Synchrotron Radiation FTIR Microspectroscopy Research Articles

Spectral histology of hair and hair follicle using infrared microspectroscopy.

Today, there is only limited knowledge of the spatial organization of hair chemistry. Infrared microspectroscopy is a well-established tool to provide such information and has significantly contributed to this field. In this study, we present new results combining multiple infrared microspectroscopy methods at different length scales to create a better chemical histology of human hair, including the hair follicle, hair shaft, hair medulla and hair cuticle. We used hyperspectral IR imaging & spectroscopy (HIRIS) and synchrotron-radiation FTIR microspectroscopy (SR-μFTIR) to measure transversal hair sections and SR-μFTIR to obtain high-resolution maps of longitudinal sections from the hair shaft and from the hair follicle. We used optical photothermal IR microspectroscopy (OPTIR) to analyse the cuticle surface of intact hairs. By mapping longitudinal sections of the human hair follicle with confocal SR-μFTIR, we report the first demonstration of glycogen presence in the outer root sheath of the hair follicle by spectroscopy, and its quantification at the micron scale. Spectral maps, combined with machine learning-based analysis, enabled us to differentiate the various layers of the hair follicle and provided insights into the chemical changes that occur during hair formation in the follicle. Using HIRIS and SR-μFTIR to analyse the hair medulla in transversal sections of human hairs, we report here, for the first time by vibrational spectroscopy methods, the detection of unsaturated lipids at very low concentrations in the medulla. By analysing longitudinal sections of the hair shaft with SR-μFTIR, we found that calcium carboxylates are present in large regions of the hair cuticle, and not just in small focal areas as previously thought. We then use OPTIR to analyse the hair cuticle of intact hairs at submicron resolution without sectioning and report the distribution of calcium carboxylates at the surface of intact hair for the first time. These new findings illustrate the potential of infrared microspectroscopy for imaging the chemical composition of human hair and may have implications for biomedical research or cosmetology.

Synchrotron Radiation FTIR Microspectroscopy Study of Biomolecular Alterations in Vincristine-Treated WRL68 Cells at the Single-Cell Level.

The toxic effect of vincristine on hepatocytes has rarely been studied. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy is a novel technique for investigating drug-cell interaction systems. In this research, the biomolecular alterations in WRL68 cells induced by vincristine treatment were investigated by SR-FTIR microspectroscopy and were further analyzed by multivariate statistical analysis and semiquantitative methods, including principal component analysis (PCA), orthogonal partial least square-discriminant analysis (OPLS-DA), and the peak area ratios of several characteristic IR bands. In vincristine-treated WRL68 cells, alterations in lipid structures and the presence of more long-chain fatty acids were found. A decrease in protein α-helical content relative to β-sheet structures in vincristine-treated WRL68 cells was identified. The nucleic acid content was decreased relative to that of lipids and proteins in WRL68 cells treated with vincristine. These results provide important information about the toxic effect of vincristine on normal liver cells. This research also provides a new approach to reveal the biomolecular alterations in drug-treated hepatocytes by combining SR-FTIR with multivariate statistical analysis and semiquantitative methods.

AFM Study of Nanoscale Membrane Perturbation Induced by Antimicrobial Lipopeptide C14 KYR.

Lipopeptides have been extensively studied as potential antimicrobial agents. In this study, we focused on the C14-KYR lipopeptide, a modified version of the KYR tripeptide with myristic acid at the N-terminus. Here, membrane perturbation of live E. coli treated with the parent KYR and C14-KYR peptides was compared at the nanoscale level using AFM imaging. AFM analyses, including average cellular roughness and force spectroscopy, revealed the severe surface disruption mechanism of C14-KYR. A loss of surface roughness and changes in topographic features included membrane shrinkage, periplasmic membrane separation from the cell wall, and cytosolic leakage. Additional evidence from synchrotron radiation FTIR microspectroscopy (SR-FTIR) revealed a marked structural change in the membrane component after lipopeptide attack. The average roughness of the E. coli cell before and after treatment with C14-KYR was 129.2 ± 51.4 and 223.5 ± 14.1 nm, respectively. The average rupture force of the cell treated with C14-KYR was 0.16 nN, four times higher than that of the untreated cell. Our study demonstrates that the mechanistic effect of the lipopeptide against bacterial cells can be quantified through surface imaging and adhesion force using AFM.

Precise correlation of macroscopic mechanical properties and microscopic structures of animal silks-using Antheraea pernyi silkworm silk as an example.

Animal silks, as one type of high performance natural material, display a unique combination of modulus, tensile strength, and extensibility that gives rise to a greater toughness than any other natural or synthetic fibers. Many previous researchers have already suggested that such excellent comprehensive mechanical properties should be closely related to their special molecular structures. In this paper, we provide more direct evidence to such an assumption by using Antheraea pernyi silkworm silk (tussah silk) as an example with synchrotron radiation FTIR microspectroscopy as a major characterization tool. Being a silkworm silk, A. pernyi silk has the same function as other silkworm silks (like common Bombyx mori silk), but on the other hand, its amino acid residue sequence is similar to that of spider dragline silk. Thus, A. pernyi silk can be a bridge between silkworm silk and spider silk that is worth investigating. Hence, in this research we designed different forcibly reeled A. pernyi silk samples by controlling the reeling rate, and subsequently tested their mechanical properties and then correlated them with their molecular structures and orientation degrees. Results show that the Young's modulus and breaking stress of forcibly reeled A. pernyi silks increased with the reeling rate, whereas the breaking strain was reduced. In the meantime, structure characterization revealed that the β-sheet content and molecular chain orientation in A. pernyi silk all increased significantly with an increase in reeling rate. In addition, the mechanical performance of A. pernyi silk can be altered from close to that of spider dragline silk to that of B. mori silkworm silk, with just a change of the reeling rate. All these phenomena clearly indicate that structural changes in A. pernyi silks contrived and controlled by reeling rate have a great effect upon their final mechanical properties. These observations further confirm that the mechanical properties of animal silks are able to be tuned by structure control during harvest time. Furthermore, the results obtained in this study may provide useful guidance when designing and producing high performance regenerated silk fibers for different applications.

Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams

Droplets with colloidal biological suspensions evaporating on substrates with defined wetting properties generate confined environments for initiating aggregation and self-assembly processes. We describe smart micro- and nanostructured surfaces, optimized for probing single droplets and residues by synchrotron radiation micro- and nanobeam diffraction techniques. Applications are presented for Ac-IVD and β-amyloid (1–42) peptides capable of forming cross-β sheet structures. Complementary synchrotron radiation FTIR microspectroscopy addresses secondary structure formation. The high synchrotron radiation source brilliance enables fast raster-scan experiments.

Taxon-specific responses of Southern Ocean diatoms to Fe enrichment revealed by synchrotron radiation FTIR microspectroscopy

Abstract. Photosynthesis by marine diatoms contributes substantially to global biogeochemical cycling and ecosystem productivity. It is widely accepted that diatoms are extremely sensitive to changes in Fe availability, with numerous in situ experiments demonstrating rapid growth and increased export of elements (e.g. C, Si and Fe) from surface waters as a result of Fe addition. Less is known about the effects of Fe enrichment on the phenotypes of diatoms, such as associated changes in nutritional value – furthermore, data on taxon-specific responses are almost non-existent. Enhanced supply of nutrient-rich waters along the coast of the subantarctic Kerguelen Island provide a valuable opportunity to examine the responses of phytoplankton to natural Fe enrichment. Here we demonstrate the use of synchrotron radiation Fourier Transform Infrared (SR-FTIR) microspectroscopy to analyse changes in the macromolecular composition of diatoms collected along the coast and plateau of Kerguelen Island, Southern Ocean. SR-FTIR microspectroscopy enabled the analysis of individual diatom cells from mixed communities of field-collected samples, thereby providing insight into in situ taxon-specific responses in relation to changes in Fe availability. Phenotypic responses were taxon-specific in terms of intraspecific variability and changes in proteins, amino acids, phosphorylated molecules, silicate/silicic acid and carbohydrates. In contrast to some previous studies, silicate/silicic acid levels increased under Fe enrichment, in conjunction with increases in carbohydrate stores. The highly abundant taxon Fragilariopsis kerguelensis displayed a higher level of phenotypic plasticity than Pseudo-nitzschia spp., while analysis of the data pooled across all measured taxa showed different patterns in macromolecular composition compared to those for individual taxon. This study demonstrates that taxon-specific responses to Fe enrichment may not always be accurately reflected by bulk community measurements, highlighting the need for further research into taxon-specific phenotypic responses of phytoplankton to environmental change.

In situ thermal denaturation of myofibre sub-type proteins studied by immunohistofluorescence and synchrotron radiation FT-IR microspectroscopy

The thermal denaturation of proteins in skeletal muscle was studied and characterised for the first time taking into account the in situ metabolic and contractile fibre types. From serial histological sections, collagen, elastin, various type I, IIa and IIx fibres and type I-IIa and IIa-IIx hybrids were identified by immunohistofluorescence. Histological sections were incubated in buffer solutions at increasing temperatures (40, 50, 60, 70 and 80°C). Protein secondary structure was investigated by synchrotron radiation FT-IR microspectroscopy on connective tissue and in muscle fibres rigorously identified for sub-type. Whatever the target protein components, increasing temperature resulted in a decrease in α-helix secondary structure and an increase in β-sheet structure. This phenomenon was more pronounced for intracellular proteins than for connective tissue. Although hybrid fibres were generally somewhat less sensitive to unfolding than the pure types, the amplitude of the thermal denaturation of intracellular proteins was practically independent of fibre type.

Imaging fossil bone alterations at the microscale by SR-FTIR microspectroscopy

Diagenetic alterations modifying fossil bones over geological time can limit their use as archaeological and paleontological proxies. The understanding of fossilization processes and the evaluation of the extent of diagenetic alterations of bones therefore constitute major issues in current research. The complex hierarchical structure of bone tissue and the spatial heterogeneity of the diagenetic alterations induce significant chemical variations in fossil bones at the microscale. We adapted a sample preparation procedure based on PMMA impregnation that allowed us to obtain bone thin sections independent of their level of degradation. As a first step to investigate the variations in bone composition at the histological scale, synchrotron radiation FTIR micro-spectroscopy (FTIRM) was applied on two fossil bone samples displaying distinct preservation states: (a) a bone from Magdalenian layers dated to 15 ka of the Bize-Tournal cave (Aude, France) and (b) another from Song Terus site (Java island, Indonesia, 60 ka). The first one is partially recrystallized but still contains zones with locally preserved collagen and biogenic carbonates, whereas the other consists of a recrystallized mineral fraction without any remaining collagen fraction. These data obtained on the microscale were compared to bulk measurement data and the relationships between different IR diagenetic parameters explored. This analytical approach allowed the characterization of diagenetic alterations such as collagen loss, carbonate uptake and mineral recrystallization in heavily altered fossil bone tissue at the histological scale. Using the presented procedure, the histological bone structures could be studied, even for brittle samples altered by an extensive loss of their collagen matrix during diagenesis.

Fabrication of a microfluidic platform for investigating dynamic biochemical processes in living samples by FTIR microspectroscopy

Here we present the optimization of fabrication steps for realizing an infrared–visible microfluidic chip to study single-living cell behaviour in physiological environment by synchrotron radiation FTIR microspectroscopy. We optimized subtractive and additive lithographic processes on CaF 2 substrate, employing X-ARP 3100/10 photoresist both as etching-mask and for the device fabrication. Using prototype microfabricated liquid cells 9 and 5 μm thick, we measured the response of small groups of THP1 monocytic cells to mechanical compression and chemical stimulation with fMLP using conventional IR globar source, aiming to evaluate biochemical rearrangements of leukocytes during the capillary circulation or recruitment processes. Stimulated monocytes have spectral features recognizable, differentiating them from un-stimulated, especially affecting the spectral region 1280–1000 cm −1, characteristic of nucleic acids and carbohydrates, and specific band ratios, such as proteins on lipids and methylene on methyl. Spectra variations have been correlated with biochemical events such as transcription, synthesis of new-proteins and variations in membrane fluidity.

Molecular basis of protein structure in proanthocyanidin and anthocyanin-enhanced Lc-transgenic alfalfa in relation to nutritive value using synchrotron-radiation FTIR microspectroscopy: A novel approach

To date there has been very little application of synchrotron radiation-based Fourier transform infrared microspectroscopy (SRFTIRM) to the study of molecular structures in plant forage in relation to livestock digestive behavior and nutrient availability. Protein inherent structure, among other factors such as protein matrix, affects nutritive quality, fermentation and degradation behavior in both humans and animals. The relative percentage of protein secondary structure influences protein value. A high percentage of β-sheets usually reduce the access of gastrointestinal digestive enzymes to the protein. Reduced accessibility results in poor digestibility and as a result, low protein value. The objective of this study was to use SRFTIRM to compare protein molecular structure of alfalfa plant tissues transformed with the maize Lc regulatory gene with non-transgenic alfalfa protein within cellular and subcellular dimensions and to quantify protein inherent structure profiles using Gaussian and Lorentzian methods of multi-component peak modeling. Protein molecular structure revealed by this method included α-helices, β-sheets and other structures such as β-turns and random coils. Hierarchical cluster analysis and principal component analysis of the synchrotron data, as well as accurate spectral analysis based on curve fitting, showed that transgenic alfalfa contained a relatively lower ( P < 0.05) percentage of the model-fitted α-helices (29 vs. 34) and model-fitted β-sheets (22 vs. 27) and a higher ( P < 0.05) percentage of other model-fitted structures (49 vs. 39). Transgenic alfalfa protein displayed no difference ( P > 0.05) in the ratio of α-helices to β-sheets (average: 1.4) and higher ( P < 0.05) ratios of α-helices to others (0.7 vs. 0.9) and β-sheets to others (0.5 vs. 0.8) than the non-transgenic alfalfa protein. The transgenic protein structures also exhibited no difference ( P > 0.05) in the vibrational intensity of protein amide I (average of 24) and amide II areas (average of 10) and their ratio (average of 2.4) compared with non-transgenic alfalfa. Cluster analysis and principal component analysis showed no significant differences between the two genotypes in the broad molecular fingerprint region, amides I and II regions, and the carbohydrate molecular region, indicating they are highly related to each other. The results suggest that transgenic Lc-alfalfa leaves contain similar proteins to non-transgenic alfalfa (because amide I and II intensities were identical), but a subtle difference in protein molecular structure after freeze drying. Further study is needed to understand the relationship between these structural profiles and biological features such as protein nutrient availability, protein bypass and digestive behavior of livestock fed with this type of forage.

Resonant Mie scattering in infrared spectroscopy of biological materials – understanding the ‘dispersion artefact’

Infrared spectroscopic cytology is potentially a powerful clinical tool. However, in order for it to be successful, practitioners must be able to extract reliably a pure absorption spectrum from a measured spectrum that often contains many confounding factors. The most intractable problem to date is the, so called, dispersion artefact which most prominently manifests itself as a sharp decrease in absorbance on the high wavenumber side of the amide I band in the measured spectrum, exhibiting a derivative-like line shape. In this paper we use synchrotron radiation FTIR micro-spectroscopy to record spectra of mono-dispersed poly(methyl methacrylate) (PMMA) spheres of systematically varying size and demonstrate that the spectral distortions in the data can be understood in terms of resonant Mie scattering. A full understanding of this effect will enable us to develop strategies for deconvolving the scattering contribution and recovering the pure absorption spectrum, thus removing one of the last technological barriers to the development of clinical spectroscopic cytology.

Synchrotron radiation FT-IR micro-spectroscopy of fluorophlogopite in the O–H stretching region

Synchrotron radiation FT-IR polarized micro-spectroscopy has been used to investigate the natural mica fluorophlogopite in order to characterize the stretching vibrations of the OH groups and their orientation relative to the mica cleavage (0 0 1) plane. A procedure to eliminate undesired interference fringes, typically observed when working with thin single-crystals, was set up using an ad hoc sample preparation, and tested. The naturally high brilliance and high polarization properties of synchrotron radiation allows to study very small flakes and hence offers new analytical possibilities in comparison to conventional powder FT-IR spectroscopy.