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A noise-free hyperspectral FT-IR imaging dataset of a pancreatic tissue core was simulated based on experimental data that allows to test the performance of various data analysis and processing algorithms. A set of experimental noise levels was also added and used for denoising approaches comparison, which due to the noise-free reference signal enables to truly observe signal distortion caused by different approaches., Measurement(s)pancreasTechnology Type(s)Fourier transform infrared spectroscopy • hyperspectral imagingFactor Type(s)nucleic acid • protein • lipid • wavelength • noise Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.9959129

The family of vibrational spectroscopic imaging techniques grows every few years and there is a need to compare and contrast new modalities with the better understood ones, especially in the case of demanding biological samples. Three vibrational spectroscopy techniques (high definition Fourier-transform infrared [FT-IR], Raman and atomic force microscopy infrared [AFM-IR]) were applied for subcellular chemical imaging of cholesteryl esters in PC-3 prostate cancer cells. The techniques were compared and contrasted in terms of image quality, spectral pattern and chemical information. All tested techniques were found to be useful in chemical imaging of cholesterol derivatives in cancer cells. The results obtained from FT-IR and Raman imaging showed to be comparable, whereas those achieved from AFM-IR study exhibited higher spectral heterogeneity. It confirms AFM-IR method as a powerful tool in local chemical imaging of cells at the nanoscale level. Furthermore, due to polarization effect, p-polarized AFM-IR spectra showed strong enhancement of lipid bands when compared to FT-IR.

The recent development of the AFM-IR technique, which combines nanoscale imaging with chemical contrast through infrared spectroscopy, opened up new fields for exploration, which were out of reach for other modalities, e.g. Raman spectroscopy. Lipid droplets (LDs) are key organelles, which are associated with stress response mechanisms in cells and their size falls into that niche. LDs composition is heterogeneous and varies depending on cancer cell type and the tumor microenvironment. Prostate cancer cells show a unique lipid metabolism manifested by an increased requirement for lipid accumulation in cytosolic LDs. In the current work, AFM-IR nanoimaging was undertaken to analyze lipids in untreated and x-ray irradiated PC-3 prostate cancer cells. Cells poor in LDs showed slightly increased lipid signal in cytoplasm close to the nucleus. On the other hand, high lipid signal coming from LDs accumulation could be found in any part of the cytoplasmic region. The observed behavior was found to be independent from irradiation and its dose. According to the band assignment of the collected AFM-IR spectra, the main components of LDs were assigned to cholesteryl esters. The size of LDs present in cells poor in lipids was found to be of less than 1 μm, whereas LDs aggregates spread out over a few microns. Analysis of AFM-IR spectra shows relative homogeneity of LDs composition in single cells and heterogeneity of LDs content within the PC-3 cell population.

Understanding the response of cancer cells to ionising radiation is a crucial step in modern radiotherapy. Raman microspectroscopy, together with Partial Least Squares Regression (PLSR) analysis has been shown to be a powerful tool for monitoring biochemical changes of irradiated cells on the subcellular level. However, to date, the majority of Raman studies have been performed using a single spectrum per cell, giving a limited view of the total biochemical response of the cell. In the current study, Raman mapping of the whole cell area was undertaken to ensure a more comprehensive understanding of the changes induced by X-ray radiation. On the basis of the collected Raman spectral maps, PLSR models were constructed to elucidate the time-dependent evolution of chemical changes induced in cells by irradiation, and the performance of PLSR models based on whole cell averages as compared to those based on average Raman spectra of cytoplasm and nuclear region. On the other hand, prediction of X-ray doses for individual cellular components showed that cytoplasmic and nuclear regions should be analysed separately. Finally, the advantage of the mapping technique over single point measurements was verified by a comparison of the corresponding PLSR models.

Owing to the high information content about the biochemical composition of the sample, the implementation of Fourier-Transform Infrared Spectroscopy (FT-IR) in the clinic is currently under investigation by many researchers. Cancer biology with the use of histopathological models is one of the most explored application areas. Most of the publications show sensitivity of the method to be above 90%, however, it is still often not enough for clinical standards. Robust denoising techniques with an optimized classification model allow to shorten the experimental acquisition times which still are a bottleneck for FT-IR translation into the clinic. The main premise of this work is to evaluate denoising impact on classification results using spectral techniques: Savitzky Golay (SG), Wavelets (WV), Principal Component Analysis (PCA) and Minimum Noise Fraction (MNF); and spatial techniques: Deep Neural Network (DNN), Median Filter. Using denoising methods, especially MNF and PCA, gave significant improvement of the classification and prediction results. Moreover, the increase in pixel level accuracy for High Definition data (1.1 μm projected pixel size) was found to be dependent on the complexity of the histopathological class and reached even 43–44% level, while core level increase reached around 28%. Moreover, we investigated the impact of denoising methods on the spectral input to better understand the mechanism of such large improvement. The results presented here highlight the benefits and the importance of proper denoising for classification purposes of FT-IR imaging data.

Many transition metal ions modulate the aggregation of different amyloid peptides. Substoichiometric zinc concentrations can inhibit aggregation, while an excess of zinc can accelerate the formation of cytotoxic fibrils. In this study, we report the fibrillization of the octarepeat domain to amyloid-like structures. Interestingly, this self-assembling process occurred only in the presence of Zn(ii) ions. The formed peptide aggregates are able to bind amyloid specific dyes thioflavin T and Congo red. Atomic force microscopy and transmission electron microscopy revealed the formation of long, fibrillar structures. X-ray diffraction and Fourier transform infrared spectroscopy studies of the formed assemblies confirmed the presence of cross-β structure. Two-component analysis of synchrotron radiation SAXS data provided the evidence for a direct decrease in monomeric peptide species content and an increase in the fraction of aggregates as a function of Zn(ii) concentration. These results could shed light on Zn(ii) as a toxic agent and on the metal ion induced protein misfolding in prion diseases.

Human prion protein is a subject of extensive study, related in particular to the molecular basis of neurodegenerative disease development and prevention. This protein has two main domains: the membrane C-terminal, structured domain as well as the unstructured N-terminal domain. While PrPC (23-231) has up to eight Cu(ii) binding sites in the N-terminal domain, it includes a characteristic, conservative octarepeat region PHGGGWGQ, which was studied by means of X-ray absorption near edge spectroscopy. The measurements were conducted at the SuperXAS beamline (SLS, PSI, Villigen). For the initial 1 : 1 protein-to-Cu(ii) ratio, the two main Cu(ii) binding modes were identified using linear combination fitting and ab initio FEFF calculations for X-ray spectra. Their electronic structures indicated that Cu(ii) coordinated by strong π-donors could effectively suppress the pre-edge structure due to the filling of empty Cu(ii) d-states. The suppression was correlated with the charge transfer effect and filling of the virtual electronic Cu(ii) states. What is more, we showed that the 1s → 4p + LMCT (Ligand-to-Metal-Charge-Transfer) multielectron transition relation with the main edge transition could be used as a marker for preliminary comparison of an unknown organic compound to a reference. The presented results permitted a possible explanation of the mechanism of choosing the preferred Cu(ii) modes in PrPC-Cu(ii) coordination processes and of the complex stability from the electronic point of view.

RSC Advances 9(39), 22211 - 22219 (2019). doi:10.1039/C9RA01510H, Many transition metal ions modulate the aggregation of different amyloid peptides. Substoichiometric zinc concentrations can inhibit aggregation, while an excess of zinc can accelerate the formation of cytotoxic fibrils. In this study, we report the fibrillization of the octarepeat domain to amyloid-like structures. Interestingly, this self-assembling process occurred only in the presence of Zn(II) ions. The formed peptide aggregates are able to bind amyloid specific dyes thioflavin T and Congo red. Atomic force microscopy and transmission electron microscopy revealed the formation of long, fibrillar structures. X-ray diffraction and Fourier transform infrared spectroscopy studies of the formed assemblies confirmed the presence of cross-β structure. Two-component analysis of synchrotron radiation SAXS data provided the evidence for a direct decrease in monomeric peptide species content and an increase in the fraction of aggregates as a function of Zn(II) concentration. These results could shed light on Zn(II) as a toxic agent and on the metal ion induced protein misfolding in prion diseases., Published by RSC Publishing, London

The integrity of the chromatin structure is essential to every process occurring within eukaryotic nuclei. However, there are no reliable tools to decipher the molecular composition of metaphase chromosomes. Here, we have applied infrared nanospectroscopy (AFM-IR) to demonstrate molecular difference between eu- and heterochromatin and generate infrared maps of single metaphase chromosomes revealing detailed information on their molecular composition, with nanometric lateral spatial resolution. AFM-IR coupled with principal component analysis has confirmed that chromosome areas containing euchromatin and heterochromatin are distinguishable based on differences in the degree of methylation. AFM-IR distribution of eu- and heterochromatin was compared to standard fluorescent staining. We demonstrate the ability of our methodology to locate spatially the presence of anticancer drug sites in metaphase chromosomes and cellular nuclei. We show that the anticancer 'rule breaker' platinum compound [Pt[N(p-HC6F4)CH2]2py2] preferentially binds to heterochromatin, forming localized discrete foci due to condensation of DNA interacting with the drug. Given the importance of DNA methylation in the development of nearly all types of cancer, there is potential for infrared nanospectroscopy to be used to detect gene expression/suppression sites in the whole genome and to become an early screening tool for malignancy.

Biological membranes are undoubtedly very interesting systems. Unfortunately, their analysis is very complicated and therefore simplified artificial models, like Langmuir monolayers, are frequently applied. In this work a novel method of label-free monomolecular films analysis based on infrared nano-spectroscopy (AFM-IR) is presented. In order to verify applicability of this approach well-defined referential system of sphingomyelin (SM), cholesterol (Chol) and cyclosporin A (CsA) is applied. AFM-IR method allows to directly and chemoselectively map the distribution of components in model lipid membranes. Additionally the orientation of Chol and SM molecules in the monolayer is determined by application of two perpendicular infrared laser polarizations. This paper is the first report of using AFM-IR to analyze LB films with extremely high sensitivity.

Raman spectroscopy was applied to the measurement of urinary and in vitro endothelium-derived extracellular vesicles (EVs) isolated by hydrostatic filtration dialysis (HFD) method. Raman spectra obtained for urinary EVs (UEVs) showed distinct differences in the fingerprint region. In contrast, average Raman spectra of endothelium-derived EVs samples were almost identical. Cluster Analysis of UEVs significantly discriminated diabetic samples from control, moreover endothelium-derived EVs revealed stronger similarity between long hyperglycemia and normoglycemia samples compared to short hyperglycemia. Results obtained from Partial Least Squares analysis corresponded well with integral intensities of selected bands. Our proof-of-concept approach demonstrates the potential for Raman spectroscopy to be used both for identification of EVs molecular signatures in urine samples from patients with type 2 diabetes mellitus and good glycemic control and unsatisfactory glycemic control as well as for in vitro hyperglycemic model. This noninvasive technique may be useful in identifying new biomarkers of diabetes and renal complications.

Metal nanoparticles, with their unique physicochemical and biological properties, have become increasingly studied for application in the medical field. However, the observed cytotoxicity of these structures is still a concerning issue. One of the factors determining the toxic effect of nanoparticles is the stabilizer that is present on the nanoparticle surface to prevent aggregation. In this study, the effect of the stabilizer on the drug/metal interaction is considered. For this purpose, surface-enhanced Raman spectroscopy (SERS) was applied for the characterization of erlotinib (drug included in non-small-cell lung cancer therapy) adsorption on two types of gold nanoparticles (AuNPs) obtained from a chemical reduction method using trisodium citrate (CT) and sodium borohydride (SB). Additionally, atomic force microscopy in combination with infrared spectroscopy (AFM-IR) was used with two polarization modulations to more locally determine the drug adsorption geometry, namely, at a nanoscale spatial resolution. The obtained results indicate that the aliphatic functional groups of erlotinib, NH, CH2, and OCH3, demonstrate a stronger interaction with AuSBNPs in comparison with AuCTNPs. These data imply that the effect of the stabilizing layer should be taken into account in the design of drug/metal nanoparticle conjugates.

The biological influence of radiation on living matter has been studied for years; however, several questions about the detailed mechanism of radiation damage formation remain largely unanswered. Among all biomolecules exposed to radiation, DNA plays an important role because any damage to its molecular structure can affect the whole cell and may lead to chromosomal rearrangements resulting in genomic instability or cell death. To identify and characterize damage induced in the DNA sugar-phosphate backbone, in this work we performed x-ray absorption spectroscopy at the P K-edge on DNA irradiated with either UVA light or protons. By combining the experimental results with theoretical calculations, we were able to establish the types and relative ratio of lesions produced by both UVA and protons around the phosphorus atoms in DNA.

Application of a compact laser plasma source of soft X-rays in radiobiology studies is demonstrated. The source is based on a laser produced plasma as a result of irradiation of a double-stream gas puff target with nanosecond laser pulses from a commercially available Nd:YAG laser. The source allows irradiation of samples with soft X-ray pulses in the “water window” spectral range (wavelength: 2.3–4.4 nm; photon energy: 280–560 eV) in vacuum or a helium atmosphere at very high-dose rates and doses exceeding the kGy level. Single-strand breaks (SSB) and double-strand breaks (DBS) induced in DNA plasmids pBR322 and pUC19 have been measured. The different conformations of the plasmid DNA were separated by agarose gel electrophoresis. An exponential decrease in the supercoiled form with an increase in linear and relaxed forms of the plasmids has been observed as a function of increasing photon fluence. Significant difference between SSB and DSB in case of wet and dry samples was observed that is connected with the production of free radicals in the wet sample by soft X-ray photons and subsequent affecting the plasmid DNA. Therefore, the new source was validated to be useful for radiobiology experiments.

In order to investigate changes in chemical state of iron in normal and pathologically altered human aortic valves X-ray absorption spectroscopy was applied. Since Fe is suspected to play detrimental role in aortic valve stenosis pathogenesis the oxidation state of this element has been determined. The experimental material consisted of 10 μm sections of valves excised during routine surgery and from autopsies. The experiment was performed at the MicroXAS beamline of the SLS synchrotron facility in Villigen (Switzerland). The Fe K-edge XANES spectra obtained from tissue samples were carefully analyzed and compared with the spectra of reference compounds containing iron in various chemical structures. The analysis of absorption edge position and shape of the spectra revealed that both chemical forms of iron are presented in valve tissue but Fe 3+ is the predominant form. Small shift of the absorption edge toward higher energy in the spectra from stenotic valve samples indicates higher content of the Fe 3+ form in pathological tissue. Such a phenomenon suggests the role of Fenton reaction and reactive oxygen species in the etiology of aortic valve stenosis. The comparison of pre-edge regions of XANES spectra for control and stenotic valve tissue confirmed no differences in local symmetry or spin state of iron in analyzed samples.

A desk-top laser-produced plasma (LPP) source of soft X-rays (SXR) has been developed for radiobiology research. The source is based on a double-stream gas puff target, irradiated with the focused beam of a commercial Nd:YAG laser. The source has been optimized to get a maximum photon emission from LPP in the X-ray “water window” spectral wavelength range from 2.3 nm (i.e., an absorption edge of oxygen) to 4.4 nm (i.e., an absorption edge of carbon) (280–540 eV in photon energy units) by using argon gas-puff target and spectral filtering by free-standing thin foils. The present source delivers nanosecond pulses of soft X-rays at a fluence of about 4.2 × 103 photons/μm2/pulse on a sample placed inside the vacuum chamber. In this paper, the source design, radiation output characterization measurements and initial irradiation experiments are described. The source can be useful in addressing observations related to biomolecular, cellular and organisms’ sensitivity to pulsed radiation in the “water window”, where carbon atoms absorb X-rays more strongly than the oxygen, mostly present in water. The combination of the SXR source and the radiobiology irradiation layout, reported in this article, make possible a systematic investigation of relationships between direct and indirect action of ionizing radiation, an increase of a local dose in carbon-rich compartments of the cell (e.g., lipid membranes), an experimental estimation of a particular role of the Auger effect (in particular in carbon atoms) in the damage to biological systems, and the study of ionization/excitation-density (LET – Linear Energy Transfer) and dose-rate effects in radiobiology.

Lipid droplets (LDs) are key organelles in cancer cells proliferation, growth, and response to stress. These nanometric structures can aggregate to reach the size of microns becoming important cell components. Although it is known that LDs contain various lipids, their chemical composition is still under investigation. Moreover, their function in cell's response to exogenous factors is also not fully understood. Raman spectroscopy, together with chemometrics, has been shown to be a powerful tool for analytical analyses of cancer cell components on the subcellular level. It provides the opportunity to analyse LDs in a label-free manner in live cells. In the current study, this method was applied to investigate LDs composition in untreated and irradiated with X-ray beams prostate cancer cells. Raman mapping technique proved lipids accumulation in PC-3 cells and allowed visualization of LDs spatial distribution in cytoplasm. A heterogeneous composition of LDs was revealed by detailed analysis of Raman spectra. Interestingly, PC-3 cells were found to accumulate either triacylglycerols or cholesteryl esters. Finally, effect of X-ray radiation on the cells was investigated using Raman spectroscopy and fluorescence staining. Significant influence of LDs in the process of cell response was confirmed and time dependence of this phenomenon was determined.

This work explores the potential of Fourier transform infrared (FTIR) spectroscopic imaging at the single-cell level combined with partial least squares (PLS) regression to investigate the cell’s response to drug/gold nanoparticles (AuNPs) conjugates. In a broader context, this study provides not only the effective differentiation between non-tumorigenic, mildly malignant and malignant cells, but above all proposes a novel approach to define the sensitivity of cancer cells to drugs. AuNPs can be widely and effectively used in biosensing and bioimaging applications. Three human breast cell lines, i.e. MCF10A (non-tumorigenic), MCF7 (metastatic, pleural effusion, mildly malignant) and MDA-MB-231 (metastatic, pleural effusion, malignant) have been exposed to different concentration of AuNPs (15 mg/L and 10 mg/L) and AuNPs/α-methyl-DL-tryptophan conjugates conjugates (10 mg/L NPs @ 3.75 × 10−6 M and 10 mg/L NPs @ 3.75 × 10−5 M). It is implied that the class values predicted by the PLS regression model indicate a very good separation between non-tumorigenic, mildly malignant and malignant cells. Furthermore, the FTIR-PLS approach shows that bands responsible for the segregation between the control cells and the cells treated with drug/AuNPs conjugate result from the changes in RNA/DNA, lipids and amide I and II vibrations. Drug/AuNPs conjugate affects the nucleic acids in all of the studied cell types. The performed analysis suggests that MCF-7 cell line is particularly sensitive to the drug treatment, which result in the drug interacting with nucleic acids and DNA conformational changes.

The correction of optical effects in infrared spectroscopy has been developing over the years with Resonant Mie Extended Multiplicative Scattering Correction marking a significant point. A new open-source algorithm of Mie Extinction Extended Multiplicative Scattering Correction has been released in 2019, giving a much needed flexibility for the user. The new approach uses a Van der Hulst approximation for absorbing spheres instead of full Mie theory, significantly speeding up the calculations. In this article a comparison of both algorithms is done in the context of biological tissue imaging with a Focal Plane Array, which has its own characteristics over single animal cell measurements or IR confocal measurements.

The paper concentrates on the role of saliva in the early diagnosis of salivary gland tumor. Due to the still not fully understood etiology of head and neck cancer, the fundamental objective of this study is to find tumor markers of salivary gland tumor progression. This work demonstrates the feasibility of Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) to track spectral variations between saliva samples derived from healthy volunteers and from salivary gland tumor (tumor mixus, TM) patients. Furthermore, a Real-Time Polymerase Chain Reaction (RT-PCR) has been used to detect a selected genes expression associated with neoplasm changes. The obtained results imply that spectral signals attributed to the amide I/II (secondary structure of protein), carbohydrates and inorganic phosphates oscillations are the most sensitive to alterations associated with the salivary gland cancer progression. Several spectroscopic biomarkers have been indicated as potential predictors of salivary gland tumor development. Additionally, the RT-PCR results reveal the increased level of Bcl-2 factor in salivary gland tumor patients' samples which may be related to inhibition of apoptosis process and increasing uncontrolled cell proliferation. Such combination of physico-chemical methods is a unique approach towards better understanding the tumor's etiology and early diagnosis problem. Based on the promising findings presented in this article, it could be concluded that saliva fluid has a great potential to be used as a first-line diagnostic tool in patients with suspicion of salivary gland tumor.

In this study, surface - enhanced Raman spectroscopy (SERS) was applied at the first time for estimation of how pH, temperature, and nanoparticle (NP) stabilizer affect an adsorption behavior of erlotinib (drug approved in a non-small cell lung cancer therapy) onto citrate-stabilized silver nanoparticles (AgNPs). Novel approach to improve cancer therapy assumes application of NPs as an efficient drug delivery system. This strategy requires designing stable drug/nanocarrier conjugates that can effectively interact in the target site. It is also important to perform deeply characterization of a drug orientation on the potential carrier surface and estimation how stable the appeared interaction is. Performed analysis, indicates that pH, temperature, presence of NP stabilizers, and time of incubation have an influence on the occurring adsorption geometry of the drug. However, the observed erlotinib/AgNP interaction remains stable regardless of the applied conditions. These considerations were supported by insightful physicochemical characteristics of the AgNPs and the erlotinib/AgNP conjugates by conducting transmission electron microscopy (TEM) imaging, determination of colloid stability conducted with the use of dynamic light scattering technique (DLS) and measurements of electrophoretic mobility. Such complex approach allows a better understanding of the stability of the erlotinib/AgNP conjugates and provides information how the investigated interaction is affected by the induced perturbations.

High performance carbon nanotube fibres and yarns with similar strength to Kevlar could now offer significant structural reinforcement and multifunctionality to 3D printed parts. However, recently printed parts which were reinforced with highly-densified CNT yarns exhibited only modest mechanical properties due to poor wettability with polymers. In this work, PMMA is proposed for infiltration of CNT fibres produced via Floating Catalyst CVD, as the polymer may be printed at a lower temperature and is industrially used in resilient fibre-reinforced thermoplastic prepregs and laminates. Nanoscale chemical imaging by AFM combined infrared spectroscopy revealed good infiltration while the PMMA/CNT fibre composite filaments exhibited pronounced increase in tensile strength and Young’s modulus.

The work presents a detailed analysis of adsorption geometry of nocodazole, an antineoplastic agent, deposited onto gold nanoparticle surface. This type of study is fundamental because of improving drugs delivery specifically to cancer cells. Simultaneously, it will revolutionize oncology, dramatically improving the effect of drugs while reducing or even eliminating adverse side effects for the patient. The analytical means are now mature to study very precisely the solid-liquid interactions that will foreshadow the development of new anti-cancer therapies. This research demonstrates the feasibility of surface-enhanced infrared absorption spectroscopy (SEIRA), surface-enhanced Raman spectroscopy (SERS) and polarization-modulated surface-enhanced infrared absorption nanospectroscopy (PM-SEIRA) to study adsorption phenomenon of a molecule at the solid/liquid interface. A novel approach combining the classical infrared spectroscopy (IR) with atomic force microscopy (AFM) is employed to more comprehensively describe the behavior of the drug adsorbed on metal surface at the nanoscale level. The performed analysis enabled to track subtle changes in nocodazole bonds arrangement upon adsorption. The observed changes provide evidence for strong interaction between the electron-rich thiophene ring and metal surface. It can also be inferred that charge-transfer mechanism contributes considerably to the SERS signal amplification.

The goal of this contribution is to present and familiarize the medical community with the method for the assessment of trace and essentials elements in prostate tissue sections.X-ray fluorescence based technique(namely Synchrotron Induced X-ray Emission (SRIXE)) is described in terms of methodology, sample preparation and the evaluation of the recorded results (spectral data sets). Materials for the samples were collected from the patients underwent radical prostatectomy due to Adenocarcinoma prostatae. Specimens were freeze-dried, cut by microtome (to the thickness of 15 μm), one slice was placed on Mylar foil (for SRIXE measurements) and adjacent one on microscopic glass (for histopathological assessment).Results presented here show the usability of SRIXE method for the evaluation of concentration of trace and essential elements in prostate tissue sections with the spatial resolution better than 15 microns.Histopathological analysis of samples, which is only focused on morphological features, is unable to reveal information about changes in biochemical signature of tissues affected by the illness. SRIXE is a powerful and promising technique to analyse even very low concentrations oat the cellular level without any labelling or separating procedures. Obtained results may be correlated with classic histopathological assessment allowing for drawing conclusions on the changes in certain elements concentrations with the progression of disease. Moreover, mentioned in this work analysis, can be performed for any type of biological tissues.

Exposure to ionizing radiation can induce cellular defense mechanisms including cell activation and rapid proliferation prior to metastasis and in extreme cases can result in cell death. Herewith we apply infrared nano- and microspectroscopy combined with multidimensional data analysis to characterize the effect of ionizing radiation on single glioblastoma nuclei isolated from cells treated with 10 Gy of X-rays or 1 and 10 Gy of protons. We observed chromatin fragmentation related to the formation of apoptotic bodies following X-ray exposure. Following proton irradiation we detected evidence of a DNA conformational change (B-DNA to A-DNA transition) related to DNA repair and accompanied by an increase in protein content related to the synthesis of peptide enzymes involved in DNA repair. We also show that proton exposure can increase cholesterol and sterol ester synthesis, which are important lipids involved in the metastatic process changing the fluidity of the cellular membrane in preparation for rapid proliferation.

Raman spectroscopy of erythrocytes provides detailed information about the structure and status of heme moiety, which can be used to provide new insights into molecular pathogenesis of several diseases. In this study, we present the first Raman spectroscopy investigations of the effect of hemoglobin oxygenation in the context of hypertensive disease. The experimental data was subjected to Logistic Regression, which indicated heme‑oxygenation status as an important risk factor alongside other clinical parameters. The 1605/1621 cm−1 band ratio was selected as an optimal Raman metric for risk assessment and along with other band ratios (1583, 1639, 1310 cm−1) related to heme status and when combined with clinical data via logistic regression gave an Area Under the Curve (AUC) >0.95 for prehypertension risk prediction. The work demonstrates the feasibility of Raman spectroscopy to distinguish between prehypertensive and normotensive states. Simultaneously, it is implied that the etiology of the high blood pressure progression may be connected with the changes in hemoglobin oxygenation.

Although anthracycline antibiotics have been successfully used for nearly half a century in the treatment of various malignancies, their use is limited by their cardiac and vascular toxicities, and the mechanisms of these toxicities are still not entirely clear. Herein, we comprehensively characterized cytotoxic effects of two structurally related anthracyclines, doxorubicin and daunorubicin. In nanomolar concentrations, both drugs induced DNA damage and increased nuclear area that were associated with their accumulation in the nucleus (doxorubicin ⩾50 nM and daunorubicin ⩾25 nM) as evidence by Raman microspectroscopy at 3820-4245 cm(-1). At low micromolar concentrations, doxorubicin (⩾5 μM) and daunorubicin (⩾1 μM) increased the generation of reactive oxygen species, decreased intracellular reduced glutathione, induced an alteration in endothelial elasticity and caused a reorganization of the F-actin cytoskeleton. In isolated mouse aortic rings, doxorubicin (⩾50 μM) was less potent than daunorubicin (⩾5 μM) in impairing the endothelium-dependent response. In summary, using a comprehensive endothelial profiling approach, we demonstrated clear-cut differences in the potencies to induce endotheliotoxic responses for two structurally similar chemotherapeutics, at a nuclear, cytosolic and membrane levels. Furthermore, our results suggest that the differences in the endothelial toxicities of doxorubicin and daunorubicin are linked to differences in their nuclear accumulation and the DNA damage-triggered response of the endothelium.

Pre-processing of Fourier transform infrared (FTIR) spectra is typically the first and crucial step in data analysis. Very often hyperspectral datasets include the regions characterized by the spectra of very low intensity, for example two-dimensional (2D) maps where the areas with only support materials (like mylar foil) are present. In that case segmentation of the complete dataset is required before subsequent evaluation. The method proposed in this contribution is based on a multivariate approach (hierarchical cluster analysis), and shows its superiority when compared to the standard method of cutting-off by using only the mean spectral intensity. Both techniques were implemented and their performance was tested in the R statistical environment - open-source platform - that is a favourable solution if the repeatability and transparency are the key aspects.

The lack of molecular mechanistic understanding of the interaction between metal complexes and biomolecules hampers their potential medical use. Herein we present a robust procedure combining resonant X-ray emission spectroscopy and multiscale molecular dynamics simulations, which allows for straightforward elucidation of the precise interaction mechanism at the atomic level. The report unveils an unforeseen hydrolysis process and DNA binding of [Pt{N(p-HC6F4)CH2}2py2] (Pt103), which showed potential cytotoxic activity in the past. Pt103 preferentially coordinates to adjacent adenine sites, instead of guanine sites as in cisplatin, because of its hydrogen bond ability. Comparison with previous research on cisplatin suggests that selective binding to guanine or adenine may be achieved by controlling the acidity of the compound.

Atomic force microscopy - infrared (AFM-IR) spectroscopy allows spectroscopic studies in the mid-infrared (mid-IR) spectral region with a spatial resolution better than is allowed by the diffraction limit. We show that the high spatial resolution can be used to perform spectroscopic and imaging studies at the subcellular level in fixed eukaryotic cells. We collect AFM-IR images of subcellular structures that include lipid droplets, vesicles and cytoskeletal filaments, by relying on the intrinsic contrast from IR light absorption. We also obtain AFM-IR absorption spectra of individual subcellular structures. Most spectra show features that are recognizable in the IR absorption spectra of cells and tissue obtained with FTIR technology, including absorption bands characteristic of phospholipids and polypeptides. The quality of the spectra and of the images opens the way to structure and composition studies at the subcellular level using mid-IR absorption spectroscopy.

Aortic valve is a part of the heart most frequently affected by pathological processes in humans what constitute a very serious health problem. Therefore, studies of morphology and molecular microstructure of the AV are needed. µSR- FTIR spectroscopy and microscopy represent unique tools to study chemical composition of the tissue and to identify spectroscopic markers characteristic for structural and functional features. Normal AV reveals a multi-layered structure and the compositional and structural changes within particular layers may trigger degenerative processes within the valve. Thus, deep insight into the structure of the valve to understand pathological processes occurring in AV is needed. In order to identify differences between three layers of human AV, tissue sections of macroscopically normal AV were studied using µSR- FTIR spectroscopy in combination with histological and histochemical stainings. Tissue sections deposited onto CaF2 substrates were mapped and representative set of IR spectra collected from fibrosa, spongiosa and ventricularis were analysed by Principal Component Analysis (PCA) in the spectral range between 1850–1000 cm−1 and 3050–2750 cm−1. PCA revealed a layered molecular structure of the valve and it was possible to identify IR bands associated to different tissue parts. Spongiosa layer was well differentiated from other two layers mainly based on IR bands characteristic for the distribution of glycosaminoglycans (GAGs) in the tissue – like 1170 cm−1 (υas(C-O-S)) and 1380 cm−1 (acetyl amino group). Additionally, it was distinguished from fibrosa and ventricularis based on 1085 cm−1 and 1240 cm−1 bands characteristic for GAGs and for carbohydrates- ν(C-O) and ν(C-O-C) respectively and nucleic acids -νsym(PO2−) and νasym(PO2−) respectively, which were less specific for this layer. The use of µSR- FTIR spectroscopy demonstrated co-localization of GAGs and lipids in spongiosa layer what may indicate their contribution in the very early phase of aortic valve calcific degeneration.

In this study we present vibrational analysis of healthy (non-affected by cataract) and cataractous human lenses by means of Raman and FTIR spectroscopy methods. The performed analysis provides complex information about the secondary structure of the proteins and conformational changes of the amino acid residues due to the formation of opacification of human lens. Briefly, the changes in the conformation of the Tyr and Trp residues and the protein secondary structure between the healthy and cataractous samples, were recognized. Moreover, the observed spectral pattern suggests that the process of cataract development does not occur uniformly over the entire volume of the lens.

Synchrotron Radiation Fourier Transform Infrared (SR-FTIR) spectra of single human prostate adenocarcinoma PC-3 cells, irradiated with a defined number of 2 MeV protons generated by a proton microbeam along with non-irradiated control cells, were analysed using multivariate methods. A number of different Principal Component Analysis (PCA) models were tested and the spectral ranges associated with nucleic acids, proteins and lipids were analysed separately. The results show a dose dependent shift of the O P O asymmetric stretching mode from 1234 cm−1 to 1237 cm−1, consistent with local disorder in the B-DNA conformation along with a change in intensity of the O P O symmetric stretching band at 1083 cm−1 indicative of chromatin fragmentation – the natural consequence of a high number of DNA Double Strand Breaks (DSBs). 2D mapping of characteristic functional groups at the diffraction limit shows evidence of lipid deposition and chromatin condensation in cells exposed to protons indicative of cell apoptosis following irradiation. These studies lay the foundation for understanding the macromolecular changes that occur to cells in response to radiation therapy, which has important implications in the treatment of tumours.

SR-FTIR in combination with Principal Component Analysis (PCA) was applied to investigate macromolecular changes in a population of melanocytes and their extracted nuclei induced by environmentally relevant fluxes of UVR (Ultraviolet Radiation). Living cells and isolated cellular nuclei were investigated post-irradiation for three different irradiation dosages (130, 1505, 15,052 Jm(-2) UVR, weighted) after either 24 or 48 hours of incubation. DNA conformational changes were observed in cells exposed to an artificial UVR solar-simulator source as evidenced by a shift in the DNA asymmetric phosphodiester vibration from 1236 cm(-1) to 1242 cm(-1) in the case of the exposed cells and from 1225 cm(-1) to 1242 cm(-1) for irradiated nuclei. PCA Scores plots revealed distinct clustering of spectra from irradiated cells and nuclei from non-irradiated controls in response to the range of applied UVR radiation doses. 3D Raman confocal imaging in combination with k-means cluster analysis was applied to study the effect of the UVR radiation exposure on cellular nuclei. Chemical changes associated with apoptosis were detected and included intra-nuclear lipid deposition along with chromatin condensation. The results reported here demonstrate the utility of SR-FTIR and Raman spectroscopy to probe in situ DNA damage in cell nuclei resulting from UVR exposure. These results are in agreement with the increasing body of evidence that lipid accumulation is a characteristic of aggressive cancer cells, and are involved in the production of membranes for rapid cell proliferation.

Spore-forming bacteria are a class of microorganisms that possess the ability to survive in extreme environmental conditions. Morphological features of spores assure their resistance to stress factors such as high temperature, radiation, disinfectants, and drying. Consequently, spore elimination in industrial and medical environments is very challenging. Ceragenins are a new class of cationic lipids characterized by a broad spectrum of bactericidal activity resulting from amphipathic nature and membrane-permeabilizing properties. To assess the impact of ceragenin CSA-13 on spores formed by Bacillus subtilis (ATCC 6051), we performed the series of experiments confirming that amphipathic and membrane-permeabilizing properties of CSA-13 are sufficient to disrupt the structure of B. subtilis spores resulting in decreased viability. Raman spectroscopy analysis provided evidence that upon CSA-13 treatment the number of CaDPA-positive spores was clearly diminished. As a consequence, a loss of impermeability of the inner membranes of spores, accompanied by a decrease in spore resistance and killing take place. In addition to their broad antimicrobial spectrum, ceragenins possess great potential for development as new sporicidal agents.

Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and optical microscopy were applied to identify pigments, binders, fillers and degradation products in the 17th century painting 'The Coronation and Assumption of the Blessed Virgin Mary’. This piece of art is located in the Convent of the Sisters of St. Catherine in Orneta (Poland). The main important aspect is the non-destructive character of the examination and the small amount of sample necessary for analysis. Individual layers of five micro-samples cross-sections and powder samples taken from discrete and representative areas of the painting were studied. The combination of a linear array detector with a micro-ATR imaging accessory provides the possibility to overcome the diffraction limit and deliver a spatial resolution of about 5 microns (pixel size at the sample equal to 6.25 μm). Noteworthy, a rich palette of white, blue, red, and black pigments was characterized in the analyzed areas. Additionally, the mapping mode provided information about the distribution of individual components in the paint cross-sections. The analysis made it possible to identify binding media, such as proteins, triglycerides, fatty acids or resin acids, and degradation products, including whewellite, moolooite, and lead soaps.

Measurement of physical properties of porous geological materials is a crucial issue in oil and gas recovery industry. A conventional experimental way to obtain information on porosity, pore size distribution, specific surface area and permeability are the intrusion porosimetry and permeameter measurement. However, in this approach Washburn's equation is usually used, thus approximation of cylindrical pore shapes is made. However, in recent times the computed microtomography (CMT) technique is more widely used in geoscience ( Appoloni et al., 2007 ). We have already reported preliminary results of investigation of elemental content, microporosity, and specific surface area of porous rocks by means of the CMT technique based on a laboratory source ( Bielecki et al., 2012 ). In this paper, results of complex study of porous rock samples with the use of X-ray CMT (laboratory-source-based facility and synchrotron radiation source) combined with permeability tensor computation by means of the Lattice Boltzmann Method (LBM) are presented. Moreover, the proton induced X-ray emission (PIXE) method was additionally used for elemental content determination of pores-filling substance.

The role of sulfur in prostate cancer progression may be significant for understanding the process of carcinogenesis. This work, based on X-ray Absorption Near Edge Structure (XANES) spectroscopy, is focused on determination of sulfur chemical species occurring in prostate cancer cell lines. The experimental material consisted of four commercially available cell lines: three from metastasized prostate cancer (PC3, LNCaP, and DU145) and one, used as a control, from the non-tumourigenic peripheral zone of the prostate (PZ-HPV-7). The experiment was performed at the SUL-X beamline of the synchrotron radiation source ANKA, Karlsruhe (Germany). The K-edge XANES spectra of sulfur were analyzed by deconvolution in order to establish sulfur species that occur in prostate cancer cells and to find out whether there are any differences in their content between various cell lines. Experimental spectra were fitted in two ways: with two Gaussian peaks and one arctangent step function, and additionally by a Linear Combination Fit with spectra of reference compounds in order to obtain quantitative chemical information. All fitting procedures were performed with the Athena code ( Ravel and Newville, 2005 ) and the results of deconvolution were used to determine the fraction of each sulfur form. The results of data analysis showed that cell lines from different metastasis had different ratio of reduced to oxidized sulfur species. The LCF analysis demonstrated that the highest content of GSH, one of the most important sulfur-bearing compounds in cells, was observed in DU145 cells. These findings may confirm the hypothesis of changes in redox balance in case of cancer initiation and progression.

The effect of ionizing radiation on single cells using a proton source was investigated using Fourier transform infrared (FTIR) microspectroscopy. The prostate cancer cells (DU-145) were irradiated by a specific number (50, 200, 400, 2000 and 4000) of protons per cell. Next after fixing the cells with 70% ethanol micro-FTIR spectra were obtained using both: (a) the synchrotron radiation source with a Mercury–Cadmium–Telluride (MCT) detector and (b) a globar source with a focal plane array (FPA) detector. FTIR spectra obtained from both instrumental configurations were analyzed independently to investigate the changes in the DNA phosphodiester region (1150–950 cm −1 ) of irradiated and control (untreated by ionizing radiation) cells. A Principal Component Analysis (PCA) scores plot revealed distinct clusters for all groups of irradiated cells, even for those irradiated by the smallest dose of protons. The dose-dependent changes in the relative intensities of DNA peak at 970 cm −1 (ribose-phosphate skeletal motions), along with a shift of the O–P–O band corresponding to the symmetric phosphodiester stretching mode at 1090 cm −1 were observed. The results demonstrate that FTIR spectroscopy is a promising tool to investigate DNA damage in single cells and may become an important tool in assessing cell damage following radiotherapy.

DNA double strand breaks (DSBs) are deadly lesions that can lead to genetic defects and cell apoptosis. Techniques that directly detect DNA DSBs include scanning electron microscopy, atomic force microscopy (AFM), and fluorescence based approaches. While these techniques can be used to identify DSBs they provide no information on the molecular events occurring at the break. Tip-enhanced Raman scattering (TERS) can provide molecular information from DNA at the nanoscale and in combination with AFM provides a new way to visualize and characterize the molecular structure of DSBs. DSBs result from cleavage at the 3'- and 5'-bonds of deoxyribose upon exposure to UVC radiation based on the observation of P-O-H and methyl/methylene deformation modes enhanced in the TERS spectra. It is hypothesized that strand fragments are hydrogen-terminated at the lesion, indicating the action of free radicals during photon exposure. Refereed/Peer-reviewed

This study was conducted to verify whether the FT-IR spectroscopy and Focal Plane Array (FPA) imaging can be successfully applied to estimate the quality of bovine blastocysts (on the basis of the concentration of nucleic acids and amides). The FT-IR spectra of inner cell mass from blastocysts of three different culture systems were examined. The spectral changes between blastocysts were analyzed in DNA (spectral range of 1240–950 cm −1 ) and protein amides (1800–1400 cm −1 ). Blastocyst 1 (BL1-HA) was developed from the fertilized oocyte cultured with low concentration of hialuronian (HA), Blastocyst 2 and 3 were developed from the oocytes cultured in standard conditions. Cleavage stage blastocyst 2 (BL2-SOF) has been cultured in SOF medium while blastocyst 3 (BL3-VERO) was cultured in co-culture with VERO cells. The multivariate statistical analysis (Hierarchical Cluster Analysis – HCA and Principal Component Analysis – PCA) of single cells spectra showed high similarity of cells forming the inner cell mass within single blastocyst. The main variance between the three examined blastocysts was related to amides bands. Differences in the intensities of the amides’ peaks between the bovine blastocysts derived from different culture systems indicated that specific proteins reflecting the appearance of a new phenotype were produced. However, for the three blastocysts, the α-helix typical peak was twice more intensive than the β-sheet typical peak suggesting that the differentiation processes had been started. Taking into account the quantitative and qualitative composition of the protein into examined blastocysts, it can be assumed, that the quality of the BL1-HA turned out much more similar to BL3-VERO than to BL2-SOF. FT-IR spectroscopy can be successfully applied in reproductive biology research for quality estimation of oocytes and embryos at varied stages of their development. Moreover this technique proved to be particularly useful when the quantity of the available material for research purposes is limited.

Extremely brilliant infrared (IR) beams provided by synchrotron radiation sources are now routinely used in many facilities with available commercial spectrometers coupled to IR microscopes. Using these intense non-thermal sources, a brilliance two or three order of magnitude higher than a conventional source is achievable through small pinholes (

Here, we present the first approach to human lenses investigations with and without cataract development changes in nanoscale resolution using AFM – IR spectroscopy. We proved that the application of this technique allowed us to better understand of structural changes connected with advancing disease process in studied lenses. The obtained results show the impact of the disease development on the secondary structure of proteins in these human tissues. The domination of the β–turn protein secondary structure is observed in the clear (non-affected by cataract) lens. While, in the case of the opaque (cataractous) samples the different degree of the degradation due to development of cataract, was recognized. Briefly, this process is associated with the protein secondary changes from β–turn/β–sheet parallel for less altered part of the lens to stable anti-parallel β–sheet for the more degraded part. Interestingly, the AFM – IR technique provided estimation of the protein secondary structure without the need for using deconvolution procedure.

The use of synchrotron radiation may shed more light on the study of prostate cancer, one of the leading diseases among men. In the presented study the microbeam setup at the PSI Swiss Light Source combined with fluorescence detected X-ray absorption spectroscopy (XAS) was applied to determine two-dimensional (2D) imaging of distributions of various chemical sulfur forms in prostate cancer tissue sections, since sulfur is considered important and essential in cancer progression. The research focused on prostate tissues obtained during routine prostatectomies on patients suffering from prostate cancer. Our previous studies using μ-XAS point measurements on prostate cancer cell lines showed the differences in fractions of various forms of sulfur between cancerous and non-cancerous cells. Therefore, in this experiment the chosen areas of prostate cancer tissues were scanned to get the full picture of the chemical composition of tissue, which is highly heterogeneous. The incident X-ray beams of energies tuned to spectroscopic features of the near-edge region of sulfur K-edge absorption spectra were used to provide contrast between chemical species presented in the tissue. Next, the relative content of the three main sulfur forms, found in biological systems, was calculated and the results are presented in a form of 2D color maps. These maps are correlated with the microscopic histological image of the scanned area. The main findings show that sulfur occurs in prostate tissue mainly in reduced form. The oxidized form of sulfur is present mostly in prostatic stroma, while sulfur in intermediate oxidation state is present in trace amount.

Trace elements concentrations of Ca, Fe, and Zn in cataractous human lenses have been determined using the proton induced X-ray emission technique (PIXE). The technique was selected due to its sensitivity and only modest requirements for sample material processing and preparation. In the study a statistically significant sample of post–surgery (phacoemulsification method) human lenses with cataract was analyzed, targeting two diseases, often accompanying the cataract: diabetes and glaucoma. Patients were split into three, not overlapping groups: control, diabetic and glaucoma. Elevated levels of Zinc (strong effect) and Iron (weak effect) were measured for fraction of diabetic patients treated with insulin or pills. Samples from fraction of patients with glaucoma showed very elevated Zinc levels, often reaching concentration values of several hundreds of ppm, about one order of magnitude higher than in remaining cases. Due to overwhelming popularity of the phacoemulsification procedure and the ease of sample preparation for PIXE, current study suggests new perspective in medical diagnostic.

In recent years, the Fourier transformed infrared spectroscopy is often applied in studies of biologicalmaterials on cellular level. Undoubted advantage of this method is high sensitivity. In presented research theFTIR microspectroscopy was used to analyse the DNA damage in single PC-3 cells (prostate cancer cell linederivedfrombonemetastases)irradiatedbycountednumberofprotons. Focusedprotonmicrobeam2MeVfromthe Van de Graaff accelerator at the Institute of Nuclear Physics, Polish Academy of Sciences, was used as anirradiation source. Four groups of single cells were irradiated with 1000, 2000, 4000, and 8000 protons per cell,respectively. Following irradiation cells were fixed in 70% ethanol and then analyzed by IR microspectroscopy.Bond analysis of IR spectra served as a base for result analysis. This research has focused on the detection ofchangesinDNAbackbonespectralrange(950–1240cm