Synchrotron Radiation Fourier Transform Infrared Research Articles

Synchrotron FTIR shows evidence of DNA damage and lipid accumulation in prostate adenocarcinoma PC-3 cells following proton irradiation

Synchrotron Radiation Fourier Transform Infrared (SR-FTIR) spectra of single human prostate adenocarcinoma PC-3 cells, irradiated with a defined number of 2MeV protons generated by a proton microbeam along with non-irradiated control cells, were analysed using multivariate methods. A number of different Principal Component Analysis (PCA) models were tested and the spectral ranges associated with nucleic acids, proteins and lipids were analysed separately. The results show a dose dependent shift of the OPO asymmetric stretching mode from 1234cm−1 to 1237cm−1, consistent with local disorder in the B-DNA conformation along with a change in intensity of the OPO symmetric stretching band at 1083cm−1 indicative of chromatin fragmentation – the natural consequence of a high number of DNA Double Strand Breaks (DSBs). 2D mapping of characteristic functional groups at the diffraction limit shows evidence of lipid deposition and chromatin condensation in cells exposed to protons indicative of cell apoptosis following irradiation. These studies lay the foundation for understanding the macromolecular changes that occur to cells in response to radiation therapy, which has important implications in the treatment of tumours.

Contraction-Induced Changes in Hydrogen Bonding of Muscle Hydration Water.

Protein–water interaction plays a crucial role in protein dynamics and hence function. To study the chemical environment of water and proteins with high spatial resolution, synchrotron radiation-Fourier transform infrared (SR-FTIR) spectromicroscopy was used to probe skeletal muscle myofibrils. Observing the OH stretch band showed that water inside of relaxed myofibrils is extensively hydrogen-bonded with little or no free OH. In higher-resolution measurements obtained with single isolated myofibrils, the water absorption peaks were relatively higher within the center region of the sarcomere compared to those in the I-band region, implying higher hydration capacity of thick filaments compared to the thin filaments. When specimens were activated, changes in the OH stretch band showed significant dehydrogen bonding of muscle water; this was indicated by increased absorption at ∼3480 cm–1 compared to relaxed myofibrils. These contraction-induced changes in water were accompanied by splitting of the amide I (C=O) peak, implying that muscle proteins transition from α-helix to β-sheet-rich structures. Hence, muscle contraction can be characterized by a loss of order in the muscle–protein complex, accompanied by a destructuring of hydration water. The findings shed fresh light on the molecular mechanism of muscle contraction and motor protein dynamics.

The state of P Onb non-bridging oxygen and proton incorporation in binary MO·P2O5 (M = Ca, Mg) phosphate glasses

Abstract Binary x MO·(100 − x )P 2 O 5 (mol%; M = Ca, Mg) glasses were prepared using a conventional melting method, and the proton conduction mechanism in the glasses was investigated. The proton conductivities of the glasses were measured under various hydrogen (H 2 ) gas concentrations from 0 to 100 vol.% using Pt electrodes. The CaO·P 2 O 5 glasses show higher proton conductivities than MgO·P 2 O 5 ones. Fourier-transform infrared (FTIR) measurements showed that protons dissociated from H 2 gas at the Pt electrodes are incorporated into the glasses, and the proton conductivity increases as a result of an increase in the carrier (proton) concentration. There is a good linear relationship between the peak wavenumber of P O nb (O nb indicates non-bridging oxygen) and the proton conductivity measured in a 100 vol.% H 2 atmosphere. Furthermore synchrotron radiation X-ray diffraction (SRXRD) and FTIR results suggest that the π-electron of P O double bond is localized significantly in MgO·P 2 O 5 compared with CaO·P 2 O 5 glasses, which is the origin of higher proton conductivity in CaO·P 2 O 5 glasses.

Synchrotron radiation Fourier-transform infrared and Raman microspectroscopy study showing an increased frequency of creatine inclusions in the rat hippocampal formation following pilocarpine-induced seizures

In the present work, synchrotron radiation Fourier-transform infrared (SRFTIR) and Raman microspectroscopies were used to evaluate a possible role of creatine in the pathogenesis and progress of pilocarpine-evoked seizures and seizure-induced neurodegenerative changes in the rat hippocampal tissue. The main goal of this study was to identify creatine deposits within the examined brain area, to analyze their frequency in epileptic animals and naive controls and to examine correlations between the number of inclusions in the hippocampal formation of epileptic rats and the quantitative parameters describing animal behavior during 6-h observation period after pilocarpine injection. The presence of creatine in the brain tissue was confirmed based on the vibrational bands specific for this compound in the infrared and Raman spectra. These were the bands occurring at the wavenumbers around 2800, 1621, 1398, and 1304 cm−1 in IR spectra and around 1056, 908 and 834 cm−1 in the Raman spectra. Creatine was detected in eight of ten analyzed epileptic samples and in only one of six controls under the study. The number of deposits in epileptic animals varied from 1 to 100 and a relative majority of inclusions were detected in the area of the Dentate Gyrus and in the multiform hippocampal layer. Moreover, the number of creatine inclusions was positively correlated with the total time of seizure activity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-5488-z) contains supplementary material, which is available to authorized users.

Characterisation of chondrogenic differentiation of human mesenchymal stem cells using synchrotron FTIR microspectroscopy

A major limiting factor in stem cell therapy is the accurate identification of the differentiation state of cells destined for transplantation. This study aimed to evaluate the potential of synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy as a novel technique to probe the differentiation state of human mesenchymal stem cells (hMSCs) to chondrocytes over a period of 7, 14 and 21 days of induction. The chondrogenic markers were determined using reverse transcription polymerase chain reaction, histology and immunohistochemistry. The changes of average spectra located near 1338-1230 and 1175-960 cm(-1) indicated increased levels of collagen and aggrecan, respectively, in chondrocyte-induced hMSCs compared with control cells. Classification of independent test spectra using partial least squares discriminant analysis (PLS-DA) could distinguish control and chondrocyte-induced cells with 100% accuracy. We conclude that the SR-FTIR microspectroscopy technique is sensitive for monitoring the differentiation state of stem cells under chondrogenic induction particularly at an early stage. It provides biochemical information that is complimentary to that obtained from conventional techniques, and may give more unambiguous results particularly at the very early stage of cellular differentiation. In addition, the spectroscopic approach is more straightforward, non-destructive and requires less sample preparation compared with the conventional methodologies.

Synchrotron FTIR micro-spectroscopy study of the rat hippocampal formation after pilocarpine-evoked seizures

In the present work, synchrotron radiation Fourier transform infrared (SRFTIR) micro-spectroscopy and imaging were used for topographic and semi-quantitative biochemical analysis of rat brain tissue in cases of pilocarpine-induced epilepsy. The tissue samples were analyzed with a beam defined by small apertures and spatial resolution steps of 10 μm which allowed us to probe the selected cellular layers of hippocampal formation. Raster scanning of the samples has generated 2D chemical cartographies revealing the distribution of proteins, lipids and nucleic acids. Spectral analysis has shown changes in the saturation level of phospholipids and relative secondary structure of proteins. Special interest was put in the analysis of two areas of the hippocampal formation (sector 3 of the Ammon's horn, CA3 and dentate gyrus, DG) in which elemental abnormalities were observed during our previous studies. Statistically significant increase in the saturation level of phospholipids (increased ratio of the absorption intensities at around 2921 and 2958 cm −1) as well as conformational changes of proteins (β-type structure discrepancies as shown by the increased ratio of the absorbance intensities at around 1631 and 1657 cm −1 as well as the ratio of the absorbance at 1548 and 1657 cm −1) were detected in pyramidal cells of CA3 area as well as in the multiform and molecular layers of DG. The findings presented here suggest that abnormalities in the protein secondary structure and increases in the level of phospholipid saturation could be involved in mechanisms of neurodegenerative changes following the oxidative stress evoked in brain areas affected by pilocarpine-induced seizures.

SR-FTIR spectroscopy of renal epithelial carcinoma side population cells displaying stem cell-like characteristics

It is hypothesized that cells with stem cell-like properties may be influential in carcinogenesis, possessing the ability to self-renew, produce differentiated daughter cells and resist environmental or therapeutic injury. This has led to a surge in interest in identifying and characterizing the tumour initiating or cancer stem cell (CSC) with the aim of discovering novel diagnostic and prognostic markers and of understanding the basic biology with the ultimate aim of generating new therapeutic approaches and biomarkers. However, a major hurdle to this process has been the lack of a truly specific cancer stem cell biomarker allied to the rarity of these cells. This has led to problems in characterising these CSCs by traditional '-omic' techniques. Using a renal carcinoma cell line model, we show that synchrotron radiation-Fourier transform infrared (SR-FTIR) spectroscopy is a suitable tool to measure discrete differences in the biochemistry of small numbers of single-cells. Using the chemometric techniques of Principal Component and Linear Discriminant Analysis (PCA and LDA) for multivariate reduction, biochemical differences between the cells from different sub-populations were evaluated. Results found lipid and phosphodiester vibrations to be particularly good discriminating markers in the spectra of these stem-like cells, relative to the more differentiated, proliferating cells that make up the majority of the cell population.

Chemical and in situ characterization of macromolecular components of the cell walls from the green seaweed Codium fragile

A comprehensive analysis of the carbohydrate-containing macromolecules from the coencocytic green seaweed Codium fragile and their arrangement in the cell wall was carried out. Cell walls in this seaweed are highly complex structures composed of 31% (w/w) of linear (1-->4)-beta-D-mannans, 9% (w/w) of pyruvylated arabinogalactan sulfates (pAGS), and low amounts of hydroxyproline rich-glycoprotein epitopes (HRGP). In situ chemical imaging by synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy and by immunolabeling using antibodies against specific cell wall carbohydrate epitopes revealed that beta-d-mannans and pAGS are placed in the middle part of the cell wall, whereas HRGP epitopes (arabinogalactan proteins (AGPs) and extensins) are located on the wall boundaries, especially in the utricle apical zone. pAGS are sulfated at C-2 and/or C-4 of the 3-linked beta-L-arabinopyranose units and at C-4 and/or C-6 of the 3-linked beta-D-galactopyranose residues. In addition, high levels of ketals of pyruvic acid were found mainly at 3,4- of some terminal beta-D-Galp units forming a five-membered ring. Ramification was found at some C-6 of the 3-linked beta-D-Galp units. In agreement with the immunolabeled AGP epitopes, a nonsulfated branched furanosidic arabinan with 5-linked alpha-L-Araf, 3,5-linked alpha-L-Araf, and terminal alpha-L-Araf units and a nonsulfated galactan structure composed of 3-(3,6)-linked beta-D-Galp residues, both typical of type-II AG glycans were found, suggesting that AGP structures are present at low levels in the cell walls of this seaweed. Based on this study, it is starting to emerge that Codium has developed unique cell wall architecture, when compared, not only with that of vascular plants, but also with other related green seaweeds and algae.

Enhanced x-ray irradiation-induced cancer cell damage by gold nanoparticles treated by anew synthesis method of polyethylene glycol modification

We explored a very interesting gold nanoparticle system—pegylated gold incolloidal solution—and analyzed its uptake by mice colorectal adenocarcinomaCT26 tumor cells and the impact on the cell’s response to x-ray irradiation.We found that exposure to polyethylene glycol (PEG) modified (‘pegylated’)4.7 ± 2.6 nm gold nanoparticles synthesized by a novel synchrotron-based method enhances theresponse of CT26 cells to x-ray irradiation. Transmission electron microscopy (TEM) andconfocal microscopy revealed that substantial amounts of such nanoparticles are takenup and absorbed by the cells and this conclusion is supported by quantitativeinduced coupled plasma (ICP) results. Standard tests indicated that the internalizedparticles are highly biocompatible but strongly enhance the cell damage induced byx-ray irradiation. Synchrotron radiation Fourier transform infrared (SR-FTIR)spectromicroscopy analyzed the chemical aspects of this phenomenon: the appearance ofC = O stretching bond spectral features could be used as a marker for cell damage and confirmedthe enhancement of the radiation-induced toxicity for cells.

SR-FTIR spectroscopic preliminary findings of non-cancerous, cancerous, and hyperplastic human prostate tissues

Synchrotron radiation Fourier transform infrared (SR-FTIR) spectroscopy belongs to modern spectroscopic techniques and has been very often used to study chemical composition of different types of tissue. A microscope setup attached to the SR-FTIR spectrometer enables tissue analysis to be done at micrometer scale. Prostate diseases including hyperplasia and cancer have recently become the most common among men and therefore there is a need for continuous analysis using several supplementary analytical techniques. In the present study, prostate samples from five patients have been analyzed. The samples have been obtained from the organs removed during prostatectomy and then classified as non-cancerous, cancerous, or hyperplastic. Classification has been based on histopathological examinations. The tissue sections have been analyzed by FTIR microspectroscopy using both globar and infrared synchrotron radiation sources. In both cases, microscopic mapping of selected areas of the tissues has been performed. In order to characterize differences between sections of the tissues containing various numbers of healthy and pathological cells, changes in relative intensity of the bands in the range of 4000–2800 cm −1 have been analyzed. Maps of absorbance intensity ratios of bands due to CH 2 and CH 3 stretching vibrations (in the region from 3000 to 2800 cm −1) and those due to CH 2 and NH stretching vibrations (in the region from 3700 to 2800 cm −1) have been created in order to analyze pathological changes in tissues. This study confirms a correlation between CH 2/CH 3 and CH 2/NH absorption intensity ratios depending on the histological state of the sample.

Advantages of the Use of SR-FT-IR Microspectroscopy: Applications to Cultural Heritage

Synchrotron radiation Fourier transform infrared (SR-FT-IR) microspectroscopy represents an advance over conventional FT-IR spectroscopy because it gives a higher signal/noise ratio at the highest spatial resolution due to the high brightness and collimation of synchrotron radiation. It has been successfully applied to the study of ancient paintings, alteration and corrosion layers which are heterogeneous microlayered materials made by complex mixtures of organic and inorganic compounds. Moreover, the high brightness attribute allows FT-IR spectra to be routinely obtained directly on the surfaces of the objects and opens the possibility for nondestructive testing of museum objects. We present in this paper a selection of applications of SR-FT-IR to the analysis of ancient paintings, alteration and corrosion layers where the technique has proven to be especially useful: first, the separation and identification of pigment microparticles from ancient Roman wall paintings; second, the determination of the binding media and the byproducts resulting from the interaction between binders and pigments from medieval altarpieces; and third, the study of the surface corrosion layers of a bronze helmet by means of direct analysis of the surface.