Volker Deckert

Nanoscale chemical analysis by tip-enhanced Raman spectroscopy

Abstract A fine metal tip brought to within a few nanometers of a molecular film is found to give strong enhancement of Raman scattered light from the sample. This new principle can be used for molecular analysis with excellent spatial resolution, only limited by the tip apex size and shape. No special sample preparation is required, and the enhancement is identical at every sample location, allowing for quantitative surface-enhanced Raman spectroscopy measurements. When scanning the tip over the sample surface, topographic information is obtained simultaneously and can be directly correlated with the spectroscopic data.

Scanning near-field optical microscopy with aperture probes: Fundamentals and applications

In this review we describe fundamentals of scanning near-field optical microscopy with aperture probes. After the discussion of instrumentation and probe fabrication, aspects of light propagation in metal-coated, tapered optical fibers are considered. This includes transmission properties and field distributions in the vicinity of subwavelength apertures. Furthermore, the near-field optical image formation mechanism is analyzed with special emphasis on potential sources of artifacts. To underline the prospects of the technique, selected applications including amplitude and phase contrast imaging, fluorescence imaging, and Raman spectroscopy, as well as near-field optical desorption, are presented. These examples demonstrate that scanning near-field optical microscopy is no longer an exotic method but has matured into a valuable tool.

Catalytic processes monitored at the nanoscale with tip-enhanced Raman spectroscopy

Heterogeneous catalysts play a pivotal role in the chemical industry, but acquiring molecular insights into functioning catalysts remains a significant challenge. Recent advances in micro-spectroscopic approaches have allowed spatiotemporal information to be obtained on the dynamics of single active sites and the diffusion of single molecules. However, these methods lack nanometre-scale spatial resolution and/or require the use of fluorescent labels. Here, we show that time-resolved tip-enhanced Raman spectroscopy can monitor photocatalytic reactions at the nanoscale. We use a silver-coated atomic force microscope tip to both enhance the Raman signal and to act as the catalyst. The tip is placed in contact with a self-assembled monolayer of p-nitrothiophenol molecules adsorbed on gold nanoplates. A photocatalytic reduction process is induced at the apex of the tip with green laser light, while red laser light is used to monitor the transformation process during the reaction. This dual-wavelength approach can also be used to observe other molecular effects such as monolayer diffusion.

Tip‐Enhanced Raman Spectroscopy of Single RNA Strands: Towards a Novel Direct‐Sequencing Method

The sequencing of DNA or proteins is procedurally complex and requires sophisticated analytical techniques. DNA sequencing while a very powerful method requires separation and visualization methods to recognize specific DNA fragments. Furthermore, all the established methods require substantial amounts of DNA and fail to directly read the base composition of the strand. A method that utilizes the inherent information of the distinct bases present in DNA or RNA without the need of further labeling is therefore desirable. Recent approaches in this direction include pulling single DNA strands through nanopores, detecting certain electric properties, and then deducing the sequence. Attempts were also made to directly sequence DNA by using a scanning tunneling microscope. The main challenge with STM methods is always the low contrast and usually a statistic approach is necessary to evaluate the data. Our studies demonstrate that by using near-field optical techniques in combination with vibrational spectroscopy gives high contrast and the direct identification of bases on a single isolated RNA strand becomes feasible. Standard Raman spectroscopy makes the identification of the base components straight forward, however, the lateral resolution and the sensitivity of the method are far from the single-strand or even single-base detection levels required for a sequencing method. Herein we show that tip-enhanced Raman scattering (TERS) provides several advantages over conventional Raman spectroscopy: in just a few seconds acquisition time it gives high sensitivity at lateral resolution down to a few tens of nucleobases. These properties allow TERS mapping along a poly(cytosine) RNA strand. The results demonstrate the potential of the method to identify and sequence the composition of polymeric biomacromolecules (DNA, RNA, peptides). TERS spectra of a single-stranded RNA homopolymer of cytosine (poly(C)) have been measured with a spatial resolution down to a few nucleobases. The basic experiment is shown in Figure 1. A standard transmission TERS setup is used to focus a laser onto a silver-coated atomic-force microscope (AFM) tip, while the sample is moved independently, the sample surface is thus always in focus. Similar arrangements have been used to enhance infrared or Raman spectra of nanoscale materials, such as polymer samples, molecular monolayers, or single carbon nanotubes. In Figure 2 the topography image of a single-stranded RNA cytosine homopolymer is shown. To avoid Raman scattering from compounds other than RNA, the use of buffer solutions and other chemicals was kept to a minimum. However, as a Figure 1. A) The tip-enhanced Raman scattering (TERS) experiment along a single strand of RNA. B) Higher magnification of the area approximately corresponding to the size of the laser spot. C) Magnification corresponding to the interaction area of the TERS probe tip.

High-quality near-field optical probes by tube etching

A method called tube etching for the fabrication of near-field optical probes is presented. Tip formation occurs inside a cylindrical cavity formed by the polymer coating of an optical fiber which is not stripped away prior to etching in hydrofluoric acid. The influence of temperature, etchant concentration, and fiber type on the tip quality is studied. A tip formation mechanism for the given geometry is proposed. The procedure overcomes drawbacks of the conventional etching techniques while still producing large cone angles: (i) tips with reproducible shapes are formed in a high yield, (ii) the surface roughness on the taper is drastically reduced, and (iii) the tip quality is insensitive to vibrations and temperature fluctuations during the etching process. After aluminum coating, optical probes with well-defined apertures are obtained. Due to the smooth glass surface the aluminum coating is virtually free of pinholes.

Near-Field Surface-Enhanced Raman Imaging of Dye-Labeled DNA with 100-nm Resolution

Raman chemical imaging on a scale of 100 nm is demonstrated for the first time. This is made possible by the combination of scanning near-field optical microscopy (SNOM or NSOM) and surface-enhanced Raman scattering (SERS), using brilliant cresyl blue (BCB)-labeled DNA as a sample. SERS substrates were produced by evaporating silver layers on Teflon nanospheres. The near-field SERS spectra were measured with an exposure time of 60 s and yielded good signal-to-noise ratios (25:1). The distinction between reflected light from the excitation laser and Raman scattered light allows the local sample reflectivity to be separated from the signal of the adsorbed DNA molecules. This is of general importance to correct for topographic coupling that often occurs in near-field optical imaging. The presented data show a lateral dependence of the Raman signals that points to special surface sites with particularly high SERS enhancement.

Near-field surface-enhanced Raman spectroscopy of dye molecules adsorbed on silver island films

Abstract We report the combined use of scanning near-field optical microscopy and surface-enhanced Raman scattering (SERS) to obtain spectral, spatial and chemical information of molecular adsorbates with subwavelength lateral resolution. Near-field SERS spectra of cresyl fast violet and rhodamine 6G on silver substrates are obtained with short exposure times (less than 100 s) and a signal-to-noise ratio of >20. Spectra from as few as ≈300 molecules or less than 10 −2 monolayers adsorbed on about ten silver nanoparticles can be recorded using 200 nm tip apertures. For the near-field spectra, a local Raman enhancement factor of 10 13 or greater can be derived from a comparison with fluorescence measurements.

Tip-Enhanced Raman Scattering (TERS) from Hemozoin Crystals within a Sectioned Erythrocyte

Tip-enhanced Raman scattering (TERS) is a powerful technique to obtain molecular information on a nanometer scale, however, the technique has been limited to cell surfaces, viruses, and isolated molecules. Here we show that TERS can be used to probe hemozoin crystals at less than 20 nm spatial resolution in the digestive vacuole of a sectioned malaria parasite-infected cell. The TERS spectra clearly show characteristic bands of hemozoin that can be correlated to a precise position on the crystal by comparison with the corresponding atomic force microscopy (AFM) image. These are the first recorded AFM images of hemozoin crystals inside malaria-infected cells and clearly show the hemozoin crystals protruding from the embedding medium. TERS spectra recorded of these crystals show spectral features consistent with a five-coordinate high-spin ferric heme complex, which include the electron density marker band ν(4) at 1373 cm(-1) and other porphyrin skeletal and ring breathing modes at approximately 1636, 1557, 1412, 1314, 1123, and 1066 cm(-1). These results demonstrate the potential of the AFM/TERS technique to obtain nanoscale molecular information within a sectioned single cell. We foresee this approach paving the way to a new independent drug screening modality for detection of drugs binding to the hemozoin surface within the digestive vacuole of the malaria trophozoite.

Structure and Composition of Insulin Fibril Surfaces Probed by TERS

Amyloid fibrils associated with many neurodegenerative diseases are the most intriguing targets of modern structural biology. Significant knowledge has been accumulated about the morphology and fibril-core structure recently. However, no conventional methods could probe the fibril surface despite its significant role in the biological activity. Tip-enhanced Raman spectroscopy (TERS) offers a unique opportunity to characterize the surface structure of an individual fibril due to a high depth and lateral spatial resolution of the method in the nanometer range. Herein, TERS is utilized for characterizing the secondary structure and amino acid residue composition of the surface of insulin fibrils. It was found that the surface is strongly heterogeneous and consists of clusters with various protein conformations. More than 30% of the fibril surface is dominated by β-sheet secondary structure, further developing Dobsons model of amyloid fibrils (Jimenez et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9196-9201). The propensity of various amino acids to be on the fibril surface and specific surface secondary structure elements were evaluated. β-sheet areas are rich in cysteine and aromatic amino acids, such as phenylalanine and tyrosine, whereas proline was found only in α-helical and unordered protein clusters. In addition, we showed that carboxyl, amino, and imino groups are nearly equally distributed over β-sheet and α-helix/unordered regions. Overall, this study provides valuable new information about the structure and composition of the insulin fibril surface and demonstrates the power of TERS for fibril characterization.

Ultraflat Transparent Gold Nanoplates—Ideal Substrates for Tip‐Enhanced Raman Scattering Experiments

Ultraflat gold nanoplates were found to be very attractive substrates for tip-enhanced Raman scattering (TERS) measurements. The transparent flat triangular or hexagonal nanoplates were synthesized by citrate reduction of HAuCl4 in aqueous solution. The obtained nanoparticles with a height of 15–20 nm had a smooth homogeneous surface with a roughness of about 100–200 pm. After spreading the gold plates on glass slides, cystine was successfully immobilized on them and the first TERS spectra of an amino acid were recorded. The spectra revealed a local variation of the attachment of cystine on the gold surface in two conformers. The production of well-defined gold particles is of general interest due to their geometry-dependent physical properties. Controlling and tuning the properties of these particles down to the nanometer regime extends their present applications, such as fluorescence quenching of organic molecules, surface-enhanced Raman scattering (SERS), and, as will be presented here, TERS. In SERS, molecules are adsorbed on rough metal substrates like colloids, electrodes, or evaporated films, resulting in a Raman signal-enhancement of several orders of magnitude. The irregular surface structure of those metallic surfaces, however, restricts the application of the SERS technique. With SERS, chemical information on low sample concentrations down to single molecules is accessible but the spatial information is lost. This argument is also valid for defined and highly reproducible SERS structures like electronbeam lithographic masks (see for instance Reference [5]) and for such structures the field enhancement invariably varies across the surfaces. Due to this lack of spatial resolution, a quantitative molecular analysis is hardly possible. If such information is required, TERS is the method of choice. In TERS a scanning tunneling microscope (STM) or an atomic force microscope (AFM) is coupled with a Raman spectrometer. Here the field-enhancing feature is confined to the very end of the probe. Usually a sharp metal tip or a single small metal particle acts as the sole source of enhancement.