Molecular Mapping Beyond the Diffraction Limit
Using Tip-Enhanced Optical Spectroscopy
- Fig. 1: (a) Schematic diagram of an AFM-TERS set-up with bottom illumination geometry. (b) Left: AFM topography image of a single-layer graphene flake on a glass substrate. Right: TERS map of the D band intensity measured at the area highlighted in the centre of the topography image . Adapted from Ref. 2 with permission from the Royal Society of Chemistry (RSC).
- Fig. 2: (a) AFM topography image of a heterogeneous Ag catalyst substrate. (b) TERS map of the 1142 cm-1 DMAB Raman band intensity measured at the area highlighted in (a) revealing positions of pMA → DMAB reaction hotspots . Adapted from Ref. 5 with permission from the RSC.
- Fig. 3: TERS maps of distribution of newly-synthesised phospholipid (NSP) molecules within a mouse pre-adipocyte cell with a spatial resolution of < 20 nm . Molecular structure of a NSP molecule is shown in the inset. Adapted from Ref. 6 with permission from the RSC.
- Fig. 4: An AFM topography image of a polymer:fullerene solar cell active layer is shown in the centre along with high-resolution maps of topography, photoluminescence, Raman and photocurrent measured simultaneously using STEOM . Adapted from Ref. 7 with permission from the RSC.
Non-destructive and label-free molecular mapping at the nanoscale under ambient conditions challenges the limits of most analytical techniques. Tip-Enhanced Optical Spectroscopy (TEOS) is one of the very few techniques capable of achieving this. In this article, we discuss the principles of TEOS, its application to nanoscale chemical charaterisation in different areas and how it can be combined with other methods to provide multi-parameter imaging at the nanoscale.
Over the last two decades tip-enhanced optical spectroscopy (TEOS) has become a powerful nanoanalytical tool for correlative topographical and chemical mapping of a surface at the nanoscale. The most widely used method has been tip-enhanced Raman spectroscopy (TERS), which has proven useful for nanoscale chemical characterization in a wide range of research areas such as biology, catalysis, polymer-blends, semiconductors, 2D and 1D materials as well as single molecule imaging . More recently, tip-enhanced photoluminescence (TEPL) spectroscopy has emerged as an effective method of probing chemical, electrical and optical properties at the nanoscale. The advantages of TEOS over other analytical techniques are the high lateral resolution (beyond the diffraction limit), the non-destructive and label-free nature of the method and the fact that it can be used under ambient conditions. TEOS works on the principle of localized surface plasmon resonance (LSPR), whereby a high intensity electric field is created at the apex of a metal-coated scanning probe microscopy (SPM) tip placed inside the focal spot of a laser beam, as schematically shown in figure 1a. The LSPR between the excitation laser and the metal nanoparticles at the tip-apex confines the electric field to a region similar to the size of the nanoparticles and enhances its intensity by several orders of magnitude thereby increasing both the spatial resolution as well as the sensitivity of optical imaging.
Visualizing Defects in Graphene and 2D Materials
Structural defects in 2D materials, such as graphene [2,3] and molybdenum disulphide  can significantly degrade their carrier transport properties reducing efficiency of potential 2D materials-based electronic devices such as high performance field effect transistors, super capacitors, photonic detectors etc.
Using TERS we have shown (fig. 1b) that it is possible to map individual structural defects with radii as small as 1.9 ± 0.1 nm in single-layer graphene , under ambient conditions and even on a rough substrate such as glass.
Mapping Catalytic Activity at the Nanoscale
Catalysts are used in more than 90% of industrial chemical processes and contribute to almost 35% of global GDP. However, conventional spectroscopy methods of catalyst charaterization typically rely upon macroscopic techniques that measure an ensemble of particles. Using TERS, we have demonstrated mapping of the catalytic activity at a heterogeneous Ag catalyst substrate employing plasmon-driven photocatalytic oxidation of p-mercaptoaniline (pMA) → p,p’-dimercaptoazobenzene (DMAB) as a model reaction as shown in figure 2 . In the TERS map, reaction hotspot of pMA → DMAB are clearly revealed with a spatial resolution of ≈ 20 nm.
Imaging Small Molecules in Biological Cells
Visualizing nanoscale distribution of small molecules present inside a biological cell is crucial for understanding structure–function relationships in sub-cellular biological nanostructures. Currently, super-resolution fluorescence microscopy (SRFM) is the most popular technique for nanoscale molecular mapping in biological cells. However, SRFM suffers from the essential requirement of fluorescent labels that can sometimes change the behavior of biomolecules they are attached to. Furthermore, only the molecules tagged with fluorescent labels can be visualized using SRFM while the others remain invisible. Recently, we have demonstrated non-destructive 2D mapping of small biomolecules within a biological cell using TERS without employing any extraneous labels . Figure 3 shows nanoscale chemical mapping of newly-synthesised phospholipid (NSP) molecules within a mouse pre-adipocyte cell. NSP distribution is mapped with < 20 nm spatial resolution using TER intensity of C-D vibration mode (2100 cm-1) of the NSP molecules.
Multi-Parameter Imaging at the Nanoscale
The ability to directly correlate structure and function is the holy grail in the development of new materials and devices. This requires multi-parameter approaches (measurements of multiple properties) which becomes more challenging as higher resolution is required. Recently we demonstrated that it is possible to combine nano-optical spectroscopy (TERS, TEPL) with electrical mode scanning probe microscopy in a single, simultaneous experiment, allowing non-destructive structure-to-function characterization with nanoscale resolution. This was achieved by developing multifunctional probes, i.e. metal-coated tips that act as both plasmonic probes and electrical contact for scanning probe measurements. Such simultaneous measurements reduce uncertainty of data analysis by facilitating registration of multiple images, diminish unwanted contamination/degradation and decrease overall measurement time. Figure 4 shows simultaneous topographical, electrical and optical microscopy (STEOM) of an organic solar cell with <20 nm spatial resolution where we used the different probe depths of each measured signal to infer the surface and subsurface molecular composition correlating it with the local photocurrent generation and used it to explain macroscopic device performance .
TEOS provides molecular information well beyond the diffraction limit and has been applied to support development of materials and devices in numerous areas, from biology to electronic devices. Recent advances in metrology [8-10] and instrumentation, such as the integration of TEOS with electrical mode SPM (STEOM) , are facilitating wider use of these methods, which we believe will guide the rational design of a range of nanomaterial-based optoelectronic devices.
 Kumar, N.; Mignuzzi, S.; Su, W.; Roy, D.: Tip-enhanced Raman spectroscopy: principles and applications. EPJ Tech. Instrum. 2015, 2, 9
 Mignuzzi, S.; Kumar, N.; Brennan, B.; Gilmore, I. S.; Richards, D.; Pollard, A. J.; Roy, D.: Probing individual point defects in graphene via near-field Raman scattering. Nanoscale 2015, 7, 19413-19418
 Su, W.; Kumar, N.; Dai, N.; Roy, D.: Nanoscale mapping of intrinsic defects in single-layer graphene using tip-enhanced Raman spectroscopy. Chem. Commun. 2016, 52, 8227-8230
 Su, W.; Kumar, N.; Mignuzzi, S.; Crain, J.; Roy, D.: Nanoscale mapping of excitonic processes in single-layer MoS 2 using tip-enhanced photoluminescence microscopy. Nanoscale 2016, 8, 10564-10569
 Kumar, N.; Stephanidis, B.; Zenobi, R.; Wain, A.; Roy, D.: Nanoscale mapping of catalytic activity using tip-enhanced Raman spectroscopy. Nanoscale 2015, 7, 7133-7137
 Kumar, N.; Drozdz, M. M.; Jiang, H.; Santos, D. M.; Vaux, D. J.: Nanoscale mapping of newly-synthesised phospholipid molecules in a biological cell using tip-enhanced Raman spectroscopy. Chem. Commun. 2017, 53, 2451-2454
 Kumar, N.; Zoladek-Lemanczyk, A.; Guilbert, A. A.; Su, W.; Tuladhar, S. M.; Kirchartz, T.; Schroeder, B. C.; McCulloch, I.; Nelson, J.; Roy, D.: Simultaneous topographical, electrical and optical microscopy of optoelectronic devices at the nanoscale. Nanoscale 2017, 9, 2723-2731
 Blum, C.; Opilik, L.; Atkin, J. M.; Braun, K.; Kammer, S. B.; Kravtsov, V.; Kumar, N.; Lemeshko, S.; Li, J. F.; Luszcz, K.; Maleki, T.; Meixner, A. J.; Minne, S.; Raschke, M. B.; Ren, B.; Rogalski, J.; Roy, D.; Stephanidis, B.; Wang, X.; Zhang, D.; Zhong, J. H.; Zenobi, R.: Tip-enhanced Raman spectroscopy - an interlaboratory reproducibility and comparison study. J. Raman Spectrosc. 2014, 45, 22-31
 Kumar, N.; Spencer, S. J.; Imbraguglio, D.; Rossi, A. M.; Wain, A. J.; Weckhuysen, B. M.; Roy, D.: Extending the plasmonic lifetime of tip-enhanced Raman spectroscopy probes. Physical Chemistry Chemical Physics 2016, 18, 13710-13716
 Kumar, N.; Rae, A.; Roy, D.: Accurate measurement of enhancement factor in tip-enhanced Raman spectroscopy through elimination of far-field artefacts. Appl. Phys. Lett. 2014, 104, 123106
Naresh Kumar1 and Fernando A. Castro1
1National Physical Laboratory, Teddington, United Kingdom
Naresh Kumar MRes MInstP MRSC
Higher Research Scientist
Chemical, Medical and Environmental Sciences
National Physical Laboratory
Teddington, United Kingdom