural history, impeding the work for both curators and conservators. Dramatically, in some cases the used mounting media can already be observed to have undergone crystallization and other decomposition processes within few years of mounting demanding immediate action in restoring as well as an imminent precaution in conservation. Therefore, an unambiguous chemical identification of the used agent as well as its current aging stage is of great interest for the biologist community. The technical demands on the analytical approach to obtain this information can be straightforwardly identified. Any used technique has to be non-destructive, yield in molecular information allowing for a chemical identification of the used mounting agents and allow for a spatially well-defined interrogation in a thin sample slice, typically through a transparent cover slip. In this contribution we present a thorough study of the applicability of Raman spectroscopy for the described task. The obtained results clearly demonstrate the successful feasibility of the chosen method for a) a clear distinction between different media, b) the elucidation of the chemical composition of a multicomponent medium and c) an unambiguous identification of real unknown samples by a distinct assignment to a previously recorded spectral library. This library database was built up by recording pure mounting agents and will be provided to the general public. In combination with a Raman spectrometer, it can be an invaluable tool for future curation and conservation endeavors devoted to microscope slide collections at natural history museums.

Silver nanoparticles were generated based on citrate reduction in the ultrastructure of the sporopollenin biopolymer of Ambrosia artemisiifolia (ragweed) and Secale cereale (rye). The nanoparticles enable the acquisition of SERS spectra and thereby a vibrational characterization of the local molecular structure of sporopollenin.

The interaction of nanomaterials with biomolecules, cells, and organisms plays an important role in cell biology, toxicology, and nanotechnology. Spontaneous Raman scattering can be used to probe biomolecules, cells, whole animals, and nanomaterials alike, opening interesting avenues to study the interaction of nanoparticles with complex biological systems. In this review we discuss work in biomedical Raman spectroscopy that has either been concerned directly with nanostructures and biosystems, or that indicates important directions for successful future studies on processes associated with nano-bio-interactions.

The interaction of nanoparticles with hemoglobin (Hb), a major constituent of red blood cells, is important in nanotoxicity research. We report SERS spectra of Hb using gold and silver nanoparticles at very small nanoparticle:Hb molecule ratios, that is, under conditions relevant for SERS-based nanotoxicity experiments with red blood cells at high sensitivity. We show that the structural information obtained from the experiment is highly dependent on the type of SERS substrate and the conditions under which the interaction of nanoparticles with Hb molecules takes place. In experiments with isolated red blood cells, we demonstrate that the dependence of the spectra on the type of nanoparticle used as the SERS substrate extends to whole red blood cells and red blood cell components. Regarding the applicability of SERS to red blood cells in vivo, evidence is provided that the molecular information contained in the spectra is highly dependent on the material and size of the nanoparticles. The results indicate specific interactions of gold and silver nanoparticles with Hb and the red blood cell membrane, and reflect the hemolytic activity of silver nanoparticles. The results of this study help improve our understanding of the interactions of silver and gold nanoparticles with red blood cells.

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 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. As a two-photon excited Raman scattering process, SEHRS provides chemical structure information based on molecular vibrations, but follows different selection rules than the normal, one-photon excited surface-enhanced Raman scattering (SERS). Therefore, simultaneous hyperspectral mapping using SEHRS excited at 1064 nm and SERS excited at 532 nm improves spatially resolved multivariate discrimination based on complementary vibrational information. The possibility to map distributions of the structurally similar dyes crystal violet and malachite green demonstrates the potential of this approach for multiplex imaging of complex systems.

Surface enhanced hyper Raman scattering (SEHRS) is the spontaneous, two-photon excited Raman scattering that occurs for molecules residing in high local optical fields of plasmonic nanostructures. Being regarded as a non-linear analogue of surface enhanced Raman scattering (SERS), SEHRS shares most of its properties, but also has additional characteristics. They include complementary spectroscopic information resulting from different selection rules and a stronger enhancement due to the non-linearity in excitation. In practical spectroscopy, this can translate to advantages, which include a high selectivity when probing molecule–surface interactions, the possibility of probing molecules at low concentrations due to the strong enhancement, and the advantages that come with excitation in the near-infrared. In this review, we give examples of the wealth of vibrational spectroscopic information that can be obtained by SEHRS and discuss work that has contributed to understanding the effect and that therefore provides directions for SEHRS spectroscopy. Future applications could range from biophotonics to materials research.