Marta Z. Pacia

Raman and infrared spectroscopy of carbohydrates: A review

Carbohydrates are widespread and naturally occurring compounds, and essential constituents for living organisms. They are quite often reported when biological systems are studied and their role is discussed. However surprisingly, up till now there is no database collecting vibrational spectra of carbohydrates and their assignment, as has been done already for other biomolecules. So, this paper serves as a comprehensive review, where for selected 14 carbohydrates in the solid state both FT-Raman and ATR FT-IR spectra were collected and assigned. Carbohydrates can be divided into four chemical groups and in the same way is organized this review. First, the smallest molecules are discussed, i.e. monosaccharides (d-(-)-ribose, 2-deoxy-d-ribose, l-(-)-arabinose, d-(+)-xylose, d-(+)-glucose, d-(+)-galactose and d-(-)-fructose) and disaccharides (d-(+)-sucrose, d-(+)-maltose and d-(+)-lactose), and then more complex ones, i.e. trisaccharides (d-(+)-raffinose) and polysaccharides (amylopectin, amylose, glycogen). Both Raman and IR spectra were collected in the whole spectral range and discussed looking at the specific regions, i.e. region V (3600-3050cm-1), IV (3050-2800cm-1) and II (1200-800cm-1) assigned to the stretching vibrations of the OH, CH/CH2 and C-O/C-C groups, respectively, and region III (1500-1200cm-1) and I (800-100cm-1) dominated by deformational modes of the CH/CH2 and CCO groups, respectively. In spite of the fact that vibrational spectra of saccharides are significantly less specific than spectra of other biomolecules (e.g. lipids or proteins), marker bands of the studied molecules can be identified and correlated with their structure.

Raman microscopy as a novel tool to detect endothelial dysfunction

Raman microscopy, a label-free method with high spatial resolution, shows growing potential in various fields of medical diagnostics. Several proof-of-concept studies related to the application of Raman microscopy to detect endothelial dysfunction are summarized in this work. Both ex vivo measurements of the tissues in the murine models of endothelial pathologies, as well as in vitro investigations of the cell cultures in the context of cellular transport, drug action and inflammation processes are discussed. The future directions in application of Raman spectroscopy-based methods in such studies are also described.

Raman, AFM and SNOM high resolution imaging of carotene crystals in a model carrot cell system

Three non-destructive and complementary techniques, Raman imaging, Atomic Force Microscopy and Scanning Near-field Optical Microscopy were used simultaneously to show for the first time chemical and structural differences of carotenoid crystals. Spectroscopic and microscopic scanning probe measurements were applied to the released crystals or to crystals accumulated in a unique, carotenoids rich callus tissue growing in vitro that is considered as a new model system for plant carotenoid research. Three distinct morphological crystal types of various carotenoid composition were identified, a needle-like, rhomboidal and helical. Raman imaging using 532 and 488 nm excitation lines provided evidence that the needle-like and rhomboidal crystals had similar carotenoid composition and that they were composed mainly of β-carotene accompanied by α-carotene. However, the presence of α-carotene was not identified in the helical crystals, which had the characteristic spatial structure. AFM measurements of crystals identified by Raman imaging revealed the crystal topography and showed the needle-like and rhomboidal crystals were planar but they differed in all three dimensions. Combining SNOM and Raman imaging enabled indication of carotenoid rich structures and visualised their distribution in the cell. The morphology of identified subcellular structures was characteristic for crystalline, membraneous and tubular chromoplasts that are plant organelles responsible for carotenoid accumulation in cells.

Lipids, hemoproteins and carotenoids in alive Rhodotorula mucilaginosa cells under pesticide decomposition – Raman imaging study

Various species of yeasts are gaining attention as producers of nutraceuticals and biofuels and due to their capacity to biodegrade chemical waste. Rhodotorula mucilaginosa is one of the most oleaginous species of yeast, an efficient de novo carotenoid producer and was reported to be capable of decomposing of organic pesticides. In this work we studied the influence of a toxic pesticide, diazinone, on production of storage (lipids) and protective (carotenoids, hemoproteins) compounds by Rh. mucilaginosa alive cells with the help of Raman imaging. It occurred that the yeast in non-oleaginous phase and aerobic environment was rich in carotenoids and their level increased significantly under incubation with diazinone, while anaerobic environment resulted in production of both carotenoids and hemoproteins and the level of the latter decreased under the influence of the pesticide. For yeasts in oleaginous phase, it was concluded that lipid production (via triggering of NAD+ accumulation and increase of the NO level) resulted in nitrosative stress leading to flavohemoprotein synthesis and was associated with the increase of the mitochondrial activity.

Raman Imaging of Biomedical Samples

Fluorescence microscopy, a gold standard in the tissue or cell imaging, has several drawbacks, with a severe one being its disability to elucidate the chemical characteristics of the sample. Confocal Raman microscopy is a tempting label free alternative to fluorescence and a prospective future method of medical diagnostics. Raman measurements of tissues can provide information about their chemical composition, i.e. main components but also specific compounds, and their changes upon the development of patholog or its treatment. The analysis is based on characteristic marker bands, provided they can be identified in the spectra, or by more common approach using chemometrics. Moreover, Raman microscopy can be used to detect small biochemical alterations and their respective distribution in a single cell or even at sub-cellular level. The impact of various factors, including the uptake of drugs, bioactive compounds or non-chemical stressors can be observed using such investigations of cells.

Raman spectroscopy as a novel tool for fast characterization of the chemical composition of perivascular adipose tissue

One of the new targets of untapped therapeutic potential is perivascular adipose tissue (pVAT). pVAT releases a plethora of pro- and anti-inflammatory agents and is involved in the inflammatory response of the vascular wall, playing a key role in various cardiovascular pathologies. Both fiber optic Raman spectroscopy with a high-spatial resolution probe and Raman microscopy were applied to study various types of adipose tissue with the emphasis on pVATs of the thoracic and abdominal aorta and the mesenteric artery, as well as epididymal and interscapular adipose tissue for comparison. Our results demonstrated that the lipid unsaturation degree was clearly distinct in various types of adipose tissue and was influenced by the age of animals. In particular, the basal unsaturation level of pVATs of the abdominal aorta and the mesenteric artery was considerably higher than that of the thoracic aorta and a significant increase of the unsaturation level of pVAT with age was observed showing that aging has a considerable impact on the pVATs chemical composition. Overall, our results show that Raman spectroscopy is a sensitive tool to determine the perivascular adipose tissue chemical composition that appears to be vascular-bed specific.