SERS microscopy as a tool for comprehensive biochemical characterization in complex samples.

Surface enhanced Raman scattering (SERS) spectra of biomaterials such as cells or tissues can be used to obtain biochemical information from nanoscopic volumes in these heterogeneous samples. This tutorial review discusses the factors that determine the outcome of a SERS experiment in complex bioorganic samples. They are related to the SERS process itself, the possibility to selectively probe certain regions or constituents of a sample, and the retrieval of the vibrational information in order to identify molecules and their interaction. After introducing basic aspects of SERS experiments in the context of biocompatible environments, spectroscopy in typical microscopic settings is exemplified, including the possibilities to combine SERS with other linear and non-linear microscopic tools, and to exploit approaches that improve lateral and temporal resolution. In particular the great variation of data in a SERS experiment calls for robust data analysis tools. Approaches will be introduced that have been originally developed in the field of bioinformatics for the application to omics data and that show specific potential in the analysis of SERS data. They include the use of simulated data and machine learning tools that can yield chemical information beyond achieving spectral classification.

Infrared spectroscopy across scales in length and time at BESSY†II.

The infrared beamline at BESSY II storage ring was upgraded recently to extend the capabilities of infrared microscopy. The endstations available at the beamline are now facilitating improved characterization of molecules and materials at different length scales and time resolutions. Here, the current outline of the beamline is reported and an overview of the endstations available is given. In particular, the first results obtained by using a new microscope for nano-spectroscopy that was implemented are presented. The capabilities of the scattering-type near-field optical microscope (s-SNOM) are demonstrated by investigating cellulose microfibrils, representing nanoscopic objects of a hierarchical structure. It is shown that the s-SNOM coupled to the beamline allows imaging to be performed with a spatial resolution of less than 30 nm and infrared spectra to be collected from an effective volume of less than 30 nm × 30 nm × 12 nm. Potential steps for further optimization of the beamline performance are discussed.

Potential Regulation for Surface-Enhanced Raman Scattering Detection and Identification of Carotenoids.

Surface-enhanced Raman scattering (SERS) is often impaired by the limited affinity of molecules to plasmonic substrates. Here, we use carbon fiber microelectrodes modified with silver nanoparticles as a plasmonic microsubstrate with tunable affinity for enrichment and molecular identification by SERS. The silver nanoparticles self-assemble by electrostatic interaction with diamine molecules that are electrochemically grafted onto the surface of the microelectrodes. ß-carotene and trans-ß-Apo-8'-carotenal, producing similar resonant SERS spectra, are employed as model molecules to study the effect of electroenrichment and SERS screening for different electrode potentials. The data show that at a characteristic electrode potential, the low affinity of polyene chains without hydrophilic groups to the substrate can be overcome. Different potentials were applied to recognize the two types of carotenoids by their typical SERS signal, and the applicability of this strategy was further confirmed in the environment of a real cell culture. The results indicate that by regulating the potential, carotenoid molecules with a similar molecular structure can be selectively quantified and identified by SERS. The developed SERS-active microelectrode is expected to help the development of portable, miniaturized point-of-care diagnostic SERS sensors.

In situ infrared imaging of the local orientation of cellulose fibrils in plant secondary cell walls.

The mechanical and chemical properties of plant cell walls greatly rely on the supramolecular assembly of cellulose fibrils. To study the local orientation of cellulose in secondary plant cell walls, diffraction limited infrared (IR) micro-spectroscopic mapping experiments were conducted at different orientation of transverse leaf section of the grass Sorghum bicolor with respect to the polarization direction of the IR radiation. Two-dimensional maps, based on polarization-sensitive absorption bands of cellulose were obtained for different polarization angles. They reveal a significant degree of anisotropy of the cellulose macromolecules as well as of other biopolymers in sclerenchyma and xylem regions of the cross section. Quantification of the signals assigned to polarization sensitive vibrational modes allowed to determine the preferential orientation of the sub-micron cellulose fibrils in single cell walls. A sample of crystalline nano-cellulose comprising both a single microcrystal as well as unordered layers of nanocrystals was used for validation of the approach. The results demonstrate that diffraction limited IR micro-spectroscopy can be used to study hierarchically structured materials with complex anisotropic behavior.

Multivariate Imaging for Fast Evaluation of In Situ Dark Field Microscopy Hyperspectral Data.

Dark field scattering microscopy can create large hyperspectral data sets that contain a wealth of information on the properties and the molecular environment of noble metal nanoparticles. For a quick screening of samples of microscopic dimensions that contain many different types of plasmonic nanostructures, we propose a multivariate analysis of data sets of thousands to several hundreds of thousands of scattering spectra. By using non-negative matrix factorization for decomposing the spectra, components are identified that represent individual plasmon resonances and relative contributions of these resonances to particular microscopic focal volumes in the mapping data sets. Using data from silver and gold nanoparticles in the presence of different molecules, including gold nanoparticle-protein agglomerates or silver nanoparticles forming aggregates in the presence of acrylamide, plasmonic properties are observed that differ from those of the original nanoparticles. For the case of acrylamide, we show that the plasmon resonances of the silver nanoparticles are ideally suited to support surface enhanced Raman scattering (SERS) and the two-photon excited process of surface enhanced hyper Raman scattering (SEHRS). Both vibrational tools give complementary information on the in situ formed polyacrylamide and the molecular composition at the nanoparticle surface.

Molecular Structure and Interactions of Lipids in the Outer Membrane of Living Cells Based on Surface-Enhanced Raman Scattering and Liposome Models.

The distribution and interaction of lipids determine the structure and function of the cellular membrane. Surface-enhanced Raman scattering (SERS) is used for selective molecular probing of the cell membrane of living fibroblast cells grown adherently on gold nanoisland substrates across their whole contact areas with the substrate, enabling mapping of the membrane's composition and interaction. From the SERS data, the localization and distribution of different lipids and their interactions, together with proteins in the outer cell membrane, are inferred. Interpretation of the spectra is mainly supported by comparison with the spectra of model liposomes composed of phosphatidylcholine, sphingomyelin, and cholesterol obtained on the same gold substrate. The interaction of the liposomes with the substrate differs from that with gold nanoparticles. The SERS maps indicate colocalization of ordered lipid domains with cholesterol in the living cells. They support the observation of ordered membrane regions of micrometer dimensions in the outer leaflet of the cell membrane that are rich in sphingomyelin. Moreover, the spectra of the living cells contain bands from the groups of the lipid heads, phosphate, choline, and ethanolamine, combined with those from membrane proteins, as indicated by signals assigned to prenyl attachment. Elucidating the composition and structure of lipid membranes in living cells can find application in many fields of research.

Fragmentation of Proteins in the Corona of Gold Nanoparticles As Observed in Live Cell Surface-Enhanced Raman Scattering.

Surface-enhanced Raman scattering (SERS) can provide information on the structure, composition, and interaction of molecules in the proximity of gold nanoparticles, thereby enabling studies of adsorbed biomolecules in vivo. Here, the processing of the protein corona and the corresponding protein-nanoparticle interactions in live J774 cells incubated with gold nanoparticles was characterized by SERS. Samples of isolated cytoplasm, devoid of active processing, of the same cell line were used as references. The occurrence of the most important SERS signals was compared in both types of samples. The comparison of signal abundances, supported by multivariate assessment, suggests a decreased nanoparticle-peptide backbone interaction and an increased contribution of denatured proteins in endolysosomal compartments, indicating an interaction of protein fragments with the gold nanoparticles in the endolysosome of the living cells. To study the protein fragmentation in a model and to confirm the assignment of specific spectral signatures in the live cell spectra, SERS data were collected from a solution of bovine serum albumin (BSA) digested by trypsin as an enzymatic model and from solutions of intact BSA and trypsin. The spectra from the enzymatic model confirm the strong interaction of protein fragments with the gold nanoparticles in the endolysosomal compartments. By proteomic analysis, using combined sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry of the extracted hard corona, we directly identified protein fragments, some originating from the culture medium. The results illustrate the use of appropriate models for the validation of SERS spectra and have potential implications for further developments of SERS as an in vivo analytical and biomedical tool.

FTIR Nanospectroscopy Shows Molecular Structures of Plant Biominerals and Cell Walls.

Plant tissues are complex composite structures of organic and inorganic components whose function relies on molecular heterogeneity at the nanometer scale. Scattering-type near-field optical microscopy (s-SNOM) in the mid-infrared (IR) region is used here to collect IR nanospectra from both fixed and native plant samples. We compared structures of chemically extracted silica bodies (phytoliths) to silicified and nonsilicified cell walls prepared as a flat block of epoxy-embedded awns of wheat (Triticum turgidum), thin sections of native epidermis cells from sorghum (Sorghum bicolor) comprising silica phytoliths, and isolated cells from awns of oats (Avena sterilis). The correlation of the scanning-probe IR images and the mechanical phase image enables a combined probing of mechanical material properties together with the chemical composition and structure of both the cell walls and the phytolith structures. The data reveal a structural heterogeneity of the different silica bodies in situ, as well as different compositions and crystallinities of cell wall components. In conclusion, IR nanospectroscopy is suggested as an ideal tool for studies of native plant materials of varied origins and preparations and could be applied to other inorganic-organic hybrid materials.

Formation of root silica aggregates in sorghum is an active process of the endodermis.

Silica deposition in plants is a common phenomenon that correlates with plant tolerance to various stresses. Deposition occurs mostly in cell walls, but its mechanism is unclear. Here we show that metabolic processes control the formation of silica aggregates in roots of sorghum (Sorghum bicolor L.), a model plant for silicification. Silica formation was followed in intact roots and root segments of seedlings. Root segments were treated to enhance or suppress cell wall biosynthesis. The composition of endodermal cell walls was analysed by Raman microspectroscopy, scanning electron microscopy and energy-dispersive X-ray analysis. Our results were compared with in vitro reactions simulating lignin and silica polymerization. Silica aggregates formed only in live endodermal cells that were metabolically active. Silicic acid was deposited in vitro as silica onto freshly polymerized coniferyl alcohol, simulating G-lignin, but not onto coniferyl alcohol or ferulic acid monomers. Our results show that root silica aggregates form under tight regulation by endodermal cells, independently of the transpiration stream. We raise the hypothesis that the location and extent of silicification are primed by the chemistry and structure of polymerizing lignin as it cross-links to the wall.

Discrimination of grass pollen of different species by FTIR spectroscopy of individual pollen grains.

Fourier-transform infrared (FTIR) spectroscopy enables the chemical characterization and identification of pollen samples, leading to a wide range of applications, such as paleoecology and allergology. This is of particular interest in the identification of grass (Poaceae) species since they have pollen grains of very similar morphology. Unfortunately, the correct identification of FTIR microspectroscopy spectra of single pollen grains is hindered by strong spectral contributions from Mie scattering. Embedding of pollen samples in paraffin helps to retrieve infrared spectra without scattering artifacts. In this study, pollen samples from 10 different populations of five grass species (Anthoxanthum odoratum, Bromus inermis, Hordeum bulbosum, Lolium perenne, and Poa alpina) were embedded in paraffin, and their single grain spectra were obtained by FTIR microspectroscopy. Spectra were subjected to different preprocessing in order to suppress paraffin influence on spectral classification. It is shown that decomposition by non-negative matrix factorization (NMF) and extended multiplicative signal correction (EMSC) that utilizes a paraffin constituent spectrum, respectively, leads to good success rates for the classification of spectra with respect to species by a partial least square discriminant analysis (PLS-DA) model in full cross-validation for several species. PLS-DA, artificial neural network, and random forest classifiers were applied on the EMSC-corrected spectra using an independent validation to assign spectra from unknown populations to the species. Variation within and between species, together with the differences in classification results, is in agreement with the systematics within the Poaceae family. The results illustrate the great potential of FTIR microspectroscopy for automated classification and identification of grass pollen, possibly together with other, complementary methods for single pollen chemical characterization.

Towards Reliable and Quantitative Surface-Enhanced Raman Scattering (SERS): From Key Parameters to Good Analytical Practice.

Experimental results obtained in different laboratories world-wide by researchers using surface-enhanced Raman scattering (SERS) can differ significantly. We, an international team of scientists with long-standing expertise in SERS, address this issue from our perspective by presenting considerations on reliable and quantitative SERS. The central idea of this joint effort is to highlight key parameters and pitfalls that are often encountered in the literature. To that end, we provide here a series of recommendations on: a) the characterization of solid and colloidal SERS substrates by correlative electron and optical microscopy and spectroscopy, b) on the determination of the SERS enhancement factor (EF), including suitable Raman reporter/probe molecules, and finally on c) good analytical practice. We hope that both newcomers and specialists will benefit from these recommendations to increase the inter-laboratory comparability of experimental SERS results and further establish SERS as an analytical tool.

High-resolution and high-repetition-rate vibrational sum-frequency generation spectroscopy of one- and two-component phosphatidylcholine monolayers.

We present broadband vibrational sum-frequency generation (VSFG) spectra of Langmuir-Blodgett monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and different mixtures of them as model systems of pulmonary surfactants. The systematic study explored the dependence of the vibrational spectra as a function of surface tension and mixture ratio in various polarization combinations. The extremely short acquisition time and the high spectral resolution of our recently developed spectrometer helped minimize sample degradation under ambient conditions throughout the duration of the measurement and allowed the detection of previously unseen vibrational bands with unprecedented signal-to-noise ratio. The dramatically improved capability to record reliable vibrational spectra together with the label-free nature of the VSFG method provides direct access to native lipid structure and dynamics directly in the monolayer. The resulting data deliver quantitative information for structural analysis of multi-component phospholipid monolayers and may aid in the development of new synthetic pulmonary surfactants.

Amorphous Carbon Generation as a Photocatalytic Reaction on DNA-Assembled Gold and Silver Nanostructures.

Background signals from in situ-formed amorphous carbon, despite not being fully understood, are known to be a common issue in few-molecule surface-enhanced Raman scattering (SERS). Here, discrete gold and silver nanoparticle aggregates assembled by DNA origami were used to study the conditions for the formation of amorphous carbon during SERS measurements. Gold and silver dimers were exposed to laser light of varied power densities and wavelengths. Amorphous carbon prevalently formed on silver aggregates and at high power densities. Time-resolved measurements enabled us to follow the formation of amorphous carbon. Silver nanolenses consisting of three differently-sized silver nanoparticles were used to follow the generation of amorphous carbon at the single-nanostructure level. This allowed observation of the many sharp peaks that constitute the broad amorphous carbon signal found in ensemble measurements. In conclusion, we highlight strategies to prevent amorphous carbon formation, especially for DNA-assembled SERS substrates.

Mapping the Inhomogeneity in Plasmonic Catalysis on Supported Gold Nanoparticles Using Surface-Enhanced Raman Scattering Microspectroscopy.

The characterization of a catalyst often occurs by averaging over large areas of the catalyst material. On the other hand, optical probing is easily achieved at a resolution at the micrometer scale, specifically in microspectroscopy. Here, using surface-enhanced Raman scattering (SERS) mapping of larger areas with micrometer-sized spots that contain tens to hundreds of supported gold nanoparticles each, the photoinduced dimerization of p-aminothiophenol (PATP) to 4,4'-dimercaptoazobenzene (DMAB) was monitored. The mapping data reveal an inhomogeneous distribution of catalytic activity in the plasmon-catalyzed reaction in spite of a very homogeneous plasmonic enhancement of the optical signals in SERS. The results lead to the conclusion that only a fraction of the nanostructures may be responsible for a high catalytic activity. The high spot-to-spot variation in catalytic activity is also demonstrated for DMAB formation by the plasmon-catalyzed reduction from p-nitrothiophenol (PNTP) and confirms that an improvement of the accuracy and reproducibility in the characterization of catalytic reactions can be achieved by microspectroscopic probing of many positions. Using SERS micromapping during the incubation of PATP, we demonstrate that the reaction occurs during the incubation process and is influenced by different parameters, leading to the conclusion of dimerization in a gold-catalyzed, nonphotochemical reaction as an alternative to the plasmon-catalyzed process. The results have implications for the future characterization of new catalyst materials as well as for optical sensing using plasmonic materials.

Chemical Mapping of Leishmania Infection in Live Cells by SERS Microscopy.

We report the direct probing of the molecular composition of Leishmania-infected macrophage cells in vitro by surface-enhanced Raman scattering (SERS). The microscopic mapping data indicate local abundance and distribution of molecular species that are very characteristic of the infection and that are observed here simultaneously. As revealed by electron microscopy, the gold nanoprobes used for SERS microspectrosopy have access to the parasitophorous vacuoles (PV) through the endosomal system. SERS nanoprobes located in the direct proximity to the parasite, in the greater volume of the PV, and in endolysosomal compartments in other cellular regions, respectively, report a characteristic chemical composition for each respective location. The data enable assessment of the distribution of ergosterol and cholesterol in the amastigote stage of the parasite and its immediate surroundings in the vacuole. Proteophosphoglycans of parasite origin, an important hallmark of the infection, are identified throughout the PV.

Different binding sites of serum albumins in the protein corona of gold nanoparticles.

The interaction of bovine serum albumin (BSA) and human serum albumin (HSA), sharing a sequence similarity of 77.5%, with gold nanoparticles of a size of ∼30 nm was investigated by surface-enhanced Raman scattering (SERS). The spectra provide information on those residues of the proteins in proximity of the nanoparticles. The SERS signals indicate an electrostatic interaction of both proteins with the citrate ligands at the nanoparticle surface via lysine residues. HSA, different from BSA also binds directly to the gold surface by particularly flexible protein segments that were identified by comparison of the vibrational bands with the known amino acid sequence of the molecule. The data suggest that both the direct binding as well as interaction with the citrate ligands determine the interaction, yet to varying extent in the two very similar serum proteins. This has implications for their use in bio-functionalization, and for the application of gold nanostructures in bioanalytics and medicine.

Vibrational sum-frequency generation spectroscopy of lipid bilayers at repetition rates up to 100 kHz.

Broadband vibrational sum-frequency generation (BB-VSFG) spectroscopy has become a well-established surface analytical tool capable of identifying the orientation and structure of molecular layers. A straightforward way to boost the sensitivity of the technique could be to increase the laser repetition rate beyond that of standard BB-VSFG spectrometers, which rely on Ti:sapphire lasers operating at repetition rates of 1-5 kHz. Nevertheless, possible thermally induced artifacts in the vibrational spectra due to higher laser average powers are unexplored. Here, we discuss laser power induced temperature accumulation effects that distort the BB-VSFG spectra of 1,2-diacyl-sn-glycero-3-phosphocholine at an interface between two transparent phases at repetition rates of 5, 10, 50, and 100 kHz at constant pulse energy. No heat-induced distortions were found in the spectra, suggesting that the increase in the laser repetition rate provides a feasible route to an improved signal-to-noise ratio or shorter data acquisition times in BB-VSFG spectroscopy for thin films on transparent substrates. The results have implications for future BB-VSFG spectrometers pushing the detection limit for molecular layers with low surface coverage.

Surface enhanced hyper Raman scattering (SEHRS) and its applications.

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.

Placement of Single Proteins within the SERS Hot Spots of Self-Assembled Silver Nanolenses.

This study demonstrates the bottom-up synthesis of silver nanolenses. A robust coating protocol enabled the functionalization of differently sized silver nanoparticles with DNA single strands of orthogonal sequence. Coated particles 10 nm, 20 nm, and 60 nm in diameter were self-assembled by DNA origami scaffolds to form silver nanolenses. Single molecules of the protein streptavidin were selectively placed in the gap of highest electric field enhancement. Streptavidin labelled with alkyne groups served as model analyte in surface-enhanced Raman scattering (SERS) experiments. By correlated Raman mapping and atomic force microscopy, SERS signals of the alkyne labels of a single streptavidin molecule, from a single silver nanolens, were detected. The discrete, self-similar aggregates of solid silver nanoparticles are promising for plasmonic applications.

Simplifying the Preparation of Pollen Grains for MALDI-TOF MS Classification.

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a well-implemented analytical technique for the investigation of complex biological samples. In MS, the sample preparation strategy is decisive for the success of the measurements. Here, sample preparation processes and target materials for the investigation of different pollen grains are compared. A reduced and optimized sample preparation process prior to MALDI-TOF measurement is presented using conductive carbon tape as target. The application of conductive tape yields in enhanced absolute signal intensities and mass spectral pattern information, which leads to a clear separation in subsequent pattern analysis. The results will be used to improve the taxonomic differentiation and identification, and might be useful for the development of a simple routine method to identify pollen based on mass spectrometry.