Applying Topographic Raman Microscopy

Damon Strom & Eleni Kallis discuss 3D chemical characterisation on curved and rough surfaces for nutritional science applications

In nutritional science and the food industry, it is necessary to examine products in their commercial state. To ensure compliance with standards of quality and to refine manufacturing processes, dietary supplements in tablet form, and food as it is served, must be investigated at high resolution over large areas.

For conventional microscopes this can be impractical as roughly textured or curved samples will present a surface that is only partially in focus. When confocal Raman imaging is employed for its precision and chemical sensitivity, this challenge is particularly acute as the technique strongly rejects all light from outside the focal plane.

WITec solved this problem by developing TrueSurface microscopy, a module for its alpha300 series that integrates an optical profilometer with Raman imaging microscopy for active focus stabilisation. The module traces the sample surface and simultaneously compensates for height differences during the Raman measurement in one-pass operation. Thus, the surface stays in focus while the sample topography is acquired along with the chemical information.

In the two following examples, TrueSurface microscopy is demonstrated with a measurement of the components of an abraded nutritional supplement tablet, and the chemical characterisation of two different substances used to print on candy.

Topographic Raman Imaging Of A Nutritional Supplement Tablet      

The tablet shown in Fig. 1 is coated with a layer of titanium dioxide (TiO2) and has a smooth but curved surface with a pronounced groove in the middle. A large-area topography image of the untreated tablet shows the surface curvature and the more than 300µm-deep cleft in the tablet (Fig. 1A). To investigate the tablet’s chemical components, the TiO2 coating was removed. The surface of the abraded tablet no longer has the cleft, but is still very roughly textured (Fig. 1B). TrueSurface kept the laser in focus during the entire Raman measurement and the topographic Raman image visualises the distribution of the ingredients on the rough surface (Fig. 1C). The nutritional supplement provides a source for magnesium (red) and calcium (green). A binder (blue) and lubricant (yellow) were also identified. 

Topographic Raman Imaging Of Candy Coatings

TiO2 has long been used as a white pigment (E 171) in the food industry. Recently it’s been the subject of increased scrutiny due to concerns that it might be carcinogenic. In May 2021, the European Food Safety Authority (EFSA) announced that the substance can no longer be considered a safe food additive. This led European food manufacturers to replace TiO2 in their products with alternative white pigments.

Raman imaging microscopy is a very effective method for analysing food additives and TiO2 is especially easy to detect as it exhibits a strong Raman signal. This study is a comparison of the same kind of candy produced for two different markets: the EU and the USA. All of the pieces have a white “S'' printed on their colourful coatings (Fig. 2A) and Raman analysis reveals the chemical identities of the pigments used. The measurements were performed with an alpha300 R equipped with a 488nm excitation laser and TrueSurface technology for topographic Raman imaging and active focus stabilisation.

Raman imaging clearly distinguished the print from the surrounding sugar coating in both candies and the topographic Raman images show their curved surfaces (Figs. 2B and 2C). The white pigments forming the S were identified by their Raman spectra as TiO2 in the USA candy (red in Figs. 2B and 2D) and calcium carbonate (CaCO3) in the EU candy (blue in Figs. 2C and 2D). Raman spectroscopy can even differentiate crystal forms of TiO2; The print in the USA candy consists of anatase (Figs. 2B and 2D).


As clearly shown in the experiments above, it is possible to generate sharp, exquisitely detailed Raman images of samples with roughly textured and very contoured surfaces. Over a large area of interest, the TrueSurface module for alpha300 microscopes can ensure a consistent focus while acquiring a Raman spectrum from every image pixel. The visualisation that results can show researchers precisely which compounds are present, their distribution and the relationship between chemical and structural properties. For nutrition and food scientists, this sort of insight is invaluable and the convenience with which it can be obtained makes the module a valuable tool that can offer great utility in their laboratories.

Damon Strom & Eleni Kallis are with WITec

Fig.1 (A): Sample topography of the untreated tablet. Scan area: 10 x 3mm².  (B): Topography of the abraded tablet. Scan area: 5 x 1.5mm². (C): Topographic Raman image of the abraded tablet, revealing the distribution of the tablet’s ingredients on the rough sample surface: magnesium compound (red), calcium compound (CaCO3, green), binder (blue) and emulsifier (yellow). Pink: fluorescence. Scan area: 5 x 1.5mm². Measurement by Andrea Richter – WITec.

Fig.2 (A): Photograph of the investigated candy. (B): Topographic Raman image of the print on the candy produced for the USA. (C): Topographic Raman image of the print on the candy produced for the EU. (D): The “S” prints are colour coded according their Raman spectra, revealing that the white pigment is TiO2 in the USA, and CaCO3 in the EU. Measurement by Dr Ievgeniia Iermak – WITec.

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