Unambiguous chemical identification of individual molecules closely packed on a surface can offer the possibility to address single chemical species and monitor their behaviour at the individual level. Such a degree of spatial resolution can in principle be achieved by detecting their vibrational fingerprints using tip-enhanced Raman scattering (TERS). The chemical specificity of TERS can be combined with the high spatial resolution of scanning probe microscopy techniques, an approach that has stimulated extensive research in the field. Recently, the development of nonlinear TERS in a scanning tunnelling microscope has pushed the spatial resolution down to ∼0.5 nm, allowing the identification of the vibrational fingerprints of isolated molecules on Raman-silent metal surfaces. Although the nonlinear TERS component is likely to help sharpen the optical contrast of the acquired image, the TERS signal still contains a considerable contribution from the linear term, which is spatially less confined. Therefore, in the presence of different adjacent molecules, a mixing of Raman signals may result. Here, we show that using a nonlinear scanning tunnelling microscope-controlled TERS set-up, two different adjacent molecules that are within van der Waals contact and of very similar chemical structure (a metal-centred porphyrin and a free-base porphyrin) on a silver surface can be distinguished in real space. In addition, with the help of density functional theory simulations, we are also able to determine their adsorption configurations and orientations on step edges and terraces.

Individual carbon nanotubes (CNTs) have been investigated by tip-enhanced Raman spectroscopy (TERS) using silver tips on the Ag(111) substrate with a low-temperature ultrahigh-vacuum scanning tunneling microscope. Thanks to the strong and highly localized plasmonic field offered by the silver nanogap, the spatial resolution of TERS on CNTs is driven down to about 0.7 nm. Such a high spatial resolution allows to visualize in real space the spatial extent of the defect-induced D-band scattering, to track the strain-induced spectral evolution, and to resolve the spectral differences between the inner and the outer sides of a bent CNT, all at the nanometer scale.

The importance of identifying DNA bases at the single-molecule level is well recognized for many biological applications. Although such identification can be achieved by electrical measurements using special setups, it is still not possible to identify single bases in real space by optical means owing to the diffraction limit. Herein, we demonstrate the outstanding ability of scanning tunneling microscope (STM)-controlled non-resonant tip-enhanced Raman scattering (TERS) to unambiguously distinguish two individual complementary DNA bases (adenine and thymine) with a spatial resolution down to 0.9 nm. The distinct Raman fingerprints identified for the two molecules allow to differentiate in real space individual DNA bases in coupled base pairs. The demonstrated ability of non-resonant Raman scattering with super-high spatial resolution will significantly extend the applicability of TERS, opening up new routes for single-molecule DNA sequencing.

Tip-enhanced Raman spectroscopy (TERS) is a powerful surface analysis technique that can provide subnanometer-resolved images of nanostructures with site-specific chemical fingerprints. However, due to the limitation of weak Raman signals and the resultant difficulty in achieving TERS imaging with good signal-to-noise ratios (SNRs), the conventional single-peak analysis is unsuitable for distinguishing complex molecular architectures at the subnanometer scale. Here we demonstrate that the combination of subnanometer-resolved TERS imaging and advanced multivariate analysis can provide an unbiased panoramic view of the chemical identity and spatial distribution of different molecules on surfaces, yielding high-quality chemical images despite limited SNRs in individual pixel-level spectra. This methodology allows us to exploit the full power of TERS imaging and unambiguously distinguish between adjacent molecules with a resolution of ~0.4 nm, as well as to resolve submolecular features and the differences in molecular adsorption configurations. Our results provide a promising methodology that promotes TERS imaging as a routine analytical technique for the analysis of complex nanostructures on surfaces.

Current molecular analysis of cells and tissues routinely relies on separation, enrichment, and subsequent measurements by various assays. Here we demonstrate a platform of hyperspectral stimulated Raman scattering microscopy for fast, quantitative and label-free imaging of biomolecules in intact tissues using spectroscopic fingerprints as the contrast mechanism.