As recently pointed out in the literature, Fourier transform infrared (FT-IR) spectroscopy is emerging as a powerful tool in stem cell research. In this work we characterized in situ by FT-IR microspectroscopy the differentiation of murine embryonic stem cells (ES) to monitor possible changes in the cell macromolecular content during the early stages of differentiation. Undifferentiated and differentiating cells at 4, 7, 9 and 14 days were measured. Data were analyzed by the principal component and subsequent linear discriminant analyses (PCA–LDA) that enabled us to segregate ES cell spectra into well separate clusters and to identify the most significant spectral changes. Important changes in the lipid (3050–2800 cm–1), protein (1700–1600 cm–1) and in the nucleic acid (1050–850 cm–1) absorption regions were observed between days 4 to 7 of differentiation, indicating the appearance – at day 7 – of the new phenotype into cardiomyocyte precursors. Also the presence of DNA/RNA hybrid bands (954 cm–1 and 899 cm–1) suggests that the transcriptional switch of the genome started at this stage of differentiation. Particularly noteworthy, we suggest that the 2936 cm–1 shoulder we observed could reflect methyl group vibrations thus allowing the detection of variations in methylation levels of the stem cell during differentiation. These infrared results were found to be in agreement with the biochemical characterization of these differentiating cells, underlying the great potential of FT-IR spectroscopy in stem cell research.

Diletta Ami

Antonino Natalello

Paolo Mereghetti

Tui Neri

Mario Zanoni

Manuela Monti

Silvia Maria Doglia

Carlo Alberto Redi

FT-IR spectroscopy supported by PCA–LDA analysis for the study of embryonic stem cell differentiation

The strain Lysinibacillus sphaericus JG-A12, isolated from the uranium mining site at Haberland, Saxony (Germany) selectively and reversibly accumulates radionuclides and toxic metals. Metal binding occurs to its surface layer (S-layer) surrounding the cells. Here, we have studied by Fourier-transform infrared (FTIR) spectroscopy the protein structure and stability as a function of AuIII binding and the subsequent reductively induced formation of Au-nanoclusters. Similar to previously studied complexes with PdII, Au-treated S-layers become resistant to acid denaturation evidenced by little response of their amide I absorption frequency. However, the strong effect of PdII exerted on the side chain carboxylate IR absorption intensity is not observed with gold. Particularly after reduction, the carboxyl absorption responds little to acidification and a fraction appears to be protonated already at neutral pH. We ascribe this to a hydrophobic environment of the carboxyl groups after formation of Au-nanoclusters. EXAFS spectra agree with the metallic Au–Au distance but the reduced coordination number indicates that the Au-nanoclusters do not exceed ~2 nm. Thus, the S-layer of L. sphaericus JG-A12 provides a biotemplate for efficient Au-nanocluster formation in an acid-resistant matrix and independently of cysteins.

Ulrike Jankowski

Mohamed L. Merroun

Sonja Selenska-Pobell

Karim Fahmy

S-Layer protein from Lysinibacillus sphaericus JG-A12 as matrix for AuIII sorption and Au-nanoparticle formation

Infrared imaging was used to investigate the effect of sub-lethal concentration of methotrexate on prostate PC-3 cancer cells. Cells were exposed for 0, 24 and 48 h to 9 nM of methotrexate, the concentration that inhibits 50% of cell growth after 72 h. Sub-lethal concentrations of methotrexate induced a reproducible signature that can be measured by infrared spectroscopy. Student t-test revealed that the process occurs in at least two steps, the first one affecting the nucleic acid region of the spectrum and the second one, the protein region. Imaging of individual cells demonstrated that the recorded differences are not due to a specific subpopulation of cells but homogenously affects all the cells. Near perfect identification of treated cells could be obtained using a supervised classification.

Allison Derenne

Régis Gasper

Erik Goormaghtigh

Monitoring of metabolism perturbation in prostate PC-3 cancer cells by sub-lethal concentrations of methotrexate

Vibrational spectroscopy is a suitable and convenient tool to probe the self-assembly of peptides, a biomedically and biotechnologically relevant process. Theoretical efforts to quantitatively analyze vibrational spectra of peptide aggregates have thus far focused on exploring the IR and to a lesser extent the vibrational circular dichroism (VCD) spectra of rather small sized planar or nearly planar β-sheet structure. The current study utilizes an algorithm based on an excitonic coupling model with experimentally or computationally determined parameters to simulate the amide I band profiles of the IR, isotropic Raman, anisotropic Raman and VCD spectra of two- and three-dimensional β-sheet structures. In agreement with earlier calculations we found that the splitting between the two prominent IR bands of an antiparallel β-sheet increases with increasing number of strands until a saturation is reached at approximately eight strands. The dominant isotropic and anisotropic Raman bands of amide I are located between the two IR bands and downshift only slightly with increasing sheet length. The VCD signal is rather weak. We also investigated the influence of sheet stacking on amide I by calculating the respective IR, Raman and VCD profiles for two in-register, facially stacked β-sheets with seven strands per sheet for an antiparallel arrangement of the sheets and six strands per sheet for a parallel arrangement. We found that stacking produces additional bands in the IR and Raman spectrum, in line with the reduced symmetry of the ideal unit cell of the β-sheet. In addition, a more pronounced VCD signal is detected. A comparison with experimental IR, Raman and VCD spectra of gelated (AAKA)4 reveals a good qualitative agreement between experimental and simulated amide I band profiles.

Reinhard Schweitzer-Stenner

Thomas J. Measey

Simulation of IR, Raman and VCD amide I band profiles of self-assembled peptides

This work presents a pilot study to illustrate the potential of Fourier transform infrared (FT-IR) imaging in breast cancer research. Using this technique, we have acquired infrared (IR) microspectroscopic images from healthy and cancerous breast tissue section from one patient. First of all, a Student t-test was applied, showing DNA/RNA spectral region (1400–1000 cm−1) as the most discriminant for the differentiation between healthy and tumor samples. Afterwards, a supervised pattern recognition method, Partial Least Squares (PLS) was used to develop an automated classifier to discriminate the two classes of data. Infrared spectra of independent IR measurements were used to test the classifier. The class identity was correlated with information obtained by histopathologic gold standard. The results showed that more than 95% of the training and validation spectra were correctly identified. We demonstrate that combination between IR microspectroscopic imaging and multivariate data analysis can be used as a complement to present diagnostic tools for breast cancer.

Audrey Bénard

Christine Desmedt

Virginie Durbecq

Ghizlane Rouas

Denis Larsimont

Christos Sotiriou

Discrimination between healthy and tumor tissues on formalin-fixed paraffin-embedded breast cancer samples using IR imaging

We have measured spectra of the absorption coefficient and refractive index of hen egg white lysozyme in the wavenumber region from 7 cm–1 to 50 cm–1 by terahertz (THz) time-domain spectroscopy. From comparison with the results of the inelastic neutron scattering experiment it is concluded that analysis of the THz spectra provides information on the vibrational density of states. We studied temperature dependence of the THz spectra as well as hydration effect on them to discuss structural fluctuation of the protein. We observed dynamical transition at around 210 K for lysozyme.

Ohki Kambara

Keisuke Tominaga

Structural fluctuation of proteins revealed by terahertz time-domain spectroscopy

Rapid-scan FTIR difference spectroscopy was used to investigate light-induced reduction of the ubiquinone QB, and the oxidation kinetics of QB&#x2013; and QH2 by external mediators in isolated reaction centers (RCs) from R. sphaeroides. As redox mediators, a Ferrocyanide/N,N,N&#x374;,N&#x374;-tetramethyl-p-phenylenediamine (TMPD) mixture and an Ascorbate/2,3,5,6-tetramethyl-p-phenylenediamine (DAD) mixture are compared.Results show that TMPDred rapidly reduces the photoproduced P870&#x2B; primary donor. The process is fast enough to record rapid-scan FTIR spectra devoid of P870&#x2B; bands down to 260 K. Results show also that TMPDox oxidises both QB&#x2013; and QH2 faster than DADox. In particular, QB&#x2013; is oxidised faster than QH2 at all temperatures studied.Results are discussed in the framework of time-resolved infrared studies on R. sphaeroides RCs, showing advantages/drawbacks of the proposed experimental approach.

Alberto Mezzetti

Rapid-scan FTIR difference spectroscopy applied to ubiquinone reduction in photosynthetic reaction centers: Role of redox mediators

This review article describes our neutron scattering experiments made in the past four years for the understanding of the single-particle (hydrogen atom) dynamics of a protein and its hydration water and the strong coupling between them. We found that the key to this strong coupling is the existence of a fragile-to-strong dynamic crossover (FSC) phenomenon occurring at around TL = 225±5 K in the hydration water. On lowering of the temperature toward FSC, the structure of hydration water makes a transition from predominantly the high density form (HDL), a more fluid state, to predominantly the low density form (LDL), a less fluid state, derived from the existence of a liquid–liquid critical point at an elevated pressure. We show experimentally that this sudden switch in the mobility of hydration water on Lysozyme, B-DNA and RNA triggers the dynamic transition, at a temperature TD = 220 K, for these biopolymers. In the glassy state, below TD, the biopolymers lose their vital conformational flexibility resulting in a substantial diminishing of their biological functions. We also performed molecular dynamics (MD) simulations on a realistic model of hydrated lysozyme powder, which confirms the existence of the FSC and the hydration level dependence of the FSC temperature. Furthermore, we show a striking feature in the short time relaxation (β-relaxation) of protein dynamics, which is the logarithmic decay spanning 3 decades (from ps to ns). The long time α-relaxation shows instead a diffusive behavior, which supports the liquid-like motions of protein constituents. We then discuss our recent high-resolution X-ray inelastic scattering studies of globular proteins, Lysozyme and Bovine Serum Albumin. We were able to measure the dispersion relations of collective, intra-protein phonon-like excitations in these proteins for the first time. We found that the phonon energies show a marked softening and at the same time their population increases substantially in a certain wave vector range when temperature crosses over the TD. Thus the increase of biological activities above TD has positive correlation with activation of slower and large amplitude collective motions of a protein.

Sow-Hsin Chen

Marco Lagi

Xiang-qiang Chu

Yang Zhang

Chansoo Kim

Antonio Faraone

Emiliano Fratini

Piero Baglioni

Dynamics of a globular protein and its hydration water studied by neutron scattering and MD simulations

It is widely accepted that FTIR spectroscopy has a huge potential in cancer diagnosis. However further work is required to bring this technique into pathology departments. One of the areas where big efforts will be required is the development of cell spectra databases to be used in the diagnosis of cancer. Presently, unstained cytology and tissue samples are studied with FTIR spectroscopy. However, it is not always possible to identify in unstained samples the types of cells present. In order to achieve this, samples need staining after FTIR spectra have been obtained. We have recently shown it is possible to obtain FTIR spectra of stained cells using a synchrotron source (10.1038/labinvest.2010.8). This allows recording FTIR spectra from cells already characterized by pathologists. In order to further this work, we have now obtained FTIR spectra from stained (Papanicolau or Haematoxylin &amp; Eosin) single cells using a benchtop spectrometer. This would be the logical step towards a clinical application in cancer diagnosis. The data here presented show that staining caused a decreased intensity of the peaks at 2920 cm−1 and 2850 cm−1, and the appearance of stronger peaks at 1374 cm−1 and 1040 cm−1. The clinical applications are discussed.

Jacek K. Pijanka

Nicholas Stone

Gianfelice Cinque

Ying Yang

Achim Kohler

Katia Wehbe

Mark Frogley

Gary Parkes

Joanne Parkes

Paul Dumas

Christophe Sandt

Daniel G. van Pittius

Gillian Douce

Ganesh D. Sockalingum

Josep Sulé-Suso

FTIR microspectroscopy of stained cells and tissues. Application in cancer diagnosis

We have developed a Raman microspectroscopy system optimised for studying supported lipid bilayers (SLB). This system combines the benefits of Raman spectroscopy with the high spatial resolution of confocal microscopy. Furthermore, the additional incorporation of an atomic force microscope (AFM) makes it possible to directly correlate chemical information with spatial features of samples at the nanoscale. We focus on the limits of this system for detecting a single SLB and imaging its microdomains, and employ surface-enhanced Raman spectroscopy (SERS) to improve the sensitivity achieved with Raman microspectroscopy.

Claire S. Sweetenham

Ioan Notingher

Raman spectroscopy methods for detecting and imaging supported lipid bilayers

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Journal of Spectroscopy publishes research into the theory and application of spectroscopy across all disciplines, including biology, chemistry, engineering, earth sciences, medicine, materials science, physics, and space science.

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Chief Editor&nbsp;Dr Daniel Cozzolino&nbsp;is based at the University of Queensland, Australia. His research focuses on the developments of chemometric and spectroscopic methods for use in agriculture and food applications.

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