Thomas Dieing, Damon Strom & Eleni Vomhof provide an overview of 3D confocal Raman imaging methodology and technical considerations along with application examples
Determining the chemical composition of samples at high spatial resolution and in three dimensions is a vital analytical capability in many fields of application. For researchers in the pharmaceutical, cosmetics or food industries, 3D confocal Raman microscopy can peer deep into emulsions and liquids to extract detailed information regarding the molecular characteristics that influence the properties of products.
3D confocal Raman imaging
In confocal Raman microscopy, a complete Raman spectrum is acquired at each pixel of a measurement and the sample components’ chemical properties are colour-coded in the resulting Raman image, visualising their spatial distribution. By recording a series of 2D Raman images at successive focal planes, 3D representations can be generated of transparent materials. Such experiments can be performed quickly, nondestructively and without requiring labelling or other specialised sample preparation.
A cosmetic cream serves here as an example. The 3D Raman image shows that the emulsion consists of two different oil phases which form droplets in the water phase (Fig. 1). Solid samples, also in other research areas, can be analysed using the same approach. For example, liquid or gas inclusions in geological samples or defects in semiconducting materials can be characterised without damaging the surrounding material.
Recording high-quality 3D Raman images requires a microscope that can achieve the highest spatial resolution, signal sensitivity, and acquisition speed – simultaneously.
For generating high-resolution 3D images, the microscope’s confocality is particularly important, as light from outside of the focal plane must be minimised. With a good confocal Raman microscope, a resolution below 300 nm laterally and 900 nm axially is achievable when using a 532 nm excitation laser.
As a 3D Raman image consists of thousands or even millions of spectra, high acquisition speed is crucial for recording the data within reasonable time. With Raman microscopes optimised for photon throughput and sensitivity, more than 1,000 spectra can be recorded per second. For example, the 3D image in Fig. 1 was recorded in under 1.5 hours.
All presented measurements were performed with a WITec alpha300 Raman microscope.
3D Raman analysis of butter
The following example illustrates how 3D Raman imaging can reveal chemical differences underlying macroscopic properties such as the consistency of butter. 3D Raman images of conventional butter and a more spreadable product were recorded for comparison (Fig. 2a, b).
Both products are water-in-oil emulsions, as expected, but the 3D views reveal differences. Compared to the spreadable butter, the water forms smaller droplets in the conventional one and the overall water content seems lower.
Additionally, the two products contain different types of fat and oil, as revealed by evaluating the Raman spectra of their fatty phases (Fig. 2C). The unsaturation level of fats can be compared by the ratio of the C=C stretching mode (approximately 1,655 cm-1) and the CH2 scissoring mode (approximately 1,444 cm-1) and this ratio was higher for the spreadable butter than for the conventional sample. This indicates a higher amount of unsaturated fatty acids in the spreadable butter, which likely contributes to the improved spreadability. Fat-spreads with a high amount of unsaturated amino acids and a reduced fat content are also considered healthier than conventional butter.
3D confocal Raman imaging is a powerful analytical tool for nondestructively characterising the molecular composition of sample volumes, as illustrated by emulsion measurements. For generating high-quality 3D chemical images, a Raman microscope that simultaneously provides high spatial resolution and confocality, signal sensitivity and acquisition speed is required.
 T. Dieing (2018). In: J. Toporski, T. Dieing, O. Hollricher (eds.) Confocal Raman Microscopy. Springer Series in Surface Sciences 66, pp. 121 – 153. Springer, Cham. DOI: 10.1007/978-3-319-75380-5_6
 K. Czamara et al. (2014) J. Raman Spectrosc. 46: 4 – 20. DOI: 10.1002/jrs.4607
Thomas Dieing, Damon Strom & Eleni Vomhof are with WITec