FTIR imaging was performed on pig ear skin samples cryo-sectioned perpendicular to the skin surface. The OH-stretch region revealed the distribution of water; the amide II band gave the protein distribution; the C═O stretch and C–H stretch regions showed the variation in lipids. Water and proteins were similarly distributed. Triglycerides were predominantly found in the deeper skin layers whereas free fatty acids and ceramides were more dominant in the upper layers. ATR-FT-Far-IR spectroscopy with synchrotron radiation was used on full thickness pig ear skin biopsies. The Far-IR spectra showed bands in the region from 100–150 cm–1 due to hydrogen bonded proteins and a band around 180 cm–1 arising from “free” water.

Tanja M. Greve

Kristine B. Andersen

Ole F. Nielsen

Anders Engdahl

FTIR imaging and ATR-FT-Far-IR synchrotron spectroscopy of pig ear skin

Focal plane arrays provide a rapid method for infrared imaging of cells and tissues. Beside statistical analyses designed to classify image features, insight into the structure of the molecules present in the sample can be obtained. The paper presents a simple way to retrieve complete information on protein secondary structure from the thousands of spectra obtained by imaging. Human PC-3 prostate cancer cells were shown here to present cell-to-cell variations in protein secondary structure which might be related to the cell cycle. Intracellular variations are small but intercellular variations can be quite significant. This observation could yield unanticipated insight in cell population analysis. Furthermore, the results demonstrate the robustness of secondary structure determination on individual pixels at a sub-cellular scale.

Erik Goormaghtigh

Allison Derenne

Audrey B&#xE9;nard

R&#xE9;gis Gasper

Vincent Raussens

Data processing in FTIR imaging of cells: Towards protein secondary structure imaging

Development of novel inverted Raman micro-spectrometer with the ability to perform multi-hours spectral measurements on living cells is presented. Our system combines a Confocal Raman Micro-Spectrometer and Fluorescence Microscope with cell incubator enclosure allowing measurement of cells in extended time period. To illustrate the feasibility of this Raman micro-spectroscopy system for in vitro time-course studies of cells we performed an experiment where the same group of cells were scanned with the laser at 2 hours intervals between the scans over 8 hours to build Raman spectral images and ensure that no changes occur due to laser damage or environmental conditions. Cell viability test was performed with fluorescence microscopy on exactly the same cells at the end of the time-course Raman measurements.

A. Zoladek

F. Pascut

P. Patel

I. Notingher

Development of Raman Imaging System for time-course imaging of single living cells

Alzheimer's disease (AD) is the most common form of dementia worldwide and represents a growing socio-economical issue. To date, no reliable diagnosis can be obtained at an early-stage of the disease, though it is now recognized that the aggregation of the amyloid β (Aβ) peptide is responsible for the onset of the disease. Recent studies have shown that soluble amyloid oligomers present in the physiological fluids were the most neurotoxic species and correlated best with the first signs of cognitive decline, which makes them good biomarkers in the development of a diagnostic tool.We describe here a new type of biosensor, based on attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, that would be able to specifically detect and quantify the presence of the different forms of the Aβ peptide in solution. The principle of the detection relies on the recognition of the peptides by specific antibodies that were previously grafted on the surface of an ATR element, consisting of a functionalized germanium crystal. We show that the BIA-ATR technology is able to detect the presence of Aβ if incubated in deuterated water and that this step is crucial in the development of our conformation-sensitive biosensor for AD.

Emilie Kleiren

Jean-Marie Ruysschaert

Development of a quantitative and conformation-sensitive ATR-FTIR biosensor for Alzheimer's disease: The effect of deuteration on the detection of the Aβ peptide

Interactions of the β-blocker drug, propranolol, with amphipathic systems have been studied using fluorescence spectroscopy. The results show a strong binding of propranolol with micelles of sodium dodecyl sulfate revealed through changes in the fluorescence spectrum and an increase in fluorescence lifetime. Quenching of propranolol fluorescence by iodide is used to demonstrate interaction with β-cyclodextrin. At high concentrations, self-quenching of propranolol fluorescence was also observed with κq=2.5×109 dm3 mol–1 s–1. Two-photon excited (630 nm) fluorescence lifetime imaging of propranolol in cells showed propranolol to be widely distributed in the cell cytoplasm, with fluorescence lifetimes shorter than in solution. The results suggest that intracellular propranolol is mainly confined within the aqueous cytoplasm and rather than membrane associated.

R. H. Bisby

S. W. Botchway

A. G. Crisostomo

J. Karolin

A. W. Parker

L. Schröder

Interactions of the β-blocker drug, propranolol, with detergents, β-cyclodextrin and living cells studied using fluorescence spectroscopy and imaging

Calf thymus DNA adsorbed on a rough gold substrate or on an atomically smooth gold (111) surface has been investigated by collecting its unique Raman fingerprints using either surface-enhanced Raman scattering (SERS) or tip-enhanced Raman scattering (TERS). A monolayer coverage of DNA strands adsorbed at both the irregular rough edges of evaporated gold grids and at gold nanoparticles is detected by SERS. Highly improved sensitivity down to single DNA strand spectroscopic determination is accomplished by TERS providing an enhancement factor of at least 1400. Based on our experimental results, we propose that TERS is a promising technique to study the DNA&#x2013;drug molecule interaction on the level of a single DNA strand.

L. E. Hennemann

A. J. Meixner

D. Zhang

Surface- and tip-enhanced Raman spectroscopy of DNA

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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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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