Raman Spectroscopy as a Potential Adjunct of Thyroid Nodule Evaluation: A Systematic Review.

The incidence of thyroid nodules (TNs) is estimated at 36.5% and 23% in females and males, respectively. A single thyroid nodule is usually detected during ultrasound assessment in patients with symptoms of thyroid dysfunction or neck mass. TNs are classified as benign tumours (non-malignant hyperplasia), benign neoplasms (e.g., adenoma, a non-invasive follicular tumour with papillary nuclear features) or malignant carcinomas (follicular cell-derived or C-cell derived). The differential diagnosis is based on fine-needle aspiration biopsies and cytological assessment (which is burdened with the bias of subjectivity). Raman spectroscopy (RS) is a laser-based, semiquantitative technique which shows for oscillations of many chemical groups in one label-free measurement. RS, through the assessment of chemical content, gives insight into tissue state which, in turn, allows for the differentiation of disease on the basis of spectral characteristics. The purpose of this study was to report if RS could be useful in the differential diagnosis of TN. The Web of Science, PubMed, and Scopus were searched from the beginning of the databases up to the end of June 2023. Two investigators independently screened key data using the terms "Raman spectroscopy" and "thyroid". From the 4046 records found initially, we identified 19 studies addressing the differential diagnosis of TNs applying the RS technique. The lasers used included 532, 633, 785, 830, and 1064 nm lines. The thyroid RS investigations were performed at the cellular and/or tissue level, as well as in serum samples. The accuracy of papillary thyroid carcinoma detection is approx. 90%. Furthermore, medullary, and follicular thyroid carcinoma can be detected with up to 100% accuracy. These results might be biased with low numbers of cases in some research and overfitting of models as well as the reference method. The main biochemical changes one can observe in malignancies are as follows: increase of protein, amino acids (like phenylalanine, tyrosine, and tryptophan), and nucleic acid content in comparison with non-malignant TNs. Herein, we present a review of the literature on the application of RS in the differential diagnosis of TNs. This technique seems to have powerful application potential in thyroid tumour diagnosis.

Elemental Composition of Skeletal Muscle Fibres Studied with Synchrotron Radiation X-ray Fluorescence (SR-XRF).

Diseases of the muscle tissue, particularly those disorders which result from the pathology of individual muscle cells, are often called myopathies. The diversity of the content of individual cells is of interest with regard to their role in both biochemical mechanisms and the structure of muscle tissue itself. These studies focus on the preliminary analysis of the differences that may occur between diseased tissues and tissues that have been recognised as a reference group. To do so, 13 samples of biopsied human muscle tissues were studied: 3 diagnosed as dystrophies, 6 as (non-dystrophic) myopathy and 4 regarded as references. From these sets of muscle biopsies, 135 completely measured muscle fibres were separated altogether, which were subjected to investigations using synchrotron radiation X-ray fluorescence (SR-XRF). Muscle fibres were analysed in terms of the composition of elements such as Br, Ca, Cl, Cr, Cu, Fe, K, Mn, P, S and Zn. The performed statistical tests indicate that all three groups (dystrophies-D; myopathies-M; references-R) show statistically significant differences in their elemental compositions, and the greatest impact, according to the multivariate discriminate analysis (MDA), comes from elements such as Ca, Cu, K, Cl and S.

Application of Factorisation Methods to Analysis of Elemental Distribution Maps Acquired with a Full-Field XRF Imaging Spectrometer.

The goal of the work was to investigate the possible application of factor analysis methods for processing X-ray Fluorescence (XRF) data acquired with a full-field XRF spectrometer employing a position-sensitive and energy-dispersive Gas Electron Multiplier (GEM) detector, which provides only limited energy resolution at a level of 18% Full Width at Half Maximum (FWHM) at 5.9 keV. In this article, we present the design and performance of the full-field imaging spectrometer and the results of case studies performed using the developed instrument. The XRF imaging data collected for two historical paintings are presented along with the procedures applied to data calibration and analysis. The maps of elemental distributions were built using three different analysis methods: Region of Interest (ROI), Non-Negative Matrix Factorisation (NMF), and Principal Component Analysis (PCA). The results obtained for these paintings show that the factor analysis methods NMF and PCA provide significant enhancement of selectivity of the elemental analysis in case of limited energy resolution of the spectrometer.

Investigation of the role and chemical form of iron in the ovarian carcinogenesis process.

BACKGROUND: Ovarian cancer is one of the most frequent types of gynaecological malignancy among women. Despite the advances in diagnostic techniques, ovarian tumours are still detected at a late stage, thus the survival rate is very low. Iron is an essential metal in the human body, yet its potential role in ovarian carcinogenesis is yet to be determined. The aim of this study was to check if iron oxidation state in tissue and cystic fluid can be treated as an indicator of the malignancy of the ovarian tumours. Another aspect of this study was to investigate the role of iron in carcinogenesis mechanism in ovarian tumour transformation. METHODS: Synchrotron radiation X-ray absorption near edge structure (SR-XANES) spectroscopy was used to analyze the human ovarian tumour tissues and cystic fluids of different types and grades of malignancy. Fresh, non-fixed, frozen samples were used to analyze the state of iron oxidation in all the biological materials. The samples were obtained from patients requiring surgical intervention. The High Energy X-ray Absorption Spectroscopy (XANES) measurements were performed at the beamline P65 at Petra III Extension, DESY - Deutsches Elektronen - Synchrotron. RESULTS: Fe XANES spectra were collected at selected points of a few different regions of the samples. For each specimen, an average of these points was probed. Having been measured, the spectra were compared with organic and inorganic reference materials. Also, the position of the absorption edge was calculated using the integration method. In all specimens, iron occurred in the oxidation states, Fe2+ and Fe3+, although the fraction of iron in the third oxidation state was substantial, especially in malignant cases. The results also show differences in the chemical form of iron in the tissue and cystic fluids of the same patient. CONCLUSIONS: The cryo-XANES measurement carried out for ovarian cancer tissues and cystic fluids showed changes in the chemical form of iron between non-malignant and malignant tumours. For both types of sample can be observed that they contain iron on second and iron on third oxidation state. Moreover, the tendency was observed that malignant tumours of the ovary contain a larger fraction of iron in the second oxidation state compared to non-malignant ones.

Combined micro-XRF and TXRF methodology for quantitative elemental imaging of tissue samples.

Local differences in structural properties of biological specimens pose a major limitation to quantitative X-ray fluorescence imaging. This is because both the various tissue compartments of different density and variation in the sample thickness upon frequently used freeze-drying come up with the different values of the sample mass per unit area to be taken into account. Even though several solutions to tackle this problem based on the home-made standards for quantification in terms of thickness- and density-independent elemental mass fractions have been proposed, this issue is not addressed enough due to the samples' heterogeneity. In our recent study, we propose a calculation scheme based on combined external-standard micro X-ray fluorescence (micro-XRF) imaging and internal-standard total reflection X-ray fluorescence (TXRF) analysis to determine the corrected elemental mass fraction distributions in commonly analysed rat tissues: kidney, liver and spleen. The results of TXRF analysis of digested large tissue sections together with the mean values of elemental masses per unit area obtained with micro-XRF were employed to determine the average masses per unit area of the samples. The correction for variation of the tissue thickness and density was done through with the use of Compton intensities. Importantly, by its versatility, our novel approach can be used to produce elemental contrast in a variety of biological specimens where local variations in either the sample density or thickness are no longer the issue.

Does titanium in ionic form display a tissue-specific distribution?

Most studies have focused on the biodistribution of titanium(IV) oxide as nanoparticles or crystals in organism. But several reports suggested that titanium is released from implant in ionic form. Therefore, gaining insight into toxicokinetics of Ti ions will give valuable information, which may be useful when assessing the health risks of long-term exposure to titanium alloy implants in patients. A micro synchrotron radiation-induced X-ray fluorescence (µ-SRXRF) was utilized to investigate the titanium distribution in the liver, spleen and kidneys of rats following single intravenous or 30-days oral administration of metal (6 mg Ti/b.w.) in ionic form. Titanium was mainly retained in kidneys after both intravenous and oral dosing, and also its compartmentalization in this organ was observed. Titanium in the liver was non-uniformly distributed-metal accumulated in single aggregates, and some of them were also enriched in calcium. Correlation analysis showed that metal did not displace essential elements, and in liver titanium strongly correlated with calcium. Two-dimensional maps of Ti distribution show that the location of the element is characteristic for the route of administration and time of exposure. We demonstrated that µ-SRXRF can provide information on the distribution of titanium in internal structures of whole organs, which helps in enhancing our understanding of the mechanism of ionic titanium accumulation in the body. This is significant due to the popularity of titanium implants and the potential release of metal ions from them to the organism.

Alterations of Bio-elements, Oxidative, and Inflammatory Status in the Zinc Deficiency Model in Rats.

Our previous study showed that dietary zinc restriction induces depression-like behavior with concomitant up-regulation of the N-methyl-D-aspartate receptor (NMDAR). Because metal ions, oxidative stress, and inflammation are involved in depression/NMDAR function, in the present study, bio-elements (zinc, copper, iron, magnesium, and calcium), oxidative (thiobarbituric acid-reactive substances; protein carbonyl content), and inflammatory (IL-1α, IL-1ß) factors were measured in serum, hippocampus (Hp), and prefrontal cortex (PFC) of male Sprague-Dawley rats subjected to a zinc-adequate (ZnA) (50 mg Zn/kg) or a zinc-deficient (ZnD) (3 mg Zn/kg) diet for 4 or 6 weeks. Both periods of dietary zinc restriction reduced serum zinc and increased serum iron levels. At 4 weeks, lowered zinc level in the PFC and Hp as well as lowered iron level in the PFC of the ZnD rats was observed. At 6 weeks, however, iron level was increased in the PFC of these rats. Although at 6 weeks zinc level in the PFC did not differ between the ZnA and ZnD rats, extracellular zinc concentration after 100 mM KCl stimulation was reduced in the PFC of the ZnD rats and was accompanied by increased extracellular iron and glutamate levels (as measured by the in vivo microdialysis). The examined oxidative and inflammatory parameters were generally enhanced in the tissue of the ZnD animals. The obtained data suggest dynamic redistribution of bio-elements and enhancement of oxidative/inflammatory parameters after dietary zinc restriction, which may have a link with depression-like behavior/NMDAR function/neurodegeneration.

New Approaches for Correction of Interlayer Absorption Effects in X-ray Fluorescence Imaging of Paintings.

The X-ray fluorescence imaging technique allows not only the imaging itself but also the identification of the hidden paint layers what makes it much more versatile as compared with X-ray radiography. One of the main disadvantages of the former method is the fact that the characteristic X-rays from the deeper paint layers are absorbed in the covering layers. This effect is manifested by some artifacts that impede a proper interpretation of the acquired images. In this work, it is shown that the methodology for correction of the interlayer absorption effects can be extended to the case of polychromatic excitation. Additionally, a new approach for determination of the optimal correction parameters has also been presented. The methodology was verified using either the test painting or the mock-up painting both measured with a table-top micro-XRF setup.

Antidepressant activity of fluoxetine in the zinc deficiency model in rats involves the NMDA receptor complex.

The zinc deficiency animal model of depression has been proposed; however, it has not been validated in a detailed manner. We have recently shown that depression-like behavior induced by dietary zinc restriction is associated with up-regulation of hippocampal N-methyl-d-aspartate receptor (NMDAR). Here we examined the effects of chronic administration of a selective serotonin reuptake inhibitor, fluoxetine (FLX), on behavioral and biochemical alterations (within NMDAR signaling pathway) induced by zinc deficiency. Male Sprague Dawley rats were fed a zinc adequate diet (ZnA, 50mg Zn/kg) or a zinc deficient diet (ZnD, 3mg Zn/kg) for 4 weeks. Then, FLX treatment (10mg/kg, i.p.) begun. Following 2 weeks of FLX administration the behavior of the rats was examined in the forced swim test (FST) and the spontaneous locomotor activity test. Twenty four hours later tissue was harvested. The proteins of NMDAR (GluN1, GluN2A and GluN2B) or AMPAR (GluA1) subunits, p-CREB and BDNF in the hippocampus (Western blot) and serum zinc level (TXRF) were examined. Depression-like behavior induced by ZnD in the FST was sensitive to chronic treatment with FLX. ZnD increased levels of GluN1, GluN2A, GluN2B and decreased pS485-GluA1, p-CREB and BDNF proteins. Administration of FLX counteracted the zinc restriction-induced changes in serum zinc level and hippocampal GluN1, GluN2A, GluN2B and p-CREB but not BDNF or pS845-GluA1 protein levels. This finding adds new evidence to the predictive validity of the proposed zinc deficiency model of depression. Antidepressant-like activity of FLX in the zinc deficiency model is associated with NMDAR complex.

Depth profiling of element concentrations in stratified materials by confocal microbeam X-ray fluorescence spectrometry with polychromatic excitation.

The confocal microbeam X-ray fluorescence technique is a well-established analytical tool that is widely used for qualitative and quantitative analysis of stratified materials. There are several different reconstruction methods dedicated to this type of samples. However, these methods are applicable with monochromatic excitation only. The full description of matrix effects and geometrical effects for polychromatic X-ray photons in confocal geometry is a demanding task. In the present paper, this problem was overcome by the use of effective energy approximation. The reduction of the whole energy dimension into one effective value eliminates the necessity of integration over the primary beam energy range for a number of basic parameters. This simplification is attainable without loss of the accuracy of analysis. The proposed approach was validated by applying it to the reconstruction of element concentration depth profiles of stratified standard samples measured with tabletop confocal microbeam X-ray fluorescence setup and by comparing the obtained results of two independent algorithms.

The oxidation states and chemical environments of iron and zinc as potential indicators of brain tumour malignancy grade - preliminary results.

Despite the enormous advances in medicine, brain tumours are still among the lesser-known types of tumours and carry the worst prognoses. Transition metals are believed to play an essential role in carcinogenesis. The aim of this study was to determine differences in the average oxidation state and trends in the changes in the chemical environment of iron and zinc contained in healthy and neoplastic tissues of the human brain. For this purpose, X-ray Absorption Spectroscopy was used, which enables the study of disordered matter. The samples were taken intraoperatively and then immediately frozen to slow down chemical processes. Sixteen tumour samples with various malignancy grades were studied as well as one control sample. For each sample four to eight spectra were recorded, with a shift between them not greater than 0.2 eV. In all of the samples, iron occurred in compounds with both Fe(2+) and Fe(3+). However, the ratio of Fe(ii) to Fe(iii) content in the tissue visibly increased with the tumour malignancy grade. The change in the oxidation state of iron did not correlate with the hypoxia level of the tissues. Analysis of EXAFS spectra of zinc atoms showed that the chemical environment of zinc atoms differed with the tumour malignancy grade. Additionally, cryogenic conditions were found to produce positive results in studies of biological samples, whose form under such conditions is close to their native state, without preparation-caused artefacts.

LabVIEW control software for scanning micro-beam X-ray fluorescence spectrometer.

Confocal micro-beam X-ray fluorescence microscope was constructed. The system was assembled from commercially available components - a low power X-ray tube source, polycapillary X-ray optics and silicon drift detector - controlled by an in-house developed LabVIEW software. A video camera coupled to optical microscope was utilized to display the area excited by X-ray beam. The camera image calibration and scan area definition software were also based entirely on LabVIEW code. Presently, the main area of application of the newly constructed spectrometer is 2-dimensional mapping of element distribution in environmental, biological and geological samples with micrometer spatial resolution. The hardware and the developed software can already handle volumetric 3-D confocal scans. In this work, a front panel graphical user interface as well as communication protocols between hardware components were described. Two applications of the spectrometer, to homogeneity testing of titanium layers and to imaging of various types of grains in air particulate matter collected on membrane filters, were presented.

First step toward the "fingerprinting" of brain tumors based on synchrotron radiation X-ray fluorescence and multiple discriminant analysis.

Synchrotron-radiation-based X-ray fluorescence was applied to the elemental microimaging of neoplastic tissues in cases of various types of brain tumors. The following cases were studied: glioblastoma multiforme, gemistocytic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, ganglioglioma, fibrillary astrocytoma, and atypical transitional meningioma. Apart from neoplastic tissue, the analysis included areas of tissue apparently without malignant infiltration. The masses per unit area of P, S, Cl, K, Ca, Fe, Cu, Zn, Br, and Rb were used to construct a diagnostic classifier for brain tumors using multiple discriminant analysis. It was found that S, Cl, Cu, Fe, K, Br, and Zn are the most significant elements in the general discrimination of tumor type. The highest similarity in elemental composition was between atypical transitional meningioma and fibrillary astrocytoma. The smallest differentiation was between glioblastoma multiforme and oligodendroglioma. The mean percentage of correct classifications, estimated according to the a posteriori probabilities procedure, was 99.9%, whereas the mean prediction ability of 87.6% was achieved for ten new cases excluded previously from the model construction. The results showed that multiple discriminant analysis based on elemental composition of tissue may be a potentially valuable method assisting differentiation and/or classification of brain tumors.

An integrated experimental and analytical approach to the chemical state imaging of iron in brain gliomas using X-ray absorption near edge structure spectroscopy.

X-ray absorption near-edge structure spectroscopy is used for human neoplastic tissues in order to investigate distributions and chemical states of iron. The specimens used in this study were obtained intraoperatively from brain gliomas of different types and various grades of malignancy and from a control subject. An integrated experimental and analytical approach toward topographic and quantitative analysis in thin freeze-dried cryo-sections is presented. The full XANES spectra at the Fe absorption K edge show the presence of both chemical forms of Fe in the analyzed points of the tissues. The main goal of the work is the chemical state imaging of Fe in tissue areas. Topographic analysis of Fe speciation in the tissues investigated with the use of the XANES technique indicates the presence of microstructures where Fe(2+) is dominant as well as those with a high abundance of the oxidized form of Fe. The quantitative analysis shows that for all cases the content of the oxidized form of Fe is significantly higher in comparison with Fe(2+). The highest level of Fe(3+) is found in the control sample, and the lowest one for the glioma of the highest grade of malignancy. The content of either Fe(2+) or Fe(3+) is increased in low grade gliomas in comparison to high-grade malignant tumors.

Early lifetime zinc supplementation protects zinc-deficient diet-induced alterations.

Preclinical and clinical data indicate the involvement of zinc in the pathophysiology and therapy of depression. A relationship between zinc-deficiency and depression symptoms was recently proposed. The present study investigated alterations in spontaneous locomotor activity and zinc concentrations in the serum, hippocampus and frontal cortex; these alterations were induced by subjecting rats to a zinc-deficient diet, prior subjected after birth to zinc-supplemented diet. Body weight was significantly reduced in animals subjected to the four-week zinc-deficient diet compared to those subjected to the zinc-adequate diet. The two-week zinc-deficient diet induced a significant increase in locomotor activity in all measured time periods (5, 30 and 60 min by 44-62%). The four-week zinc-deficient diet did not affect locomotor activity, while the six-week zinc-deficient diet resulted in a 45% increase in the 5 min time period. Serum zinc concentrations were significantly reduced (by 29%) in animals subjected to the four-week zinc-deficient diet but not in those subjected to the two- or six-week zinc-deficient diets. The zinc-deficient diet did not influence the zinc concentration in the examined brain regions regardless of the length. These results indicate that post-birth supplementation with zinc may protect zinc-deficient diet-induced rapid alterations in zinc homeostasis.

Study of Cu chemical state inside single neurons from Parkinson's disease and control substantia nigra using the micro-XANES technique.

Parkinson's disease (PD) is referred to as idiopathic disorder, which means that its causes have not been found yet. However, a few processes such as oxidative stress, protein aggregation and mitochondrial dysfunction are suspected to lead to the atrophy and death of substantia nigra (SN) neurons in case of this neurodegenerative disorder. Cu is a trace element whose role in the pathogenesis of PD is widely discussed. The investigation of Cu oxidation state inside single nerve cells from SN of PD and control cases may shed some new light on the role of this element in PD. The differences in Cu chemical state were investigated with the use of X-ray absorption near edge structure (XANES) spectroscopy. The least-square fitting method was applied for the analysis of XANES spectra. The comparison of the positions of white line, multiple scattering and pre-edge peak maximum at the energy scale did not reveal the existence of differences in Cu chemical state between PD and control samples. However, it was found that most of the Cu inside SN neurons occurs in tetrahedral environment and probably as Cu(II).

Biomolecular investigation of human substantia nigra in Parkinson's disease by synchrotron radiation Fourier transform infrared microspectroscopy.

Synchrotron radiation based-Fourier transform infrared microspectroscopy was used for preliminary investigation of the chemical composition and morphologies of the human substantia nigra of brain between normal and Parkinson's diseased tissues. The studies were carried out for thin tissue sections, focusing more particularly on nerve cell bodies, that are affected in Parkinson's disease (PD). The major spectral differences between normal (control) and PD tissues were identified at the following vibrational frequencies: 2930, 2850, 1655, 1380, 1236, 1173 and 1086 cm(-1). The infrared imaging of these biochemical markers show that for control cases the protein and nucleic acids functional groups (bands at: approximately 3300, approximately 3100, approximately 1655, approximately 1545, approximately 1240, approximately 1080 cm(-1)) are located mainly in the cell body. The spatial distribution of the band at 1740 cm(-1) (ester carbonyl stretching band) is quite dissimilar to the others, while it exhibits a minimal concentration in the cell body area. Contrarily, in PD samples, no clear evidence of variation of any of the vibrational fingerprint between cell body and the surrounding was noticed. Moreover, decrease of protein to lipid ratio as well as increase of amide I/amide II ratio were observed for PD case. The preliminary results strengthen the hypothesis that PD is a multietiological disorder. Moreover, the reported results clearly indicate that, in addition to a distinct visual observation, the diseased nerve cells exhibits change of their biochemical composition. It suggests that disturbances of normal functioning of SN neurons appear before their morphological atrophy.

Investigations of differences in iron oxidation state inside single neurons from substantia nigra of Parkinson's disease and control patients using the micro-XANES technique.

X-ray absorption near edge structure spectroscopy was applied in order to investigate differences in iron chemical state between the nerve cells of substantia nigra (SN) representing Parkinson's disease (PD) and those of control cases. Autopsy samples were cut using a cryotome, and were not fixed and not embedded in paraffin. The comparison of the absorption spectra near the iron K-edge measured in melanized neurons from SN of PD and control samples did not show significant differences in iron oxidation state. Measurements of inorganic reference materials containing iron in the second and third oxidation states indicate that most of the iron in all the nerve cell bodies examined was oxidized and occurred as trivalent ferric iron (Fe(3+)).

Classification of nerve cells from substantia nigra of patients with Parkinson's disease and amyotrophic lateral sclerosis with the use of X-ray fluorescence microscopy and multivariate methods.

The causes of Parkinson's disease and amyotrophic lateral sclerosis are still not known, but there is evidence that metal ions can be involved in processes leading to degeneration and atrophy of neurons in the case of these two neurodegenerative disorders. A synchrotron microbeam X-ray fluorescence technique was applied for topographic and quantitative analyses of selected elements on central nervous system tissue. The thin slices of brain were measured on the undulator beamline ID 22 at the European Synchrotron Radiation Facility in Grenoble, France. The polychromatic beam with the dimension of 5 microm x 2 microm (horizontal x vertical) was used in measurements. Tissues of substantia nigra representing Parkinson's disease, amyotrophic lateral sclerosis, and the control case were scanned. The results obtained indicated that accumulation of some elements depends on the case that the substantia nigra represents. Some variability in the elemental distribution for a given case was noticed as well. To investigate if present differences in the elemental accumulation between analyzed cases are statistically significant, multivariate methods were used. Cluster and discriminant analyses confirmed the significance of the differences in elemental accumulation in biological structures representing the examined cases. The methods used let us classify these structures in separate groups and determine elements, which play the greatest role in the differentiation of the biological structures for each case.