Confocal and surface-enhanced Raman spectroscopy (SERS) are powerful techniques for molecular characterization; however, they suffer from the drawback of diffraction-limited spatial resolution. Tip-enhanced Raman spectroscopy (TERS) overcomes this limitation and provides chemical information at length scales in the tens of nanometers. In contrast to alternative approaches to nanoscale chemical analysis, TERS is label free, is non-destructive, and can be performed in both air and liquid environments, allowing its use in a diverse range of applications. Atomic force microscopy (AFM)-based TERS is especially versatile, as it can be applied to a broad range of samples on various substrates. Despite its advantages, widespread uptake of this technique for nanoscale chemical imaging has been inhibited by various experimental challenges, such as limited lifetime, and the low stability and yield of TERS probes. This protocol details procedures that will enable researchers to reliably perform TERS imaging using a transmission-mode AFM-TERS configuration on both biological and non-biological samples. The procedure consists of four stages: (i) preparation of plasmonically active TERS probes; (ii) alignment of the TERS system; (iii) experimental procedures for nanoscale imaging using TERS; and (iv) TERS data processing. We provide procedures and example data for a range of different sample types, including polymer thin films, self-assembled monolayers (SAMs) of organic molecules, photocatalyst surfaces, small molecules within biological cells, single-layer graphene and single-walled carbon nanotubes in both air and water. With this protocol, TERS probes can be prepared within ~23 h, and each subsequent TERS experimental procedure requires 3–5 h.
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N.K., A.J.W. and A.J.P. acknowledge funding from the National Measurement System of the Department of Business, Energy & Industry Strategy (BEIS), UK. B.M.W. acknowledges support from the Netherlands Center for Multiscale Catalytic Energy Conversion (MCEC), an NWO Gravitation programme funded by the Ministry of Education, Culture and Science of the Government of The Netherlands.
The authors declare no competing interests.
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Key references using this protocol
Kumar, N. et al. Nanoscale 10, 1815–1824 (2018): https://doi.org/10.1039/c7nr08257f
van Schrojenstein Lantman, E. M., Deckert-Gaudig, T., Mank, A. J. G., Deckert, V. & Weckhuysen, B. M. Nat. Nanotechnol. 7, 583–586 (2012): https://doi.org/10.1038/nnano.2012.131
Kumar, N., Drozdz, M. M., Jiang, H., Santos, D. M. & Vaux, D. J. Chem. Commun. 53, 2451–2454 (2017): https://doi.org/10.1039/C6CC10226C
Mignuzzi, S. et al. Nanoscale 7, 19413–19418 (2015): https://doi.org/10.1039/C5NR04664E
Integrated supplementary information
Supplementary Figure 1 Nanoscale imaging of a single-layer graphene flake using TERS.
a, Overlay image of confocal Raman maps (60 × 60 pixels) obtained using the intensity of 2D (red), D (green) and G (blue) Raman bands measured from a single-layer graphene flake. Integration time: 1 s. Pixel size: 25 nm. Scale bar: 300 nm. b, Overlay image of TERS maps (1.5 µm × 1.5 µm) obtained using the intensity of the 2D (red), D (green) and G (blue) bands from the single-layer graphene area shown in a. Integration time: 1 s. Pixel size: 30 nm. Scale bar: 300 nm. In the TERS image in b, a localized D band signal is observed at the edge of the graphene flake, clearly delineating the boundary of the single-layer flake. Furthermore, a defect signal from the D-peak is observed within the single-layer flake, which cannot be clearly distinguished in the confocal Raman image due to the lower sensitivity and spatial resolution. Adapted with permission from Su et al.60, Royal Society of Chemistry.
Supplementary Figure 2 TERS measurement of a PEDOT:PSS thin film on glass.
Tip-in (TERS) and tip-out (far-field) Raman spectra measured on a PEDOT:PSS thin film sample. Integration time: 60 s. Laser power: 50 µW. TERS contrast of 1444 cm-1 PEDOT Raman band is calculated to be 1.4 ± 0.1. Spectra are vertically shifted for easier visualisation.
Supplementary Figure 3 TERS measurement of a SAM of BPT on Au.
Tip-in (TERS) and tip-out (far-field) Raman spectra measured on a BPT SAM sample, where the BPT Raman peak is observed only for the TERS measurement. Integration time: 1 s. Laser power: 110 µW. Spectra are vertically shifted for easier visualisation.
Supplementary Figure 4 Monitoring of pNTP → DMAB photocatalytic process using TERS.
Time-dependent TERS spectra monitored before and after pNTP → DMAB reaction. Before starting the reaction, the TERS signal of a pNTP SAM was monitored for at least 1 min using 633 nm excitation (5 s integration time, 380 µW) as shown in part a. pNTP → DMAB was induced by switching to a 532 nm excitation laser (700 µW) for 30 s as shown in part b. Finally, the TERS signal of DMAB was monitored by switching back to the 633 nm laser excitation as shown in part c, where the characteristic Raman bands of DMAB appear at 1142 cm-1, 1390 cm-1 and 1437 cm-1. Reproduced from van Schrojenstein Lantman et al.67, Springer Nature.
Supplementary Figure 5 Nanoscale imaging of pATP → DMAB photocatalytic reaction using TERS.
a, AFM topography image of a nanostructured Ag substrate. Scale bar: 200 nm. b, TERS image of the dashed rectangle region marked in a, showing the locations of pATP → DMAB via variation of the 1142 cm−1 DMAB Raman band intensity. Pixel size: 12.5 nm. Scale bar: 50 nm. c, TERS spectra measured at the positions marked in b. Characteristic Raman bands of DMAB are visible at 1142 cm-1, 1390 cm-1 and 1437 cm-1 in the TERS spectra measured at pATP→ DMAB reaction hotspot locations 1–3, whereas these bands are absent in the TERS spectra from locations 4–6. Integration time: 0.5 s. Adapted from Kumar et al.68, Royal Society of Chemistry.
Supplementary Figure 6 Imaging of newly synthesized phospholipid molecules in biological cells using TERS.
a, TERS image of C–D band intensity of a 1 µm × 1 µm cellular region containing newly-synthesised phospholipids (NSPs). Pixel size: 50 nm. Scale bar: 200 nm. b, TERS image of NSPs (obtained using C–D band intensity) in the 100 nm × 200 nm area marked by a dashed rectangle in a. Pixel size: 13 nm. Scale bar: 50 nm. c, TERS spectra measured at the positions marked in b. The spectra are vertically shifted for easier visualisation. TERS spectra measured at locations 1–3 clearly show the presence of a C-D band at 2100 cm-1, whereas the C-D band is absent in the TERS spectra measured at locations 4–6. Integration time: 10 s. Laser power at the sample: 350 µW. Adapted from Kumar et al.52 under a Creative Commons Attribution 3.0 license (https://creativecommons.org/licenses/by/3.0/legalcode).
Supplementary Figure 7 Nanoscale chemical imaging of SWCNTs in air and water using TERS.
a, AFM topography image showing distribution of SWCNTs over a glass substrate. Scale bar: 500 nm. b, Confocal Raman spectrum of a SWCNT showing characteristic Raman bands. Integration time: 10 s. c, TERS image of a SWCNT in water, obtained using the 1591 cm-1 Raman band intensity. Scale bar: 50 nm. Integration time for TERS imaging: 1 s; pixel size: 10 nm; laser power at the sample: 170 µW. d, Intensity profile along the line marked in c with Gaussian curve fits corresponding to the near-field (green) and far-field (red) contributions. Spatial resolution of the TERS image in c is estimated from the FWHM of the green Gaussian curve to be ≈28 nm. Adapted with permission from Kumar et al.29, Royal Society of Chemistry.
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Kumar, N., Weckhuysen, B.M., Wain, A.J. et al. Nanoscale chemical imaging using tip-enhanced Raman spectroscopy. Nat Protoc 14, 1169–1193 (2019). https://doi.org/10.1038/s41596-019-0132-z
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