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

We report on the temperature dependence, at microwave (mw) frequency, of the imaginary part of the dielectric constant (&#x3b5;&#x2033;) in myoglobin powder samples with different hydration levels (h). The measurements have been performed by the cavity perturbation technique, in the range of temperature 80&#8211;345&#x2009;K. The sample is located inside a glass capillary along the axis of a cylindrical copper cavity, resonating in the TE011 mode at 9.6&#x2009;GHz, where the mw electric field has a node. By measuring the variation of the quality factor of the resonant cavity, one can extract the imaginary part of the dielectric constant. At temperatures higher than 230&#x2009;K we observe an evident increase of the dielectric losses with increasing temperature; the effect scales almost linearly with hydration, indicating that it must be attributed to a relaxation of water in the hydration shell of the protein. Furthermore, at h&#x2265;0.18, we observe a clear peak in the &#x3b5;&#x2033; vs. T curve, that shifts towards lower temperatures upon increasing hydration; this shows that the activation enthalpy of the hydration water relaxation decreases with hydration. More in general, our data show that the technique of microwave cavity perturbation allows one to study the dynamics of water molecules in the hydration shell of proteins and to extend information obtained with dielectric techniques to the mw frequencies.

M. Bonura

G. Schir&#242;

A. Cupane

Dielectric properties of myoglobin at 10&#x2009;GHz by microwave cavity perturbation measurements

By terahertz (THz) time-domain spectroscopy we have measured low-frequency spectra of amino acid (glycine; Gly), short peptides ((Gly)3 and (Gly)4), six globular proteins and bacteriorhodopsin (BR). From the analysis of the THz spectra we defined and obtained the reduced absorption cross sections for these cases, which are proportional to the vibrational density of states. We observed anharmonic behaviors in the low-frequency modes of the short peptides. The globular proteins we investigated show a universal feature in the low-frequency spectra. BR shows the dynamical transition in the temperature dependence of the THz spectrum when the sample is hydrated.

Shintaro Kawaguchi

Ohki Kambara

Carlito S. Ponseca Jr.

Mikihiro Shibata

Hideki Kandori

Keisuke Tominaga

Low-frequency dynamics of biological molecules studied by terahertz time-domain spectroscopy

Structure determination of multidomain proteins or protein–membrane complexes is one of the most challenging tasks in modern structural biology. High-resolution techniques, like NMR or X-ray crystallography, are limited to molecules of moderate size or those that can be crystallized easily. Both methods encounter serious technical obstacles in structural analysis of protein–membrane systems. This work describes an emerging biophysical technique that combines segmental isotope labeling of proteins with Fourier transform infrared (FTIR) spectroscopy, which provides site-specific structural information on proteins and allows structural characterization of protein–membrane complexes. Labeling of a segment of the protein with 13C results in infrared spectral resolution of the labeled and unlabeled parts and thus allows identification of structural changes in specific domains/segments of the protein that accompany functional transitions. Segmental isotope labeling also allows determination of the precise configuration of protein–membrane complexes by polarized attenuated total reflection FTIR (ATR–FTIR) spectroscopy. These new developments offer solutions to functionally important site-specific structural changes in proteins and protein–membrane complexes that are hard to approach using conventional methods.

Suren A. Tatulian

Structural analysis of proteins by isotope-edited FTIR spectroscopy

Fourier transform infrared spectroscopy combined with multivariate analysis was performed on a series of bone samples in order to investigate possible differences in the molecular fingerprint based on classification either between different sex and bone sites. Cortical femoral, tibia and ulna samples from five male and five female normal Wistar rats were analysed. PCA analysis classified successfully sex-related spectra since the first three principal components explained 95% of the total variance. This was not the case for bone site-related spectra. In all cases, an important aspect is the ionic exchange of phosphate and carbonate ions.

Nikolaos Kourkoumelis

Margaret Tzaphlidou

Multivariate statistical evaluation of bone site and sex as parameters for the Fourier transform infrared spectroscopic study of normal bone

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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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