Krzysztof Czamara

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.

Unsaturated lipid bodies as a hallmark of inflammation studied by Raman 2D and 3D microscopy

Endothelial HMEC-1 cells incubated with pro-inflammatory cytokine TNF-α for 6 and 24 hours were studied as a model of inflammation using Raman imaging. Striking changes in distribution, composition and concentration of cellular lipids were observed after exposure to TNF-α compared to the control. In particular, 3D Raman imaging revealed a significant increase in the amount of lipid entities formed under inflammation. Lipid bodies were randomly distributed in the cytoplasm and two types of droplets were assembled: more saturated one, in spectral characteristics resembling phosphatidylcholine and saturated cholesteryl esters, observed also in the control, and highly unsaturated one, containing also cholesterols, being a hallmark of inflamed cells. The statistical analysis showed that the number of lipid bodies was significantly dependent on the exposure time to TNF-α. Overall, observed formation of unsaturated lipid droplets can be directly correlated with the increase in production of prostacyclins - endogenous inflammation mediators.

Vascular diseases investigated ex vivo by using Raman, FT-IR and complementary methods

This work shows the application of vibrational spectroscopy supported by other complementary techniques in analysis of tissues altered by vascular diseases, in particular atherosclerosis. The analysis of atherosclerotic plaque components, as well as label-free imaging of vessels and identification of biochemical markers of endothelial dysfunction are reported. Additionally, the potential of vibrational spectroscopy imaging in following the disease progression (including calcification) and pathological changes in heart valves is described. The presented research shows the effectiveness of techniques used in the biochemical studies of altered tissues and summarizes their capabilities in research on vascular diseases. The scope of the paper is to collect previously published work connected with the application of Raman spectroscopy, FT-IR spectroscopy and complementary methods for the investigation of vascular diseases ex vivo and presenting it in a comprehensive overview.

Polypyridyl substituted BODIPY derivatives; water switchable imaging probes that exhibit halogen substituent dependent localisation in live cells

The synthesis and photophysical properties of water responsive 1,10-phenanthrolyl and 2,2′-bipyridyl substituted BODIPY derivatives prepared as lipid probes for cell imaging are reported. These compounds exhibit intense emission in non-aqueous media that is reversibly extinguished in aqueous media. Halogen substitution at the BODIPY indacene core decreases the emission quantum yields and causes red spectral shifts of emission maxima of the order H > Br > I. The emission was quenched on binding of the phenanthrolyl or bipyridyl to cations Fe2+, Cu2+ and Zn2+. The origin of the water switching effect and the impact of halogen substitution was investigated by modelling the electronic structure of the fluorophore using DFT methods. All compounds showed excellent permeability to live cells and were found, under imaging conditions, to generally exhibit low cytotoxicity. The absence of emission in the aqueous environment facilitated the collection of high contrast images from membranous regions and lipid droplets in live cells. The staining pattern in HeLa cells was found to depend on halogen substitution. Across both bpy and phen derivatives the halogenated probes showed the strongest targeting of lipid droplets within cells whilst the parent unsubstituted compounds were more widely dispersed in the cytoplasm. Resonance Raman imaging was used to map the distribution of probes within the cell and confirmed that the compounds showed strong co-localisation with lipid rich regions of the cell.

Small and Large Molecules Investigated by Raman Spectroscopy

This chapter presents selected techniques of Raman spectroscopy, i.e. Raman imaging, Raman optical activity (ROA), and surface-enhanced Raman spectroscopy (SERS), and gives an overview on their biomedical applications. The current state of the art in the research on chiroptical compounds of biomedical importance, as well as the study on early apoptosis and inflammation processes occuring in the endothelium, is presented. The pathophysiology of the endothelium is discussed based on the example of Raman imaging results for primary cells and cell cultures. Moreover, the comparison of classical Raman imaging, application of optical fiber probes, and immuno-SERS nanosensors in detection of marker proteins in ex vivo studies is discussed.

Impact of cell cycle dynamics on pathology recognition: Raman imaging study

Confocal Raman imaging combined with fluorescence-activated cell sorting was used for in vitro studies of cell cultures to look at biochemical differences between the cells in different cell phases. To answer the question what is the impact of the cell cycle phase on discrimination of pathological cells, the combination of several factors was checked: a confluency of cell culture, the cell cycle dynamics and development of pathology. Confluency of 70% and 100% results in significant phenotypic cell changes that can be also diverse for different batches. In 100% confluency cultures, cells from various phases become phenotypically very similar and their recognition based on Raman spectra is not possible. For lower confluency, spectroscopic differences can be found between cell cycle phases (G0 /G1 , S and G2 /M) for control cells and cells incubated with tumor necrosis factor alpha (TNF-α), but when the mycotoxin cytochalasin B is used the Raman signatures of cell phases are not separable. Generally, this work shows that heterogeneity between control and inflamed cells can be bigger than heterogeneity between cell cycle phases, but it is related to several factors, and not always can be treated as a rule.

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.