Application Notes
Published: 01 Nov 2024 · Last updated: 03 Aug 2026
The mechanical isolation of single-layer graphene with the help of adhesive tape in 2004 kicked off the rapid growth in research on materials consisting of only one or a few layers of atoms, defined as 2D materials. Different from bulk matter, they display interesting semiconducting characteristics that inspired an extensive exploration of their structural, electronic and optical properties. While graphene is still the most prominent example of a single-layered semiconducting material, other 2D materials such as transition metal dichalcogenides (TMDs or TMDCs) and black phosphorus also possess sizable bandgaps. In this application note we will describe both confocal Raman microscopy as a stand-alone technique and correlative Raman microscopy for the comprehensive investigation of 2D materials.
Confocal Raman imaging is an ideal method for studying 2D thin films, revealing their molecular characteristics through a non-destructive and fast procedure. It is used to discern the orientation of their layers and to investigate defects, strain and functionalization, as their Raman properties are determined by molecular bonds, relative orientation and number of layers. Their morphological details can be visualized with other high-resolution microscopy methods such as Scanning Electron Microscopy (SEM). By correlating the information of both approaches, 2D materials can be even more thoroughly analyzed.
WITec has developed microscopes that integrate confocal Raman imaging and optionally Atomic Force Microscopy (AFM), Scanning Near-field Optical Microscopy (SNOM) or SEM into one instrument for highly sensitive and efficient correlative microscopy.
The Raman effect is based on the inelastic scattering of light by the molecules of gaseous, liquid or solid materials. The interaction of a molecule with photons causes vibrations of its chemical bonds, leading to specific energy shifts in the scattered light. Thus, any given chemical compound produces a particular Raman spectrum when excited and can be easily identified by this individual "fingerprint."
Raman spectroscopy is a well-established, label-free and non-destructive method for analyzing the molecular composition of a sample.
In Raman imaging, a confocal microscope is combined with a spectrometer and a Raman spectrum is recorded at every image pixel. The resulting Raman image visualizes the distribution of the sample's compounds. Due to the high confocality of WITec Raman systems, volume scans and 3D images can also be generated.
The Raman effect is extremely weak, so every Raman photon is important for imaging. Therefore WITec Raman imaging systems combine an exceptionally sensitive confocal microscope with an ultra-high throughput spectrometer (UHTS). Precise adjustment of all optical and mechanical elements guarantees the highest resolution, outstanding speed and extraordinary sensitivity — simultaneously!
This optimization allows the detection of Raman signals of even weak Raman scatterers and extremely low material concentrations or volumes with the lowest excitation energy levels. This is an unrivaled advantage of WITec systems.
RISE Microscopy is the combination of confocal Raman Imaging and Scanning Electron Microscopy (SEM). It incorporates the sensitivity of the non-destructive, spectroscopic Raman technique along with the high resolution of electron microscopy.
In Figure 1 the SEM (A) and RISE (C) images of a CVD-grown graphene sheet deposited on a Si/SiO₂ substrate are presented. Layer numbers were deduced from the Raman spectra (Figure 1B). The colors used represent a single layer (blue, dark green) with different grades of defects and more than one layer (light green, red). The RISE image highlights the large amount of defects in the blue area, in good agreement with the high-resolution SEM image.
Fig. 1 — RISE microscopy of graphene. (A) SEM image of a graphene sample. (B) Raman spectra of graphene. (C) SEM image overlaid with the color-coded confocal Raman image. The colors display the graphene layers and wrinkles. Image parameters: 20 x 20 µm², 150 x 150 pixels = 22,500 spectra, integration time: 0.05 s/spectrum, 1 layer (blue, dark green), more than 1 layer (light green, red), Si (yellow).
RISE microscopy reveals the structure as well as the crystalline and exciton dynamics of few-layered transition metal dichalcogenides (TMDs).
CVD-grown monolayers of MoS₂ form triangular two-dimensional crystals. Twin crystals of MoS₂ on SiO₂/Si appear in the SEM image as star-shaped forms (Figure 2B). The Raman spectra of MoS₂ monolayers show the characteristic E'/E₂g and A'₁/A₁g Raman band modes of MoS₂ at 385 and 403 relative wavenumbers (cm⁻¹), respectively (Figure 2A). With an increasing number of layers the two Raman bands drift apart due to in-plane and out-of-plane (inter-layer) vibrations.
Here the green spectrum indicates monolayer MoS₂ while the blue and red spectra signify edges and defects, respectively.
The spectral information was used to create a Raman image which was overlaid on the SEM image to result in a RISE image (Figure 2C). The grain boundaries visible in the SEM image correlate perfectly with the areas where Raman signals indicate wrinkled or overlapping edges, highlighting the effectiveness of RISE imaging for characterizing MoS₂ crystals.
Fig. 2 — RISE microscopy of MoS₂ crystals. (A) Raman spectra of MoS₂: green = 1L MoS₂, blue and red = edges and defects. (B) SEM image of MoS₂ crystals. (C) Color-coded Raman image derived from the spectral information overlaid on the SEM image to produce the RISE image. Sample courtesy of Ting Yu, Nanyang Technological University, Singapore.
WS₂ crystals CVD-grown on a Si/SiO₂ substrate form triangular islands. Atomic Force Microscopy (AFM) was used to measure their layer thickness (Figure 3A). The flakes are comprised of 1, 2 and more layers. The Raman spectrum of WS₂ displays a typical in-plane (E'/Eg) mode at 352 relative wavenumbers (cm⁻¹) and an out-of-plane (A'₁/A₁g) mode at 416 relative wavenumbers (cm⁻¹) [1]. The Raman image of the intensity at 416 relative wavenumbers (cm⁻¹) shows that this signal is strongest in those parts of the crystals that consist of two or more layers and defects, respectively (Figure 3B).
The number of layers and the type of layer stacking influences not only Raman peaks' intensities and positions but also the intensity of photoluminescence peaks and their positions. Due to its direct bandgap, monolayer WS₂ shows a strong PL signal around 635 nm when excited with a 532 nm laser (Figure 3C). This PL signal decreases significantly with increasing layer numbers because the direct bandgap that is present solely in single-layered TMDs allows for direct recombination of electrons and holes (Figure 3D). Also the PL peak position varies and PL lifetime changes with thickness: the more layers the more PL shifts to the red (Figure 3E) and PL lifetime grows shorter (Figure 3F).
Scanning Near-field Optical Microscopy (SNOM) with down to 60 nm spatial resolution was also used for TMD characterization [2,3]. Here, a strong PL signal indicates edge defects in the material, as shown in a high-resolution SNOM image of another WS₂ crystal (Figure 3G). These can also be deduced from PL peak position changes (Figure 3H).
Fig. 3 — Correlative AFM-SNOM-PL imaging of WS₂. (A) AFM image of CVD-grown WS₂ flakes on a Si/SiO₂ substrate with varying numbers of layers (1, 2, >2). (B) Peak intensity map of the 416 cm⁻¹ Raman mode. (C) PL spectrum showing strong signal around 635 nm upon 532 nm excitation. (D) PL intensity map at 635 nm corresponding to material thickness. (E) Position map of PL maxima. (F) PL lifetime map. (G) High-resolution SNOM-PL image of a WS₂ flake revealing edge defects. (H) SNOM-PL peak position image showing edge defects. Sample courtesy of Ting Yu, Nanyang Technological University, Singapore.
Here we present the analysis and RISE microscopy imaging of a multi-layered WS₂ crystal. It shows the typical triangular shape of the material, as well as the heterogeneity of the surface.
A strong WS₂ Raman signal was recorded at approximately 350 relative wavenumbers (cm⁻¹) [4]. Researchers were able to divide this peak into three sub-peaks [1]. They associated a signal at 344 relative wavenumbers (cm⁻¹) with the in-plane E' Raman mode. Using this spectral band we generated a Raman intensity map. Overlaying it with the SEM data produced a RISE microscopy image (Figure 4B).
The Raman signals show that this flake is not homogenously layered. It contains 1L layers and areas with more layers and also defects. The Raman image corresponds very well to the structures revealed by SEM.
Figure 4C shows the overlay of the SEM image and an intensity peak map of PL. The WS₂ crystal displays strong and homogenous PL at about 640 nm only in the 1L areas due to the direct bandgap in monolayer WS₂. Defects seen in the SEM image exhibit PL intensity variations rich in detail. PL intensity corresponds to the intensity of the Raman signal.
Fig. 4 — Correlative RISE and SEM-PL microscopy of WS₂ crystals. (A) SEM image of a WS₂ flake revealing defects on the surface and probably adsorbed molecules. (B) The correlative RISE microscopy image composed of the SEM image and the corresponding Raman intensity map over the 344 cm⁻¹ Raman band, identifying 1L (cyan), 2L (blue) and 3L (red) areas. (C) Overlay of the SEM and PL intensity images showing that the 3L area lacks PL. Excitation wavelength for Raman and PL imaging = 532 nm. Sample courtesy of Ting Yu, Nanyang Technological University, Singapore.
Not every transition metal dichalcogenide (TMD) can be adequately characterized by its vibrational modes between 200 and 500 relative wavenumbers. In some materials typical in-plane (E'/E₂g) and out-of-plane (A'/A'₁g) modes, though changing in intensity with increasing thickness of the material, do not show peak shifts. Also, these primary Raman peaks are highly sensitive to strain and folding [5] and therefore cannot be used to unambiguously determine the number of layers.
However, low-frequency in-plane shear modes (SM) and out-of-plane layer-breathing modes (LBM) are much more informative [6]. To measure Raman spectra close to the laser's excitation wavelength at low wavenumbers between 10 and 50 cm⁻¹, efficient blocking of the Rayleigh line while still transmitting adjacent light is necessary. WITec has developed the RayShield coupler with exactly these features that can be combined with every WITec Raman microscope and is available for 488, 532, 633 and 785 nm lasers.
All measurements of Figure 5 were carried out by Maria O'Brien, Nigel McEvoy and colleagues (Trinity College, Dublin). The low-frequency spectrum shows that 1L MoSe₂ has no Raman peak in this range (Figure 5A). With increasing layer numbers SM and LBM peaks occur, shifting in positions and intensities. Accordingly, the flakes that appear to be the thinnest in the optical image (Figure 5B) are almost invisible in the Raman image, whereas thicker material can be detected by their Raman modes (Figure 5C).
The type of layer stacking also influences the Raman peaks' intensities and positions. In stable, semiconducting MoSe₂ with trigonal prismatic coordination the individual layers can combine in two arrangements, called H and R stacking. These so-called polytypes cannot be distinguished from each other in optical images but can be by their distinctive Raman peaks. Intensity changes in Raman modes at 19 and 24 relative wavenumbers (cm⁻¹) reflect differences in the layer stacking. 2L MoSe₂ displays a typical Raman signal at 19 relative wavenumbers (cm⁻¹) that is more intense in the 2H than in the 3R stacking configuration (Figure 5D).
From the peak maximum map at 24 relative wavenumbers (cm⁻¹) it is clear that 3L MoSe₂ shows the highest intensity in the 3R3R configuration followed by 2H3R and inverted 3R3R stacking (Figure 5E). O'Brien and colleagues concluded that "... low-frequency Raman mapping can distinguish between different stacking configurations rapidly and non-destructively, allowing TMDs in different stacking configurations to be identified and studied without the need for high-resolution imaging."
Fig. 5 — Low-frequency Raman modes of MoSe₂. (A) Low-frequency Raman spectra of 1L, 2L and 3L MoSe₂ with various layer configurations (H and R). (B) Optical image of CVD-grown MoSe₂ revealing differences in thickness and structure. (C) Map of position of maximum peak intensity over the range of 10 to 40 relative wavenumbers (cm⁻¹). (D) Peak intensity map of 2L MoSe₂ at 19 relative wavenumbers (cm⁻¹). (E) Peak intensity map of 3L MoSe₂ at 24 relative wavenumbers (cm⁻¹). Images courtesy of O'Brien, McEvoy and colleagues, Trinity College, Dublin, Ireland.
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