Fourier-transform Infrared Microspectroscopy Spectra Research Articles

Model-based correction algorithm for Fourier Transform infrared microscopy measurements of complex tissue-substrate systems

Model-based algorithms have recently attracted much attention for data pre-processing in tissue mapping and imaging by Fourier transform infrared micro-spectroscopy (FTIR). Their versatility, robustness and computational performance enabled the improvement of spectral quality by mitigating the impact of scattering and fringing in FTIR spectra of chemically homogeneous biological systems. However, to date, no comprehensive algorithm has been optimized and automated for large-area FTIR imaging of histologically complex tissue samples. Herein, for the first time, we propose a unique, integrated and fully-automated Multiple Linear Regression Multi-Reference (MLR-MR) method for correcting linear baseline effects due to diffuse scattering, for compensating substrate thickness inhomogeneity and accounting for sample chemical heterogeneity in FTIR images. In particular, the algorithm uses multiple-reference spectra for histologically heterogeneous biological samples. The performance of the procedure was demonstrated for FTIR imaging of chemically complex rat brain frontal cortex tissue samples, mounted onto Ultralene® films. The proposed MLR-MR correction algorithm allows the efficient retrieval of “pure” absorbance spectra and greatly improves the histological fidelity of FTIR imaging data, as compared with the one-reference approach. In addition, the MLR-MR algorithm here presented opens up the possibility for extracting information on substrate thickness variability, thus enabling the indirect evaluation of its topography. As a whole, the MLR-MR procedure can be easily extended to more complex systems for which Mie scattering effects must also be eliminated.

Documenting the degradation of animal-tissue residues on experimental stone tools: a multi-analytical approach

In lithic residue analysis, the identification of degraded animal tissues on stone tools is challenging due to many factors, not least of which is the fact that residues are complex, heterogeneous mixtures of many different kinds of molecules. In order to aid in their identification, a reference library of infrared spectra of residues collected using Fourier-transform infrared microspectroscopy (FTIRM) has recently been published (Monnier et al J Archaeol Sci: Rep 18:806–823, 2018). The goal of the present study is to explore the effects of decomposition on residues. Accordingly, we buried flakes with residues in compost for 1 year, then excavated them and documented both their appearance (using visible-light microscopy (VLM) and scanning electron microscopy (SEM)) and molecular composition (using FTIRM). The results show that while some residues (like meat and blood) disappeared entirely, others (fat and skin) were preserved on the bottoms of flakes buried in deep layers within the compost. Although the residues were damaged by microbial activity, their FTIRM spectra were clearly interpretable. Residues containing hydroxyapatite (bone and fish scales) and keratin (feather barbules, hair, and skin) were relatively well preserved. Their structures were in many cases recognizable, and their FTIRM spectra were entirely consistent with the FTIRM spectra of the standards. The results of the experiment show that the decay of animal tissues in compost proceeds primarily as a result of microbial activity, which appears to remove the tissues before they have a chance to oxidize or experience other biochemical changes. We conclude that if ancient residues have not been removed by microbial action, they can be identified using FTIR standards based upon fresh residues, such as those published in Monnier et al J Archaeol Sci 78:158–178, (2017), Monnier et al. 2018, J Archaeol Method Theory 25(1):1-44.

FTIR microspectroscopy reveals fatty acid-induced biochemical changes in C. elegans

Fourier transform infrared microspectroscopy (FTIR) is a promising method for the analysis of biological samples. Recent studies reported that FTIR imaging allows determination of the distribution of several biomolecules in a sample with no staining or extraction. In this study, FTIR was used to monitor biochemical changes in C. elegans nematodes cultured in nematode maintenance media (CeMM) without supplementation and with supplementation with either a long chain polyunsatured omega 3 fatty acid, eicosapentaenoic acid (EPA) or a saturated fatty acid, palmitic acid (PA) at 100 μM. EPA is an omega 3 fatty acid with documented health benefits while PA is generally consumed in diets. Worms were placed on BaF2 slides, and FTIR spectra were collected from single worms in transmission mode using a focal plane array detector. Principal component analysis grouped the FTIR spectra into three clusters corresponding to spectra of worms cultured with no supplementation, worms cultured with supplementation with EPA, and worms cultured with supplementation with PA. The major differences between the FTIR spectra reside in the vibrations corresponding to unsaturated fatty acids (3008 cm−1), lipids (2928, 2848, and 1744 cm−1), and proteins (1680, 1648, and 1515 cm−1). This indicates that supplementation with EPA or PA leads to biochemical alterations related to unsaturated fatty acids, lipids, and proteins. Furthermore, supplementing mutant strains (tub-1 and fat-3) CeMM with PA resulted in the appearance of the vibration 3008 cm−1, an increase in the intensity of the vibration 1744 cm−1, and a new vibration at 1632 cm−1, which is assigned to the amide I of β-pleated sheet component of proteins, in the spectra of tub-1 and fat-3 mutant strains. The results illustrated the potential use of FTIR alongside other techniques such as gas chromatography and staining techniques to investigate lipid metabolism and fat accumulation as well as induced changes in protein structures.

Developing FTIR Microspectroscopy for the Analysis of Animal-Tissue Residues on Stone Tools

The analysis of microscopic residues on stone tools provides one of the most direct ways to reconstruct the functions of such artifacts. However, new methods are needed to strengthen residue identifications based upon visible-light microscopy. In this work, we establish that reflectance Fourier-transform infrared microspectroscopy (FTIRM) can be used to document IR spectra of animal-tissue residues on experimental stone tools. First, we present a set of reflectance FTIRM standards for the most commonly identified animal-tissue residues on stone tools: skin, meat, fat, hair, blood, feather barbules, fish scales, and bone. We provide spectral peak assignments for each residue and demonstrate that high-quality reflectance FTIRM spectra can be generated under ideal circumstances. Second, we document the spectra for these residues when they are located on a stone substrate such as flint or obsidian. We discuss procedures for correcting spectra that are affected by specular reflection and explain the effects of spectral interference from the stone. Our results show that reflectance FTIRM is sensitive to small intra-sample differences in composition. This means that it will record the effects of decomposition in ancient residues. The methodological developments we present here will help lithic residue analysts incorporate in situ reflectance FTIRM into their analysis protocols to strengthen identifications.

Developing FTIR microspectroscopy for analysis of plant residues on stone tools

The analysis of residues on stone tools can yield important insights into the tool-using behaviors of Paleolithic hominins. The ambiguity of residue identifications using visible-light microscopy (VLM) has led to the development of additional techniques for their characterization. Reflectance-based Fourier-transform infrared microspectroscopy (FTIRM) is a technique with great potential to aid in non-destructive residue identifications. Recent applications of the technique, however, have been hampered by methodological challenges, causing the infrared signals to be dominated by the stone rather than the residues. We address this problem by systematically testing the limits of FTIRM on five categories of experimental plant residues (wood bark, wood pith, grass leaves, starch, and resin). We demonstrate that it is possible to obtain FTIRM spectra of in situ plant residues wherein the effect of the stone is virtually eliminated. We also generate reflectance FTIRM spectral standards for each plant residue investigated and provide peak assignments for the major peaks in all spectra. The sensitivity of the technique means that slight differences in sample preparation can result in spectral differences as well. This means that archaeological application of the technique will require (1) careful, peak-by-peak analyses of the results, (2) extensive spectral libraries, and (3) research into the effects of decomposition.

From synthetic to biogenic Mg-containing calcites: a comparative study using FTIR microspectroscopy

The formation mechanism of the thermodynamically unstable calcite phase, very high Mg calcite, in biological organisms such as sea urchin or corallina algae has been an enigma for a very long time. In contrast to conventional methods such as KBr pellet Fourier Transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD), FTIR microspectroscopy (FTIRM) provides additional information about a local disorder such as an amorphous phase or the occlusion of Mg ions in the calcite lattice. In this work, we characterise for the first time systematically synthetic and biogenic Mg-containing calcium carbonate samples (especially sea urchin teeth--SUT) in detail by using two FTIRM instruments and compare these samples with KBr pellet FTIR measurements. Furthermore, we present spectra from geogenic calcite and dolomite minerals, recorded with both FTIRM systems, as well as KBr pellet FTIR spectra as references. We analyse the spectra by applying multi-peak curve fitting on the in-plane-bending (ν(4)) and out-of-plane (ν(2)) bands. Based on the obtained results we attribute the two singlet bands at ∼860-865 cm(-1) and ∼695-704 cm(-1) observed in the SUT FTIRM spectra to the existence of amorphous calcium carbonate (ACC), and report for the first time the existence of ACC at the mature end of SUT. In the other three studied biominerals, however, we did not find any ACC. Also, based on the FTIRM results, we observe that not only ν(4), but also ν(2) shifts to higher wavenumbers if more calcium ions are replaced by magnesium ions in the calcite lattices.

Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae

In this study Fourier transform infrared micro-spectroscopy (FTIR) was used to determine lipid and carbohydrate content over time in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus subspicatus grown in batch culture in limiting concentrations of nitrogen (N). Both algae exhibited restricted cell division and increased cell size following N-limitation. FTIR spectra of cells in N-limited media showed increasing lipid:amide I and carbohydrate:amide I ratios over time. The use of lipid- and starch-staining dyes confirmed that the observed ratio changes were due to increased lipid and carbohydrate synthesis. These results demonstrate rapid metabolic responses of C. reinhardtii and S. subspicatus to changing nutrient availability, and indicate the efficiency of FTIR as a reliable method for high-throughput determination of lipid induction.

Biochemical Alterations in Human Cells Irradiated with α Particles Delivered by Macro- or Microbeams

Irradiation of individual cell nuclei with charged-particle microbeams requires accurate identification and localization of cells using Hoechst staining and UV illumination before computer-monitored localization of each cell. Using Fourier-transform infrared microspectroscopy (FT-IRM), we investigated whether the experimental conditions used for cell recognition induce cellular changes prior to irradiation and compared biochemical changes and DNA damage after targeted and nontargeted irradiation with alpha particles delivered by macro- or microbeams, using gamma radiation as a reference. Molecular damage in single HaCaT cells was studied by means of FT-IRM and comet assay (Gault et al., Int. J. Radiat. Biol. 81, 767-779, 2005). Hoechst 33342-stained HaCaT cells were exposed to single doses of 2 Gy (239)Pu alpha particles from a broad-beam irradiator, five impacted alpha particles from a microbeam irradiator, or 6 Gy gamma rays from (137)Cs, each of which resulted in about 5% clonogenic survival. FT-IRM of control cells indicated that Hoechst binding to nuclear DNA induced subtle changes in DNA conformation, and its excitation under UV illumination induced a dramatic shift of the DNA conformation from A to B as well as major DNA damage as measured by the comet assay. Comparison of the FT-IRM spectra of cells exposed to gamma rays or alpha particles specifically targeted to the nucleus, alpha particles from a broad-beam irradiator revealed spectral changes corresponding to all changes in constitutive bases in nucleic acids, suggesting oxidative damage in these bases, as well as structural damage in the deoxyribose-phosphate backbone of DNA and the osidic structure of nucleic acids. Concomitantly, spectral changes specific to protein suggested structural modifications. Striking differences in IR spectra between targeted microbeam- and nontargeted macrobeam-irradiated cells indicated greater residual unrepaired or misrepaired damage after microbeam irradiation. This was confirmed by the comet assay data. These results show that FT-IRM, together with the comet assay, is useful for assessing direct radiation-induced damage to nucleic acids and proteins in single cells and for investigating the effects of radiation quality. Significantly, FT-IRM revealed that Hoechst 33342 binding to DNA and exposure to UV light induce a dramatic change in DNA conformation as well as DNA damage. These findings suggest that fluorochrome staining should be avoided in studies of ionizing radiation-induced bystander effects based on charged-particle microbeam irradiation. An alternative cell nucleus recognition system that avoids nuclear matrix damage and its possible contribution to propagation of biological effects from irradiated cells to neighboring nontargeted cells needs to be developed.

Infrared analysis of the mineral and matrix in bones of osteonectin-null mice and their wildtype controls.

Osteonectin function in bone was investigated by infrared analysis of bones from osteonectin-null (KO) and wildtype mice (four each at 11, 17, and 36 weeks). An increase in mineral content and crystallinity in newly formed KO bone and collagen maturity at all sites was found using FTIR microspectroscopy and imaging; consistent with osteonectin's postulated role in regulating bone formation and remodeling. Mineral and matrix properties of tibias of osteonectin-null mice and their age- and background-matched wildtype controls were compared using Fourier-transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) at 10- and 7-mm spatial resolution, respectively. The bones came from animals that were 11, 17, and 36 weeks of age. Individual FTIRM spectra were acquired from 20 x 20 microm areas, whereas 4096 simultaneous FTIRI spectra were acquired from 400 x 400 microm areas. The FTIRM data for mineral-to-matrix, mineral crystallinity, and collagen maturity were highly correlated with the FTIRI data in similar regions. In general, the osteonectin-null mice bones had higher mineral contents and greater crystallinity (crystal size and perfection) than the age-matched wildtype controls. Specifically, the mineral content of the newly forming periosteal bone was increased in the osteonectin-null mice; the crystallinity of the cortical bone was decreased in all but the oldest animals, relative to the wildtype. The most significant finding, however, was increased collagen maturity in both the cortical and trabecular bone of the osteonectin-null mice. These spectroscopic data are consistent with a mechanism of decreased bone formation and remodeling.

A physical, chemical, and mechanical study of lumbar vertebrae from normal, ovariectomized, and nandrolone decanoate-treated cynomolgus monkeys ( macaca fascicularis)

Ovariectomized cynomolgus monkeys have previously been investigated as a nonhuman primate model of postmenopausal osteoporosis (Jerome et al., Bone Miner 9:527–540; 1994). In the present study, Fourier transform infrared microspectroscopy (FTIRM) was used to verify that differences in bone mineral quality and quantity in the vertebrae of mature intact (INT) and ovariectomized (ovx) monkeys were analogous to those seen in osteoporotic and nondiseased human bones. FTIRM spectra were acquired from 15 trabeculae per vertebra from three ovx and three INT adult monkeys (mean age 8 years). These spectra were compared with those of both trabecular and previously reported osteonal bone obtained from 3 “normal” and 11 postmenopausal osteoporotic human subjects. While variations in the mineral:matrix ratio (mineral content), carbonate:phosphate ratio, and crystallinity are typical for trabecular bone from iliac crests of normal human subjects, the values of these parameters were relatively static for trabecular bone from postmenopausal osteoporotic human subjects. In general, trabecular bone from postmenopausal osteoporotic human subjects exhibited decreased mineral content (1.0 ± 0.5 vs. 2.9 ± 0.6), increased crystallinity, and increased carbonate:phosphate relative to controls. Similarly, trabecular bone from ovariectomized monkeys exhibited lower mineral content (5.8 ± 0.2) compared with the INT group (6.2 ± 0.2; p ≤ 0.05) and contained larger/more perfect apatite crystals (increased crystallinity) with increased carbonate:phosphate ratios. Variations in absolute values were attributable to site differences (ilium vs. vertebrae). To appreciate the importance of mineral properties on mechanical properties, compression testing was performed using cores of monkey L-3 and L-4 vertebral bodies from a separate group of monkeys. Treating monkeys with the anabolic steroid nandrolone decanoate (ND) immediately after ovariectomy and for the next 24 months (ND group), or beginning 12 months after ovariectomy (dND group), increased the ultimate stress compared with an ovx treatment group, despite large interanimal variations in bone architecture and mechanical properties. These data support the hypothesis that ovariectomized adult monkeys are an excellent model for postmenopausal osteoporosis, and can be used for the evaluation of therapeutic modalities.

Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin

Osteocalcin, the γ-carboxyglutamic acid-containing protein, which in most species is the predominant noncollagenous protein of bone and dentin, has been postulated to play roles in bone formation and remodeling. Recently, genetic studies showed that osteocalcin acts as an inhibitor of osteoblast function. Based on von Kossa staining and measurement of mineral apposition rates in tetracycline-labeled bones, osteocalcin knockout animals were reported to have no detectable alterations in bone mineralization. To test the hypothesis that, in addition to regulating osteoblastic activity, osteocalcin is involved in regulating mineral properties, a more sensitive assay of mineralization, Fourier transform infrared microspectroscopy (FT-IRM) was used to study thin sections of femora of 4-week-, 6-month- (intact and ovariectomized), and 9-month-old wild-type and osteocalcin-knockout mice. FT-IRM spectra provided spatially resolved measures of relative mineral and carbonate contents, and parameters indicative of apatite crystal size and perfection. No differences were detected in the mineral properties of the 4-week-old knockout and wild-type mice indicating that the mineralization process was not altered at this time point. Six-month-old wild-type animals had higher mineral contents (mineral:matrix ratios) in cortical as compared with trabecular bones; mineral contents in knockout and wild-type bones were not different. At each age studied, carbonate:phosphate ratios tended to be greater in the wild-type as compared with knockout animals. Detailed analysis of the phosphate ν1,ν3 vibrations in the spectra from 6-month-old wild-type animals indicated that the crystals were larger/more perfect in the cortical as opposed to the trabecular bones. In contrast, in the knockout animals’ bones at 6 months, there were no differences between trabecular and cortical bone in terms of carbonate content or crystallite size and perfection. Spectral parameters of the cortical and trabecular bone of the knockout animals resembled those in the wild-type trabecular bone and differed from wild-type cortical bone. In ovariectomized 6-month-old animals, the mineral content (mineral:matrix ratio) in the wild-type cortices increased from periosteum to endosteum, whereas, in the knockout animals’ bones, the mineral:matrix ratio was constant. Ovariectomized knockout cortices had lower carbonate:phosphate ratios than wild-type, and crystallite size and perfection resembled that in wild-type trabeculae, and did not increase from periosteum to endosteum. These spatially resolved data provide evidence that osteocalcin is required to stimulate bone mineral maturation.

FTIR-Microspectroscopy andDSC Studies of Poly(vinylidene fluoride)

Poly(vinylidene fluoride) (PVDF) samples, obtained by casting from tetrahydrofuran solutions and submitted to various thermal treatments, have been examined by Fourier transform-infrared microspectroscopy (FTIR-M) and differential scanning calorimetry (DSC). This kind of analysis allowed us to examine microdomains of samples with different morphological characteristics and to obtain an indication of the polymorphism of PVDF. In some cases the simultaneous presence of two or three forms has been evidenced thanks to the comparison of FTIR-M spectra and DSC traces. Vibrational spectra of single crystalline forms can be recorded by FTIR-M on phase homogeneous microdomains.