Fourier Transform Infrared Microspectroscopy Analysis Research Articles

Skin wound healing improvement in diabetic mice through FTIR microspectroscopy after implanting pluripotent stem cells.

Diabetes is a chronic degenerative disease that carries multiple complications. One of the most important complications is the diabetic cutaneous complications, such as skin lesions, ulcerations, and diabetic foot, which are present in 30%-70% of the patients. Currently, the treatments for wound healing include growth factors and cytokines, skin substitutes, hyperbaric oxygen therapy, and skin grafts. However, these treatments are ineffective due to the complex mechanisms involved in developing unhealed wounds. Considering the aforementioned complications, regenerative medicine has focused on this pathology using stem cells to improve these complications. However, it is essential to mention that there is a poor biomolecular understanding of diabetic skin and the effects of treating it with stem cells. For this reason, herein, we investigated the employment of pluripotent stem cells (PSC) in the wound healing process by carrying out morphometric, histological, and Fourier-transform infrared microspectroscopy (FTIRM) analysis. The morphometric analysis was done through a photographic follow-up, measuring the lesion areas. For the histological analysis, hematoxylin & eosin and picrosirius red stains were used to examine the thickness of the epidermis and the cellularity index in the dermis as well as the content and arrangement of collagen type I and III fibers. Finally, for the FTIRM analysis, skin cryosections were obtained and analyzed by employing a Cassegrain objective of 16× of an FTIR microscope coupled to an FTIR spectrometer. For this purpose, 20 mice were divided into two groups according to the treatment they received: the Isotonic Salt Solution (ISS) group and the PSCs group (n = 10). Both groups were induced to diabetes, and six days after diabetes induction, an excisional lesion was made in the dorsal area. Furthermore, using microscopy and FTIRM analysis, the skin healing process on days 7 and 15 post-skin lesion excision was examined. The results showed that the wound healing process over time, considering the lesion size, was similar in both groups; however, the PSCs group evidenced hair follicles in the wound. Moreover, the histological analysis evidenced that the PSCs group exhibited granulation tissue, new vessels, and better polarity of the keratinocytes. In addition, the amount of collagen increased with a good deposition and orientation, highlighting that type III collagen fibers were more abundant in the PSCs. Finally, the FTIR analysis evidenced that the PSCs group exhibited a faster wound healing process. In conclusion, the wounds treated with PSCs showed a more rapid wound healing process, less inflammatory cellular infiltration, and more ordered structures than the ISS group.

Application of Glycyrrhiza glabra L. Root as a Natural Antibacterial Agent in Finishing of Textile

The use of plant resources in different areas is increasing day by day. One of the uses of these resources is textile dyeing. In this study, Glycyrrhiza glabra L. roots were used for dyeing woolen fabrics. It is also aimed to give these dyed fabrics naturally antibacterial properties. In the dyeing process, copper (II) sulfate, tin chloride, iron (II) sulfate, zinc chloride and potassium aluminum sulfate (alum) were used as mordant. In addition, dyeing experiments were carried out without the use of mordant and the dyed fabric samples were washed and dried at room temperature. After that, CIEL*a*b* (international commission on illumination) and K/S (color efficiency) measurements of the dyed fabrics, SEM (scanning electron microscopy) images, ICP-MS (inductively coupled plasma mass spectrometry) analysis, antibacterial effect tests, FTIR (fourier transform infrared microspectroscopy) analysis, light and washing fastness tests were also performed. As a result of these processes, it has been determined that Glycyrrhiza glabra L. roots can be used for dyeing woolen fabrics. In addition, it was found that dyed fabric samples showed good antibacterial effect against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria.

Does the molecular and metabolic profile of human granulosa cells correlate with oocyte fate? New insights by Fourier transform infrared microspectroscopy analysis.

Does the molecular and metabolic profile of human mural granulosa cells (GCs) correlate with oocyte fate? A close relation between the metabolic profile of mural GCs and the fate of the corresponding oocyte was revealed by the analysis of selected biomarkers defined by GC Fourier transform infrared microspectroscopy (FTIRM) analysis. In ART, oocyte selection is mainly based on the subjective observation of its morphological features; despite recent efforts, the success rate of this practice is still unsatisfactory. FTIRM is a well-established vibrational technique recently applied to evaluate oocytes quality in several experimental models, including human. GCs retrieved from single-follicle aspirates were obtained with informed consent from 55 women undergoing controlled ovarian stimulation for IVF treatment. GCs were analysed by FTIRM to retrospectively correlate their spectral features with the fate of the companion oocytes. The study has been conducted between March 2016 and September 2017. Patients were selected according to the following inclusion criteria: age <40 years; non-smokers; no ovarian infertility diagnosis (only tubal, idiopathic and male infertility); regular ovulatory menstrual cycles (25-30 days) with FSH < 10 IU/I on Day 3 of the menstrual cycle; sperm sample with a total motility count after treatment ≥300.000; number of retrieved oocytes ≥8. Based on the clinical outcome of the corresponding oocyte, GCs were retrospectively classified into the following experimental groups: clinical pregnancy (CP), fertilization failure (FF), embryo development failure (EDF) and implantation failure (IF). All samples were analysed by the FTIRM technique. The spectral biomarker signature of different oocyte fates was derived by several feature selection procedures ('Leave-one-out' method on factorial discriminant analysis (FDA), variable characterization method and logistic regression method with the multinomial Logit model). ANOVA, permutational multivariate ANOVA, FDA and canonical analysis of principal co-ordinates statistical tools were also applied to validate the identified spectral biomarkers. In total, 284 GCs samples were retrieved and retrospectively classified as FF: (N = 92), EDF (N = 113), IF (N = 56) and CP (N = 23). From the spectral profiles of GCs belonging to CP, FF, EDF and IF experimental groups, 17 spectral biomarkers, were identified by several feature selection procedures (P < 0.0001). These biomarkers were then validated by applying multivariate tools, to evaluate their ability to segregate GCs samples into the four experimental groups. FDA showed a clear separation along the F1-axis (62.75% of discrimination) between GCs from oocytes able (CP, IF groups) or not (FF, EDF groups) to develop into embryos; the F2-axis (24.14% of discrimination) segregated the embryos that gave pregnancy (CP) from those that failed implantation (IF). The confusion matrix (total percentage of correctness = 80.25%) obtained from this analysis pinpointed that GCs from oocytes unable to develop into embryos (FF, EDF) were better characterized than those from oocytes able to give viable embryos (CP, IF). ANOVA (P < 0.05) analysis pinpointed that: each experimental group showed specific macromolecular traits, ascribable to different biological and metabolic characteristics of GCs; these metabolic features were likely associated with different oocytes fates, but not to patient characteristics, since from the same patient we obtained GCs with different metabolic profiles. The study is based on a small sample size but provides proof of concept that the GCs' metabolic profile is associated with the companion oocyte fate. The generated model should be further tested on a larger cohort of patients, classified in a similar manner, to assess the potential predictive value of this approach. Ultimately, validity of the proposed approach should be tested in a RCT. For the first time, the FTIRM analysis of human GCs has demonstrated an approach to better understand the molecular crosstalk between follicular cells and oocytes and has identified potential spectral biomarkers for improving human IVF success rate. The study was funded by GFI 2014 grant. The authors declare that there is no conflict of interest.

Cellular and Pectin Dynamics during Abscission Zone Development and Ripe Fruit Abscission of the Monocot Oil Palm.

The oil palm (Elaeis guineensis Jacq.) fruit primary abscission zone (AZ) is a multi-cell layered boundary region between the pedicel (P) and mesocarp (M) tissues. To examine the cellular processes that occur during the development and function of the AZ cell layers, we employed multiple histological and immunohistochemical methods combined with confocal, electron and Fourier-transform infrared (FT-IR) microspectroscopy approaches. During early fruit development and differentiation of the AZ, the orientation of cell divisions in the AZ was periclinal compared with anticlinal divisions in the P and M. AZ cell wall width increased earlier during development suggesting cell wall assembly occurred more rapidly in the AZ than the adjacent P and M tissues. The developing fruit AZ contain numerous intra-AZ cell layer plasmodesmata (PD), but very few inter-AZ cell layer PD. In the AZ of ripening fruit, PD were less frequent, wider, and mainly intra-AZ cell layer localized. Furthermore, DAPI staining revealed nuclei are located adjacent to PD and are remarkably aligned within AZ layer cells, and remain aligned and intact after cell separation. The polarized accumulation of ribosomes, rough endoplasmic reticulum, mitochondria, and vesicles suggested active secretion at the tip of AZ cells occurred during development which may contribute to the striated cell wall patterns in the AZ cell layers. AZ cells accumulated intracellular pectin during development, which appear to be released and/or degraded during cell separation. The signal for the JIM5 epitope, that recognizes low methylesterified and un-methylesterified homogalacturonan (HG), increased in the AZ layer cell walls prior to separation and dramatically increased on the separated AZ cell surfaces. Finally, FT-IR microspectroscopy analysis indicated a decrease in methylesterified HG occurred in AZ cell walls during separation, which may partially explain an increase in the JIM5 epitope signal. The results obtained through a multi-imaging approach allow an integrated view of the dynamic developmental processes that occur in a multi-layered boundary AZ and provide evidence for distinct regulatory mechanisms that underlie oil palm fruit AZ development and function.

Glycation and secondary conformational changes of human serum albumin: study of the FTIR spectroscopic curve-fitting technique

The aim of this study was attempted to investigate both the glycation kinetics and protein secondary conformational changes of human serum albumin (HSA) after the reaction with ribose. The browning and fluorescence determinations as well as Fourier transform infrared (FTIR) microspectroscopy with a curve-fitting technique were applied. Various concentrations of ribose were incubated over a 12-week period at 37 ± 0.5 ^oC under dark conditions. The results clearly shows that the glycation occurred in HSA-ribose reaction mixtures was markedly increased with the amount of ribose used and incubation time, leading to marked alterations of protein conformation of HSA after FTIR determination.<br /> In addition, the browning intensity of reaction solutions were colored from light to deep brown, as determined by optical observation. The increase in fluorescence intensity from HSA–ribose mixtures seemed to occur more quickly than browning, suggesting that the fluorescence products were produced earlier on in the process than compounds causing browning. Moreover, the predominant α-helical composition of HSA decreased with an increase in ribose concentration and incubation time, whereas total β-structure and random coil composition increased, as determined by curve-fitted FTIR microspectroscopy analysis. We also found that the peak intensity ratios at 1044 cm⁻¹/1542 cm⁻¹markedly decreased prior to 4 weeks of incubation, then almost plateaued, implying that the consumption of ribose in the glycation reaction might have been accelerated over the first 4 weeks of incubation, and gradually decreased. This study first evidences that two unique IR peaks at 1710 cm⁻¹ [carbonyl groups of irreversible products produced by the reaction and deposition of advanced glycation end products (AGEs)] and 1621 cm⁻¹ (aggregated HSA molecules) were clearly observed from the curve-fitted FTIR spectra of HSA-ribose mixtures over the course of incubation time. This study clearly suggests that FTIR spectroscopic curve-fitting technique may be easily used to allow determining the marked changes in the secondary conformational structure and protein aggregation of HSA during ribosylation as well as the production of AGEs.

Bone material properties in premenopausal women with idiopathic osteoporosis.

Idiopathic osteoporosis (IOP) in premenopausal women is characterized by fragility fractures at low or normal bone mineral density (BMD) in otherwise healthy women with normal gonadal function. Histomorphometric analysis of transiliac bone biopsy samples has revealed microarchitectural deterioration of cancellous bone and thinner cortices. To examine bone material quality, we measured the bone mineralization density distribution (BMDD) in biopsy samples by quantitative backscattered electron imaging (qBEI), and mineral/matrix ratio, mineral crystallinity/maturity, relative proteoglycan content, and collagen cross-link ratio at actively bone forming trabecular surfaces by Raman microspectroscopy and Fourier transform infrared microspectroscopy (FTIRM) techniques. The study groups included: premenopausal women with idiopathic fractures (IOP, n = 45), or idiopathic low BMD (Z-score ≤ -2.0 at spine and/or hip) but no fractures (ILBMD, n = 19), and healthy controls (CONTROL, n = 38). BMDD of cancellous bone showed slightly lower mineral content in IOP (both the average degree of mineralization of cancellous bone [Cn.Ca(Mean) ] and mode calcium concentration [Cn.Ca(Peak) ] are 1.4% lower) and in ILBMD (both are 1.6% lower, p < 0.05) versus CONTROL, but no difference between IOP and ILBMD. Similar differences were found when affected groups were combined versus CONTROL. The differences remained significant after adjustment for cancellous mineralizing surface (MS/BS), suggesting that the reduced mineralization of bone matrix cannot be completely accounted for by differences in bone turnover. Raman microspectroscopy and FTIRM analysis at forming bone surfaces showed no differences between combined IOP/ILBMD groups versus CONTROL, with the exceptions of increased proteoglycan content per mineral content and increased collagen cross-link ratio. When the two affected subgroups were considered individually, mineral/matrix ratio and collagen cross-link ratio were higher in IOP than ILBMD. In conclusion, our findings suggest that bone material properties differ between premenopausal women with IOP/ILBMD and normal controls. In particular, the altered collagen properties at sites of active bone formation support the hypothesis that affected women have osteoblast dysfunction that may play a role in bone fragility.

Specific antibody protection of the extracellular cartilage matrix against collagen antibody–induced damage

The type II collagen (CII)-specific monoclonal antibodies (mAb) M2139 and CIIC1 induce arthritis in vivo and degrade bovine cartilage explants in vitro, whereas mAb CIIF4 is nonarthritogenic and prevents arthritis development when given in combination with M2139 and CIIC1. To determine the nature of the protective capacity of CIIF4 antibody, we examined the effects of adding CIIF4 to cartilage explants cultured in vitro with M2139 and CIIC1. Bovine cartilage explants were cultured in the presence of M2139 and CIIC1, with or without CIIF4. Histologic changes were examined, and chemical changes to collagens and proteoglycans were assessed by Fourier transform infrared microspectroscopy (FTIRM). Fresh cartilage and cartilage that had been freeze-thawed to kill chondrocytes cultured with or without the addition of GM6001, a broad-spectrum inhibitor of matrix metalloproteinases (MMPs), were compared using FTIRM analysis. M2139 and CIIC1 caused progressive degradation of the cartilage surface and loss of CII, even in the absence of viable chondrocytes. CIIF4 did not cause cartilage damage, and when given with the arthritogenic mAb, it prevented their damage and permitted matrix regeneration, a process that required viable chondrocytes. Inhibition of MMP activity reduced cartilage damage but did not mimic the effects of CIIF4. CII-reactive antibodies can cause cartilage damage or can be protective in vivo and in vitro, depending on their epitope specificity. Since CII antibodies of similar specificity also occur in rheumatoid arthritis in humans, more detailed studies should unravel the regulatory mechanisms operating at the effector level of arthritis pathogenesis.

In Situ Localization of Polyisoprene in Eucommia ulmoides

The localization of polyisoprene in the young stem tissues of Eucommia ulmoides was investigated by histochemical staining and Fourier transform infrared microspectroscopy (FT-IR). The fibrous structures were stained using Oil Red O. FT-IR microspectroscopy analysis proved that the fibrous structures were indeed trans-polyisoprene. The presence of granular structures clearly stained by the dye and having a characteristic absorption at 2960 cm-1 in the FT-IR analysis suggested that trans-polyisoprene accumulated in the vicinity of the cambium layer. We have successfully demonstrated the localization of trans-polyisoprene in plant tissues for the first time, and our histological investigation has allowed us to deduce the main sites of biosynthesis and rubber accumulation.

Complementary information on bone ultrastructure from scanning small angle X-ray scattering and Fourier-transform infrared microspectroscopy

Scanning small angle X-ray scattering (scanning SAXS) and Fourier-transform infrared microspectroscopy (FT-IRM) have previously been utilized independently to characterize the structural properties of bone in an anatomical position-resolved fashion. Whereas SAXS provides a direct measure of the physical characteristics of apatitic crystals, FT-IRM assesses structure of both mineral and organic matrix at the molecular level. In the present study both methods were applied to examine the same developing bone tissue from the L-4 vertebra of a 14-month-old (accidental death). A 200-μm-thick section was processed for examination by scanning electron microscopy and SAXS. Spectra were collected at 200 μm spatial resolution at specific locations in cortical and cancellous bone. Parameters determined included total SAXS intensity, crystal thickness ( T), and degree and direction of predominant crystal orientation. For FT-IRM analysis, a section 4 μm thick was cut longitudinally from the top of the sample. Spectra of regions 100 × 100 μm 2 were acquired from the same locations as the SAXS spectra. Integrated areas of the phosphate ν 1,3 collagen amide I, and carbonate ν 2 absorbances, were calculated to obtain mineral:matrix and carbonate:mineral ratios. The relative quantities of types A, B, and labile carbonate (substituted for apatite hydroxyl, phosphate, and surface positions, respectively) were also evaluated. Polarized FT-IRM data were collected to determine molecular orientation of the apatite and collagen components. The results of this study show that the information obtained from the two techniques is complementary. Both SAXS and FT-IRM data revealed that the crystals were significantly larger in the cancellous region compared with the cortical region, that mineralization was greater in the cortex, and that the crystals were oriented to a larger degree in the cancellous compared with the cortical bone. The scanning SAXS measure of crystal thickness was significantly correlated to the FT-IRM measures of crystallinity, type A carbonate substitution, and crystal orientation. In conclusion, it was found that the combined use of SAXS and FT-IRM provides valuable, unique information on structural changes in bone at both the microstructural and ultrastructural level. Although each method can be used individually, the combination of techniques provides additional insights into the mechanism of bone crystal maturation.

Transmission FT-IR microspectroscopy of mineral phases in calcified tissues

Fourier-transform infrared microspectroscopy (FT-IRM) was used to study bone mineralization processes in an in vivo model and in enamel in osteogenesis imperfecta. Finally, the ability of FT-IRM to map new bone formed in implanted macroporous calcium phosphate biomaterial from sections was reported for the first time. FTIRM allowed the correlation of the microstructure of bone formation in the in vivo model with modifications in carbonate and phosphate environments of the mineral phases during maturation. FT-IRM analysis on enamel sections revealed changes in the mineral environment of carbonate and phosphate ions and probably in the size of enamel crystals. These modifications contributed to the fragility of enamel in osteogenesis imperfecta. The infrared functional group imaging of a part of implanted biomaterial and the bone ingrowth provided the visualization of chemical modifications occurring in biomaterial implants at 20 μm spatial resolution. The use of FT-IRM, in conjunction with appropriate sampling methods and data analysis should provide further insight into the molecular structure of mineral phases of calcified tissues and help to elucidate mineralization processes, skeletal disorders and properties of the biomaterials used as bone substitute.

Determination of the Relative Amount of Nucleic Acids and Proteins in Leukemic and Normal Lymphocytes by Means of Fourier Transform Infrared Microspectroscopy

Previous Fourier transform infrared (FT-IR) spectroscopic studies on neoplastic and normal cells have shown different band profiles and intensity associated with absorptions of proteins and nucleic acids. In the present study, an interpretation of such differences has been attempted by comparing the spectra of DNA/RNA/protein mixtures with the spectra, particularly, obtained for lymphocytes from B-chronic lymphatic leukemia (B-CLL) patients and normal donors. FT-IR microspectroscopy analysis showed a good agreement between the intensity and the band profile of the spectra of leukemic lymphocytes and those of the binary mixture made up of 75% human serum albumin and 25% DNA. The addition of small amounts of RNA (1–5%) modified the band shape, making it more similar to the spectrum of normal lymphocytes. An attempt was also made to estimate the relative amounts of DNA and RNA. The results demonstrated an increase in the DNA/RNA ratio value in neoplastic lymphocytes with respect to that reported in literature for normal ones.