Malgorzata Baranska

Identification of secondary metabolites in medicinal and spice plants by NIR-FT-Raman microspectroscopic mapping

This paper demonstrates the special potential of vibrational NIR FT Raman microspectroscopy for the study of fennel fruits, chamomile inflorescence and curcuma roots to obtain detailed information about their microstructure and chemical composition. Microscopic Raman maps of fennel fruits demonstrate that anethole, which is the main essential oil component, is present in the whole mericarp with highest concentration at the top of the fruit. In situ measurements obtained of the essential oil cells are dominated by two bands observed at 1657 cm(-1) and 1609 cm(-1) which are characteristic for anethole. Raman images of chamomile inflorescence show that spiroethers, identified by significant bands between 2150 and 2250 cm(-1), are accumulated in the middle part of the flower head. Due to the intense curcumin bands in the Raman spectrum of curcuma root, the distribution of this dyeing substance can be clearly determined; highest concentration of curcumin was observed on the core of the root.

In situ Raman imaging of astaxanthin in a single microalgal cell

Raman imaging is shown to be a highly selective and sensitive method of studying in situ and in vivo astaxanthin distribution, concentration and molecular structure in the cyst form of the unicellular microalgae Haematococcus pluvialis.

Spatial tissue distribution of polyacetylenes in carrot root

The presented results show the usefulness of Raman spectroscopy in the investigation of polyacetylenes in carrot root. The components are measured directly in the plant tissue without any preliminary sample preparation. Compared with the strong polyacetylene signals the spectral impact of the surrounding biological matrix is weak, except for carotenoids, and therefore it does not contribute significantly to the obtained results. Three different Raman mapping techniques applied here have revealed essential information about the investigated compounds. Using point acquisition several spectra have been measured to demonstrate the complex composition of the polyacetylene fraction in carrot root. The molecular structures of falcarinol, falcarindiol and falcarindiol 3-acetate are similar but their Raman spectra exhibit differences demonstrated by the shift of their -C triple bond C- mode. Line mapping performed along the diameter of transversely cut carrot roots has been used to investigate the relative concentration of polyacetylenes and carotenoids. An area map provides detailed information regarding the distribution of both components. It has been found that high accumulation of polyacetylenes is located in the outer section of the root, namely the pericyclic parenchyma, and in the phloem part close to the secondary cambium. The highest concentration of carotenes is seen in the immediate vicinity to polyacetylene conglomerates.

Structural Changes of Carotenoid Astaxanthin in a Single Algal Cell Monitored in Situ by Raman Spectroscopy

The changes of structure of astaxanthin (AXT), a superpotent antioxidant, upon thermal stress were investigated in unicellular microalgae Haematococcus pluvialis by measuring Raman spectra in situ and analyzing obtained results with DFT calculations. Although no visual changes are observed in the Haematococcus cells upon heating, discernible changes in Raman spectra occur from -100 °C systematically up to 150 °C. The exponential increase of the Raman shift of the ν C═C band at ca. 1520 cm(-1) along with the change of the intensity ratio of bands at 1190 and 1160 cm(-1) is observed, that correlates with the changes predicted by calculations for astaxanthin conformers ordered by decreasing energy. It is assumed that AXT molecules, initially in the form of H-aggregates with the trans conformations of the end-rings, interconvert toward more stable gauche forms upon thermal stress of the algae. The applied approach enables one to follow structural changes of the carotenoid upon temperature stress both in a single algal cell and in a multicellular sample in situ. Obtained information might be of use to improve the industrial process of extraction of AXT in its most bioavailable form.

Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models.

Comparisons of 2D and 3D cell culture models in literature have indicated differences in cellular morphology and metabolism, commonly attributed the better representation of in vivo conditions of the latter cell culture environment. Thus, interest in the use of 3D collagen gels for in vitro analysis has been growing. Although comparative studies to date have indicated an enhanced resistance of cells on collagen matrices against different toxicants, in the present study it is demonstrated that non-adapted protocols can lead to misinterpretation of results obtained from classical colorimetric dye-based cytotoxic assays. Using the well established Alamar blue assay, the study demonstrates how the transfer from 2D substrates to 3D collagen matrices can affect the uptake of the resazurin itself, affecting the outcome of the assay. Using flow cytometry, it is demonstrated that the cell viability is unaffected when cells are grown on collagen matrices, thus the difference seen in the fluorescence is a result of a dilution of the resazurin dye in the collagen matrix, and an increased uptake rate due to the larger cell surface exposed to the surrounding environment, facilitating more effective diffusion through the cellular membrane. The results are supported by a rate equation based simulation, verifying that differing uptake kinetics can result in apparently different cell viability. Finally, this work highlights the feasibility to apply classical dye-based assays on collagen based 3D cell culture models. However, the diffusion and bioavailability of test substances in 3D matrices used in in vitro toxicological assays must be considered and adaption of the protocols is necessary for direct comparison with the traditional 2D models. Moreover, the observations made based on the resazurin dye can be applied to drugs or nanoparticles which freely diffuse through the collagen matrices, thus affecting the effective concentration exposed to the cells.

Raman Imaging Providing Insights into Chemical Composition of Lipid Droplets of Different Size and Origin: In Hepatocytes and Endothelium

In this work, 3D linear Raman spectroscopy was used to study lipid droplets (LDs) ex vivo in liver tissue and also in vitro in a single endothelial cell. Spectroscopic measurements combined with fluorescence microscopy and/or histochemical staining gave complex chemical information about LD composition and enabled detailed investigations of the changes occurring in various pathological states. Lipid analysis in fatty liver tissue was performed using a dietary mouse model of liver steatosis, induced by a high fat diet (HFD). HFD is characterized by a high percentage of calories from saturated fat (60%) and reflects closely the detrimental effects of dietary habits responsible for increased morbidity due to obesity and its complications in well-developed Western societies. Such diets lead to obesity, hyperlipidemia, insulin resistance, and steatosis that may also be linked to endothelial dysfunction. In the present work, Raman spectroscopy was applied to characterized chemical composition of lipid droplets in hepatocytes from mice fed HFD and in the endothelium treated with exogenous unsaturated free fatty acid (arachidonic acid). The results demonstrate the usefulness of Raman spectroscopy to characterize intracellular lipid distribution in 2D and 3D images and can be used to determine the degree of saturation. Raman spectroscopy shows the potential to be a valuable tool for studying the role of LDs in physiology and pathology. The method is generally applicable for the determination of LDs of different size, origin, and composition. Moreover, for the first time, the process of LD formation in the endothelium was detected and visualized in 3D.

Visualization of the biochemical markers of atherosclerotic plaque with the use of Raman, IR and AFM.

In this work, we describe a methodology to visualize the biochemical markers of atherosclerotic plaque in cross sections of brachiocephalic arteries (BCA) taken from ApoE/LDLR(-/-) mice. The approach of the visualization of the same area of atherosclerotic plaque with the use of Raman, IR and AFM imaging enables the parallel characterisation of various features of atherosclerotic plaques. This support to the histochemical staining is utilized mainly in studies on mice models of atherosclerotic plaques, where micro and sub-micro resolutions are required. This work presents the methodology of the measurement and visualization of plaque features important for atherosclerosis development and plaques vulnerability analysis. Label-free imaging of cholesterol, cholesteryl esters, remodeled media, heme, internal elastic lamina, fibrous cap and calcification provides additional knowledge to previously presented quantitative measurements of average plaque features. AFM imaging enhanced the results obtained with the use of vibrational microspectroscopies with additional topographical information of the sample. To the best of our knowledge, this is the first work which demonstrates that co-localized measurement of atherosclerotic plaque with Raman, IR and AFM imaging provides a comprehensive insight into the biochemical markers of atherosclerotic plaques, and can be used as an integrated approach to assess vulnerability of the plaque.

FT-IR and FT-Raman study of selected pyridinephosphonocarboxylic acids

In this work we present FT-IR and FT-Raman spectra of three acids: pyridine-2-phosphono-4-carboxylic (MC1), pyridine-2phosphono-5-carboxylic (MC2), and pyridine-2-phosphono-6-carboxylic (MC3) that possess potential neuroactive abilities. Their molecular structures and vibrational frequencies are calculated with ab initio Hartree–Fock and density functional theory methods (DFT) using the local (SVWN) and hybrid (B3LYP, B3PW91) exchange functionals. Here we discuss and compare differences in geometrical structures and vibrational patterns obtained by ab initio and DFT methods used in this work. The best agreement between the experimental and calculated spectra was obtained at the B3PW91/6-31G �� level. Assignments of Raman and IR bands for all studied acids are made on the basis of potential energy distribution (PED). We also show how the arrangements of phosphonato and carboxylic substituents on the pyridine ring change the vibrational structure of pyridine. # 2003 Elsevier Science B.V. All rights reserved.

In situ Raman and IR spectroscopic analysis of indigo dye

Non-destructive analysis of indigo dye in biological and textile samples is presented. Using different but complementary techniques of vibrational spectroscopy (FT-Raman and ATR-IR) detection of indigo in various samples is performed. This work shows the possibility of both vibrational analytical methods to identify indigo directly in plant tissue, commercially available pigments and cotton and silk textiles. The experimental work is supported by quantum-chemical calculations at the B3LYP/6-311++g(d,p) level of theory. Additionally, in order to follow the process of indigo formation upon leaf drying Raman mapping of Polygonum tinctorium is demonstrated.

Recent Advances in Raman Analysis of Plants: Alkaloids, Carotenoids, and Polyacetylenes

This paper demonstrates the special potential of Raman spectroscopy for the study of selected plant metabolites. Carotenoids, which are beneficial components in fruits and vegetables, have been shown to be a significant factor in lowering the risk of various types of cancer and ischemic heart diseases. On the other hand, alkaloids may have various effects on human health, e.g. caffeine is a mild stimulant of the central nervous system and as a result it can influence human behaviour. Polyacetylenes are highly cytotoxic against numerous cancer cell lines and demonstrate antifungal, anti-inflammatory and anti-platelet-aggregatory properties. In most cases, vibrational measurements can be performed directly on plant tissues as well as on fractions isolated from the plant material by hydro-distillation or solvent extraction. Raman spectroscopy techniques allow obtaining spectra which present some characteristic key bands of individual components. Based on such markers related to individual plant substances, spectroscopic analyses in principle allow the discrimination of different species, and even chemotypes among the same species. Moreover, Raman microspectroscopy provides 2- and 3-dimensional images of the investigated plant samples. These maps can be directly compared to the corresponding visual images obtained from a light microscope and offer additional detailed information regarding the local distribution of specific compounds in the surface layers of the analyzed plant tissue.