MICROSOCOPY & IMAGING
IN-DEPTH ANALYSIS
Thomas Dieing, Damon Strom & Eleni Vomhof provide an overview of 3D confocal Raman imaging methodology and technical considerations along with application examples
D
etermining 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. Te 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.
TECHNICAL CONSIDERATIONS Recording high-quality 3D Raman images requires a microscope that can
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Figure 1: 3D confocal Raman image of a moisturising hand cream. Blue: water phase; red, green: two oil phases with different moisturising ingredients dissolved in them. Image parameters: 40 x 40 x 15 µm³ generated from 15 images with 200 x 200 pixels each; 5 ms per spectrum
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[1]
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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 Te 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
Fig. 2 (a): 3D Raman image of conventional butter. Red: fat phase; blue: water phase. 12 x 12 x 4 µm³ generated from 6 images with 200 x 200 pixels each
Fig. 2 (b): 3D Raman image of spreadable butter. Green: fat phase; blue: water phase. 12 x 12 x 3.3 µm³ generated from 5 images with 200 x 200 pixels each
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). Te unsaturation level of fats can be compared by the ratio of
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