Map of the Month – 3D Raman Imaging of Butter Emulsions
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Welcome to Edinburgh Instruments’ blog celebrating our work in Raman, Photoluminescence, and Fluorescence Lifetime Imaging. Every month, we aim to highlight our pick for Map of the Month to show how our Raman and fluorescence spectrometers can be used to reveal all the hidden secrets in your samples.??
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The Edinburgh Instruments RM5 Confocal Microscope can image the distribution of components within a 3D volume. Raman spectra are recorded throughout an X-Y-Z cuboid, which provides detailed 3D structural and chemical information. In this Map of the Month, the RM5 is used to image the distribution of components within butter and determine the underlying morphological and chemical differences between spreadable and non-spreadable products.?
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Butter is a water-in-oil (w/o) emulsion made from churning milk or cream. This process allows butterfat, present as microscopic globules in milk and cream, to amalgamate, which creates a semi-solid product that can be separated from buttermilk. Variations in the production method will make butter with different fat compositions and, therefore, different consistencies. To probe the underlying chemical differences between butter products with different consistencies, 3D Raman imaging was used. Volumetric images of the samples were acquired with a 532 nm laser, a 100 μm confocal pinhole and using the FastMAP (0.1 s per Raman spectrum) feature of the Ramacle software. The confocal pinhole in the RM5 is essential for detailed 3D imaging, spatially filtering the Raman scatter by reducing the scatter collection volume within the sample.??
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The first butter analysed was a 78% butterfat spreadable product. The 3D map in (a) shows that the butter microstructure consists of numerous large water globules (blue) dispersed throughout an organic phase (red). These assignments were made based on vibrational fingerprints in the spectra recorded at each point in the map. An 83% butterfat non-spreadable product was then imaged, (b). The water content is the most significant morphological difference between the non-spreadable and spreadable products. The non-spreadable butter, which is semi-solid at room temperature, contains fewer and smaller microscopic water globules than the more easily spreadable product, which is closer to a liquid at room temperature.??
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The spectra from the organic phase were dominant in spectral signatures attributable to fats and proteins, (c). This phase was imaged using the intensity of the high-wavenumber region between 2800 cm-1 and 3000 cm-1, typical of CH2, CH, and CH3 vibrations.? The water globules were imaged using the intensity of a broad vibrational mode at approximately 3450 cm-1, characteristic of O-H stretching.??
Raman microscopy can also detect differences in the fatty acid content between different butter products. To investigate differences between the spreadable and non-spreadable butter, spectra were recorded at multiple points with a 2 mm confocal pinhole diameter applied for bulk analysis. Panel (d) shows the fingerprint regions of Raman spectra measured from the spreadable (red) and non-spreadable (green) butter. Differences in the fatty acid content can be determined using the ratio between the C=C stretching band ca. 1656 cm-1 and the CH2 twisting band ca. 1442 cm-1. This is often termed the unsaturation ratio because unsaturated fatty acids have a higher proportion of C=C bonds than saturated fatty acids compared to CH2 groups. The spreadable butter had a higher concentration of C=C bonds than the non-spreadable butter. The increased malleability of this butter can, therefore, also be attributed to an increased amount of unsaturated fatty acids. Interestingly, the non-spreadable butter had a higher concentration of carotenoids, which was determined using the increased intensity of the signature resonant Raman bands at 1158 cm-1 and 1524 cm-1.4 These are fat-soluble nutrients that are linked to the type of feed the cattle graze on and are associated with various health benefits such as improved cognitive function, eye health, and increased antioxidant levels.??