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Application Notes
Author: Admir Masic
Published: 01 Mar 2025 · Last updated: 03 Aug 2026
The Raman effect is based on light interacting with the chemical bonds of a sample. Due to vibrations in the chemical bonds this interaction causes a specific energy shift in the back scattered light which appear in a unique Raman spectrum through which the molecular components of a sample can be detected. The confocal Raman Imaging technique combines Raman spectroscopy with a confocal microscope. Thus the spatial distribution of the chemical components within the sample are imaged. High-resolution confocal Raman microscopes acquire the information of a complete Raman spectrum at every image pixel and achieve a lateral resolution at the diffraction limit (circa λ/2 of the excitation wavelength). A confocal microscope setup is furthermore characterized by an excellent depth resolution and features the generation of 3D Raman images and depth profiles.
Raman topographic imaging is enabled with the TrueSurface® option. 3D chemical characterization on rough, inclined or irregularly-shaped samples can be carried out precisely along a surface without requiring sample preparation.
Confocal Raman microscopes strongly reject out of focus light. The TrueSurface® module allows the surface of even large or coarsely textured samples to remain in constant focus, delivering the full benefits of the technique in terms of nanometer depth resolution and stray light suppression. As the sensor actively monitors and maintains a set distance between the objective and sample surface, its closed-loop operation can eliminate any thermally-induced variations during measurements with long integration times.
The Raman effect is extremely weak, so every Raman photon is important for imaging. WITec Raman imaging systems combine highest resolution, outstanding speed and extraordinary sensitivity – simultaneously.
In archeological research, various specimens such as paintings, mummies, antique objects or fabrics are regularly investigated. Those samples are often unique and require specialized treatment when analyzed. This application note introduces topographic confocal Raman Imaging as a flexible, noninvasive and non-destructive tool for the chemical and molecular characterization of archaeological specimens.
Raman spectroscopy has long been employed as a spectroscopic tool for materials characterization in the field of cultural heritage. Its non-invasive and non-destructive nature make it an attractive candidate for both material constituents and damage assessment and monitoring. However, specific requirements associated with objects of interest such as the impossibility of sampling as well as frequent autofluorescence associated with ageing related degradation products, made the extraction of useful Raman information extremely difficult.
In this application note it is shown how to overcome some of the mentioned challenges by applying the most advanced Raman imaging techniques. The sampling issue was addressed by applying a large-area imaging approach in correlation with the state-of-the-art profilometric measurement. This patent-pending method was recently developed by WITec. In such a way large objects (in terms of dimensions) can be automatically scanned and surface chemical images acquired.
The fluorescence problem is also an important one and cannot be easily solved. Recent advances in surface-enhanced Raman spectroscopy (SERS) enable the extraordinary enhancement of the Raman signal that can be seen above the autofluorescence. However, the limitation of the enhancement to only some types of the chemical compounds makes SERS specific to a limited number of applications. Here we suggest another approach to address the fluorescence issue: It combines the collection of the non-resonant Raman signal with a diffraction-limited spatial resolution. This approach allows the spatial separation of fluorescence affected degradation products and fluorescence-free intact particles.
All results were acquired with a WITec alpha500 R microscope for large-area confocal Raman imaging. Furthermore an Ultra-High-Throughput Spectrometer UHTS300 with a 600 g/mm grating (BLZ 750 nm) in combination with a 785 nm excitation laser, a deep depletion CCD camera, and a 100x NA 0.9 air objective was used. The TrueSurface® topographic confocal Raman imaging extension was directly integrated with the objective turret, which allows the acquisition of topographic and Raman data without any sample repositioning.
A piece of an ancient wall painting served as the specimen for all investigations. The fragment, part of a private collection, displays several layers of original polychrome paintings in relatively precarious state of preservation. The sample is shown in Figure 1 with the areas of interest that have been characterized indicated by the arrows. For the investigation of the area of interest 1 the sample was not prepared prior to the measurements. Due to the absorption of the sample the laser light is unable to penetrate deep through the layers. Therefore a small section of the sample was additionally prepared and examined in order to generate vertical depth-profiles of the sample layers in the z-direction.
Fig. 1 — Photograph of the sample with the areas of interest labelled.
It is clear from Figure 1 that the area of interest 1 shows a strong topography and can therefore not be measured by conventional confocal Raman microscopy. Therefore TrueSurface® confocal Raman imaging was used to gather the information from this area. In this mode the topography is first recorded using an optical profilometer. By simply rotating the objective turret the system is changed to Raman mode and the recorded surface can then be used to ensure that the sample always stays in focus.
Prior to the measurement a white light stitching image was recorded to locate the area of interest. The white light image is shown in Figure 2, the area from which the TrueSurface® confocal Raman imaging was consecutively recorded is marked with a black rectangle.
Fig. 2 — White light stitching image of the area of interest 1 of the sample.
Figure 3 shows the sample's topography recorded over an area of 5×10 mm2 with the TrueSurface® profilometric sensor. Please note that a change of over 500 µm in the focus position was observed. This variation in height would normally prevent measurements in standard confocal Raman imaging mode due to the surface being frequently out of focus.
Fig. 3 — Topography of the sample recorded using the TrueSurface® extension of the alpha300 microscope series.
Following the recording of the surface a large area confocal Raman image was recorded using the topographic information to trace the z-position. The scanned area in Raman imaging mode was again 5×10 mm2 with 150×300 Raman spectra recorded. The integration time per spectrum was 1 s.
The most prominent red painting layer in the analyzed area could easily be identified as cinnabar (HgS). Cinnabar is a precious red pigment characteristic of the Roman period. Minium, a ground version of cinnabar, was considered the Romans' most valuable pigment and it was used in many ambitious works, including some Pompeiian masterpieces, and demonstrated great wealth. In fact, the price of Spanish minium used in Pompeii was capped by law at 150 sesterces per kilogram to stop it rising out of all proportion (Pigment Compendium: A Dictionary and Optical Microscopy of Historical Pigments, Eastaugh et al., 2008, Elsevier). The location at which cinnabar was detected by confocal Raman imaging is overlaid in red onto the white light image in Figure 4A. By comparing the location of the cinnabar with the red painted areas shown in Figure 4B, it can be clearly seen that the distribution of cinnabar is closely correlated. Furthermore an appearance of cinnabar can be detected at the edges of the layers indicating that a cinnabar-based preparation had been applied which evinces the importance and richness of the analyzed fragment.
Fig. 4 — (A) White light image of the area of interest 1 with the locations at which cinnabar pigment could be found overlaid in red. (B) Photograph of the area of interest 1.
In addition to cinnabar, other components of the investigated area can be identified by their unique Raman spectra. Figure 5 shows the primary spectral components of the Raman analysis. Thus calcite, carbon black, and an unknown phase can be additionally detected.
Analyzing the entire data set allowed the generation of a color-coded image which could then be overlaid onto the topography as shown in Figure 6. The colors of the areas correspond to the colors of the spectra shown in Figure 5.
Fig. 5 — Spectral components Raman analysis. The spectra could be identified as: Red = Cinnabar; Green = White Chalk (CaCO
3
, Calcite); Blue = Carbon Black; Turquoise = unknown phase.
Fig. 6 — Color-coded Raman image overlaid onto the topography of the area of interest 1. The colors of the image correspond to the colors in Figure 5.
Next the area of interest 2 was investigated. Here a distinctly different red color can be seen compared to the bright red color investigated in area of interest 1. This observation could be confirmed by the spectral analysis. The Raman spectra shown in Figure 7 identifies red ocher as the main chemical component of the dark red color of area of interest 2. For comparison the spectrum of cinnabar is also shown in Figure 7.
Fig. 7 — Raman Spectra of the bright red color (identified as minium) and of the dark red area (identified as red ocher).
For the generation of a confocal Raman image of area of interest 2 an area of 70×70 µm2 was measured with an integration time of 133 ms per spectrum. The resulting Raman image consists of 70×70 pixels with the information of a complete Raman spectrum at every image pixel.
The color-coded Raman image was overlaid onto the white light image as shown in Figure 8. The colors correspond to the colors of the spectra shown in Figure 9 with ocher crystals shown in red and calcium carbonate binder in blue and green, indicating a high level of fluorescence.
Fig. 8 — Color-coded Raman image overlaid on the white light image of the area. Color coding: Ocher crystals – Red; Calcium carbonate binder mixed with ocher – Blue; High level of fluorescence – Green.
Fig. 9 — Spectra extracted from the confocal Raman image scan on area of interest 2. The colors correspond to the colors shown in Figure 8.
As outlined earlier, the penetration depth of the laser light into the material is very limited. For this reason cross sections were prepared. The cross sections allow the visualization of the succession of individual layers in z-direction (vertically). Therefore a small sampling is necessary and the fragment taken is embedded in a resin then polished perpendicular to the plane of the painting surface. In this way a suitable specimen cross section is obtained for observation of the individual layers by confocal Raman microscopy.
Figure 10 shows the overlay of the combined Raman image onto the white light image. The Raman image was recorded with 175×175 µm2 (175×175 pixels). The integration time was 133 ms per spectrum and the light was collected through a pinhole with a diameter of 100 µm. The gray frame in Figure 10 shows the zoomed area that was recorded in a second scan. The colors of the overlay image correspond to the colors of the Raman spectra in Figure 11. The spectra in Figure 11 show calcite characteristic profiles in two different configurations/orientations (green and blue spectra). The minium is again identified as the red pigment used (red spectrum) as well as a very weak signal of amorphous carbon originating from a thin layer at the surface (yellow color).
Fig. 10 — Color-coded confocal Raman image overlaid on a white light image of a cross section of the sample. The colors correspond to the colors of the spectra in Figure 11. The frame indicates the area at which a consecutively second scan was performed.
Fig. 11 — Spectra extracted from the confocal Raman image scan shown in Fig. 10. Green and blue: calcite in two different configurations/orientations; Red: Minium; Yellow: Amorphous carbon.
With a high resolution scan (marked with a gray frame in Figure 10) the area at which the minium color particles can be prominently seen was imaged again. Figure 12 shows the combined color-coded Raman image and Figure 13 the corresponding spectra. In this area a further component of unknown origin (blue color) could be extracted from the spectra using spectral de-mixing. From the image it can be seen that the color particles (in red) cluster into larger and smaller agglomerates and the smallest detectable in the image are on the order of half a micrometer (marked with an arrow in Figure 12) which corresponds to the resolution limit for a 785 nm laser used in conjunction with a 100x NA 0.9 objective (0.51 × λ/NA ≈ 445 nm [FWHM]).
Fig. 12 — Combined Raman image of the zoomed scan of the cross section. The colors correspond to the colors of the spectra shown in Fig. 13. The arrows mark the small agglomerates on the order of half a micrometer.
Fig. 13 — Raman Spectra extracted from the zoomed scan of the cross section. Red: Minium; Blue: Unknown origin; Green: Calcite.
The recent instrumental developments in Raman microscopy enable remarkable new applications in archaeological sciences. The possibility given by TrueSurface® of non-invasively and non-destructively studying large areas of ancient objects with irregular surfaces is a breakthrough in the field of cultural heritage research. It further allows investigation of extremely precious samples in situ without the need for any sampling or sample preparation. When sampling is possible, the sub-micron resolution large area imaging of a cross section further improves the quality of the analytical output and allows detailed characterization of primary material constituents on the micron scale. In conclusion, confocal Raman imaging is a valuable tool for the identification of minute quantities of a material that can provide the archaeologist, conservator and other researchers with invaluable information regarding provenance, manufacturing technology, state of preservation, and in some instances, authenticity.
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