Raman imaging of heme metabolism in situ in macrophages and Kupffer cells.

Herein, we provide the Raman imaging results for different stages of erythrophagocytosis of senescent red blood cells executed by isolated murine primary Kupffer cells and a murine macrophage cell line (RAW 264.7). Images were recorded with the use of 488 and 532 nm excitation lines. The use of Resonance Raman spectroscopy allowed the visualization of the heme metabolism and tracking of the systemic iron recycling process inside isolated murine Kupffer cells and RAW.264 cells. Because of the application of the different experimental assays, the erythrophagocytosis in two types of cells was presented and associated with the presence of different forms of oxidized and degradative derivatives of hemoglobin species. Moreover, we observed an increase of lipid level and later formation of lipid droplets during the erythrophagocytosis process inside RAW 264.7 macrophages and murine Kupffer cells.

Label-free spectroscopic characterization of live liver sinusoidal endothelial cells (LSECs) isolated from the murine liver.

Liver sinusoidal endothelial cells (LSECs) represent a highly specialized and unique type of endothelial cell in terms of their morphology and function. The biochemical and functional characterization of LSECs in vitro is restrained by the rapid change of LSECs' phenotype upon culturing under classical experimental conditions. In this work, we present a novel approach to characterize the biochemical content of murine LSECs, freshly isolated from the liver, with the use of microspectroscopic analysis. For comparison, hepatocytes and Hepatic Stellate Cells (HSCs) were analyzed. Our approach, based on label-free confocal Raman imaging of live cells combined with chemometric analysis, provided insight into the biochemical content of freshly isolated LSECs on a subcellular level. LSECs were featured by a distinct biochemical signature in comparison with other major cell types of the liver. Based on our work we claim that the non-invasive and non-destructive confocal Raman imaging may assist in obtaining chemical information spatially distributed within the cells that characterize the phenotype of primary LSECs as well as other types of liver cells. Furthermore, our approach provides a unique insight into LSECs' morphology and chemical composition that may help to understand their functions.

A new insight into the interaction of cisplatin with DNA: ROA spectroscopic studies on the therapeutic effect of the drug.

Raman optical activity (ROA) spectroscopy has been applied for the first time to study the interaction of cisplatin with DNA. The knowledge about the structure of DNA-metal ion cross-links and hence the mechanism of the drug action is fundamental for the development of new antitumor drugs. At the same time, there is an urgent need to search for new methods for monitoring of this effect at the therapeutic dose of a drug. We have demonstrated that ROA spectroscopy is a sensitive technique with the capability to follow the structural alteration of the whole DNA molecule upon drug binding via a direct observation of transformation undergoing within chiral sugar moieties. A ROA profile delivers clear evidence of a partial transition from the B-DNA to the A-form due to the formation of cisplatin-DNA cross-links.

FT-IR imaging for quantitative determination of liver fat content in non-alcoholic fatty liver.

In this work we apply FT-IR imaging of large areas of liver tissue cross-section samples (∼5 cm × 5 cm) for quantitative assessment of steatosis in murine model of Non-Alcoholic Fatty Liver (NAFLD). We quantified the area of liver tissue occupied by lipid droplets (LDs) by FT-IR imaging and Oil Red O (ORO) staining for comparison. Two alternative FT-IR based approaches are presented. The first, straightforward method, was based on average spectra from tissues and provided values of the fat content by using a PLS regression model and the reference method. The second one ­ the chemometric-based method ­ enabled us to determine the values of the fat content, independently of the reference method by means of k-means cluster (KMC) analysis. In summary, FT-IR images of large size liver sections may prove to be useful for quantifying liver steatosis without the need of tissue staining.

Raman spectroscopic studies of vitamin A content in the liver: a biomarker of healthy liver.

Confocal Raman microspectroscopy was used in this study to identify hepatic stellate cells (HSCs) from healthy mice and mice with untreated and treated liver steatosis. We have identified the main form of occurrence of vitamin A in healthy liver and confirmed its absence in the pathological state. Additionally, we have reported the reappearance of vitamin A in the tissue after treatment of liver steatosis.

Biochemical changes of the endothelium in the murine model of NO-deficient hypertension.

The main spectral differences between the biochemical compositions of the vascular endothelium of control, hypertensive NO-deficient, and NO-deficient mice supplemented with nitrate were studied using Raman microimaging. A significantly different Raman signature of the endothelium in these three groups in the 1200-1400 cm(-1) region was assigned to the α-helix and ß-sheet alterations in the protein secondary structure upon the development of hypertension. The second pronounced biochemical marker of endothelium alterations was the lipid to protein ratio. A lower intensity of the band at 2940 cm(-1) relative to the feature at 1007 cm(-1) in the endothelium in hypertension compared to the control indicated a decrease of the lipid content relative to proteins during the progress of the pathology. The nitrate-based treatment partially reversed the effects of hypertension. The nitrate supplementation restored the lipid to protein ratio in the endothelium to the control level, while the changes in the secondary structure of proteins were irreversible upon nitrate administration.

Identification of a biochemical marker for endothelial dysfunction using Raman spectroscopy.

In the present work, we propose the spectroscopic approach to identify biochemical alterations in endothelial dysfunction. The method is based on the quantification of the ratio of phenylalanine (Phe) to tyrosine (Tyr) contents in the endothelium. The synthesis of Tyr from Phe requires the presence of tetrahydrobiopterin (BH4) as a cofactor of phenylalanine hydroxylase (PAH). Limitation of BH4 availability in the endothelium is a hallmark endothelial nitric oxide synthase (eNOS) dysfunction that may also lead to PAH dysfunction and a fall in Tyr contents. Using Raman spectra, the ratio of marker bands of Tyr to Phe was calculated and the pathological state of the endothelium was detected. We provide evidence that Phe/Tyr ratio analysis by Raman spectroscopy discriminate endothelial dysfunction in ApoE/LDLR(-/-) mice as compared to control mice.

Simultaneous intracellular redox potential and pH measurements in live cells using SERS nanosensors.

Intracellular redox potential is a highly regulated cellular characteristic and is critically involved in maintaining cellular health and function. The dysregulation of redox potential can result in the initiation and progression of numerous diseases. Redox potential is determined by the balance of oxidants and reductants in the cell and also by pH. For this reason a technique for quantitative measurement of intracellular redox potential and pH is highly desirable. In this paper we demonstrate how surface enhanced Raman scattering (SERS) nanosensors can be used for multiplexed measurement of both pH and redox potential in live single cells.

Nuclear accumulation of anthracyclines in the endothelium studied by bimodal imaging: fluorescence and Raman microscopy.

Anthracycline antibiotics display genotoxic activity towards cancer cells but their clinical utility is limited by their cardiac and vascular toxicity. The aim of this study was to develop a Raman-based methodology to study the nuclear accumulation of anthracyclines in the endothelium. For this purpose bimodal confocal Raman and fluorescence imaging was used to monitor cellular composition changes as a result of anthracycline exposure on endothelial cells (EA.hy926), and nuclear drug accumulation, respectively. Simultaneously effects of anthracyclines on endothelium viability were investigated by caspases-3 and -7 and MTT assays. We demonstrated that nuclear accumulation of DOX and EDOX was similar; however, EDNR accumulated in endothelial nuclei at concentrations 10 times higher than DNR. In turn, epimers of DOX or DNR were both consistently less toxic on the endothelium as compared to their congeners as evidenced by MTT and caspase assays. In summary, bimodal Raman and fluorescence-based nucleus profiling proves to be a valuable tool to study structure-activity relationship of nuclear accumulation and toxicity of anthracyclines in endothelium.

Raman microimaging of murine lungs: insight into the vitamin A content.

The composition of the lung tissue of mice was investigated using Raman confocal microscopy at 532 nm excitation wavelength and was supported with various staining techniques as well as DFT calculations. This combination of experimental and theoretical techniques allows for the study of the distribution of lung lipofibroblasts (LIFs), rich in vitamin A, as well as the chemical structure of vitamin A. The comparison of the Raman spectra derived from LIFs with the experimental and theoretical spectra of standard retinoids showed the ability of LIFs to store all-trans retinol, which is partially oxidized to all-trans retinal and retinoic acid. Moreover, we were able to visualize the distribution of other lung tissue components including the surfactant and selected enzymes (lipoxygenase/glucose oxidase).

Stimulated Raman scattering microscopy in chemistry and life science - Development, innovation, perspectives.

In this review, we present a summary of the basics of the Stimulated Raman Scattering (SRS) phenomenon, methods of detecting the signal, and collection of the SRS images. We demonstrate the advantages of SRS imaging, and recent developments, but also the limitations, especially in image capture speeds and spatial resolution. We also compare the use of SRS microscopy in biological system studies with other techniques such as fluorescence microscopy, second-harmonic generation (SHG)-based microscopy, coherent anti-Stokes Raman scattering (CARS), and spontaneous Raman, and we show the compatibility of SRS-based systems with other discussed methods. The review is also focused on indicating innovations in SRS microscopy, on the background of which we present the layout and performance of our homemade setup built from commercially available elements enabling for imaging of the molecular structure of single cells over the spectral range of 800-3600 cm-1. Methods of image analysis are discussed, including machine learning methods for obtaining images of the distribution of selected molecules and for the detection of pathological lesions in tissues or malignant cells in the context of clinical diagnosis of a wide range of diseases with the use of SRS microscopy. Finally, perspectives for the development of SRS microscopy are proposed.

The role of N-acetyltransferase 2 polymorphism in the etiopathogenesis of inflammatory bowel disease.

BACKGROUND AND PURPOSE: Inflammatory bowel disease (IBD) consists of ulcerative colitis (UC) and Crohn's disease (CD), which are complex genetic disorders resulting from the interplay between several genetic and environmental risk factors. The arylamine N-acetyltransferase 2 (NAT2) enzyme detoxifies a wide spectrum of naturally occurring xenobiotics including carcinogens and drugs. Acetylation catalyzed by NAT2 is an important process in metabolic activation of arylamines to electrophilic intermediates that initiate carcinogenesis. The aim of our study was to determine whether there is any association between the susceptibility to inflammatory bowel disease among the variations of NAT2 genotypes. METHODS: This study was carried out in 80 patients with IBD. The control group consisted of 100 healthy volunteers. The most common mutations found in the Caucasian population are at the positions 481T, 803G, 590A and 857A on the NAT2 gene. This was determined using the polymerase chain reaction-restriction fragment length polymorphism method with DNA extracted from peripheral blood. RESULTS: Risk of IBD development was 3.86 for the carriers of the NAT2*5/NAT2*7 genotype and 2.53 for the carriers with NAT2*6/NAT2*7, but it was not statistically significant. A statistically significant correlation between the NAT2*7 allele prevalence and the risk for developing IBD was found (OR = 5.8; P = 0.005). CONCLUSIONS: Higher prevalence of the NAT2*7 allele in patients with IBD and the obtained OR values could suggest that this mutation has the effect of increasing IBD development. Future studies are needed to confirm our assumptions on larger group of patients.

Genetic polymorphisms of CYP2D6 oxidation in patients with inflammatory bowel disease.

Inflammatory bowel disease (IBD) consists of ulcerative colitis and Crohn's disease, both of which are associated with increased colorectal cancer risk. The relationship between genetically determined polymorphic metabolism of exogenous substances by oxidation catalyzed by CYP2D6 isoenzyme and susceptibility to cancer has aroused great interest. We determined whether there was an association between susceptibility to inflammatory bowel disease and particularly to CYP2D6 genotypes. The study was carried out in 39 patients with IBD. The control group consisted of 129 healthy volunteers. The CYP2D6 genotypes were analyzed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method with DNA extracted from peripheral blood. Among 39 patients with inflammatory bowel disease, extensive metabolizer (EM) genotype constituted 97.4%. One patient (2.6%) was poor metabolizer with CYP2D6*4/CYP2D6*4 genotype. Results obtained in the inflammatory bowel disease group did not differ significantly from those of the control group. Although the odds ratio for EM metabolizers was about 3.8-fold greater in the group of patients with inflammatory bowel disease, this association was not statistically significant. This data also showed no overall statistically significant association between alleles and incidence risk of inflammatory bowel disease [odds ratio (OR) of 1.36 for CYP2D6*1 allele, 0.83 for CYP2D6*3 allele, and 0.74 for CYP2D6*4 allele]. The present results suggest that EM genotype may be the risk factor of inflammatory bowel disease. Future studies are needed to confirm our assumptions on larger group of patients.

Fixed versus live endothelial cells: The effect of glutaraldehyde fixation manifested by characteristic bands on the Raman spectra of cells.

This work shows an impact of glutaraldehyde (GA) fixation on endothelial cells. Raman spectroscopy imaging was used as a method to monitor biochemical content of the cells due to GA fixation since this is an approach frequently used for studying cells by means of Raman imaging. To get a deeper insight into the changes and to understand them better the measurements of live and fixed cells were performed using two lasers, i.e. 488 and 532 nm. It has been demonstrated that GA fixation affects lipids, proteins, nucleic acid and carbohydrates to small extent. The application of 488 nm laser line seems to be more efficient for live cells due to the small impact of cytochrome resonance on Raman spectra, however 532 nm line is more beneficial for fixed cells due to higher quantum efficiency of the detector, thus leading to higher intensity of Raman bands. Generally, the changes due to fixation are not pronounced but cannot be ignored and the knowledge about them can help in a proper interpretation of data collected for fixed versus live cells.

Vibrational spectroscopy-based quantification of liver steatosis.

The liver plays a central role in lipid metabolism, and abnormal lipid accumulation in the liver is a key feature of Non-Alcoholic Fatty Liver Disease. In experimental studies, quantification of liver steatosis is commonly based on lipids staining or biochemical analysis. Here, we present a spectroscopic approach for quantitative analysis of the lipid content in the freeze-dried liver. The method is based on vibrational spectroscopy (Raman and infrared) measurements applied for Partial Least Squares (PLS) regression modeling. The obtained PLS models show a good correlation of the spectroscopic data with the reference histological evaluation of steatosis based on Oil Red O (ORO)-stained images of liver cross sections. Vibrational spectroscopy with PLS-based modeling described here represents a useful approach for the fast assessment of the liver steatosis in a small sample of freeze-dried liver tissue. In conclusion, our work demonstrates the easy-to-use method that can be applied in laboratory routine as a beneficial alternative to the established ORO staining.

Association of pesticide exposure, vaccination response, and interleukin-1 gene polymorphisms.

We performed a cross-sectional study involving workers from four European countries in which exposure to pesticides and immune parameters were evaluated over a short period of time. The total study population consisted of 238 workers occupationally exposed to pesticides and 198 nonoccupationally exposed workers. The study showed that pesticide exposure at levels encountered by workers under different conditions in Europe did not affect the ability of the immune system to respond to vaccination. We could, however, identify individuals within the group of pesticide exposed workers who were genetically characterized by the 2.2 IL-1alpha polymorphism and who showed a lower antibody response, pointing out the importance of the understanding of genetic variability and the interaction between genetic and environmental factors in the identification of high-risk individuals, which may eventually lead to preventive measures.

Pre-resonance enhancement of exceptional intensity in Aggregation-Induced Raman Optical Activity (AIROA) spectra of lutein derivatives.

Recently reported new phenomenon of Aggregation-Induced Raman Optical Activity is demonstrated here for the first time in the pre-resonance conditions for lutein diacetate and 3'-epi-lutein supramolecular self-assembles. We demonstrate that minor alterations in the lutein structure (e.g. acetylation of hydroxyl groups or different configuration at one of the chiral center) can lead to definitely different spectral profiles and optical properties due to formation of aggregates of different structure and type. Lutein forms only H-aggregates, lutein diacetate only J-aggregates, while 3'-epi-lutein can occur in both forms simultaneously. Variety of aggregates' structures is so large that not only the type of aggregation is different, but also their chirality. It is remarkable that even in the pre-resonance conditions, aggregation of lutein derivatives can lead to the intense ROA signal, and moreover, 3'-epi-lutein demonstrated the highest resonance ROA CID ratio that has ever been reported.

Unsaturated lipid bodies as a hallmark of inflammation studied by Raman 2D and 3D microscopy.

Endothelial HMEC-1 cells incubated with pro-inflammatory cytokine TNF-α for 6 and 24 hours were studied as a model of inflammation using Raman imaging. Striking changes in distribution, composition and concentration of cellular lipids were observed after exposure to TNF-α compared to the control. In particular, 3D Raman imaging revealed a significant increase in the amount of lipid entities formed under inflammation. Lipid bodies were randomly distributed in the cytoplasm and two types of droplets were assembled: more saturated one, in spectral characteristics resembling phosphatidylcholine and saturated cholesteryl esters, observed also in the control, and highly unsaturated one, containing also cholesterols, being a hallmark of inflamed cells. The statistical analysis showed that the number of lipid bodies was significantly dependent on the exposure time to TNF-α. Overall, observed formation of unsaturated lipid droplets can be directly correlated with the increase in production of prostacyclins - endogenous inflammation mediators.

Constructive influence of the induced electron pairing on the Kondo state.

Superconducting order and magnetic impurities are usually detrimental to each other. We show, however, that in nanoscopic objects the induced electron pairing can have constructive influence on the Kondo effect originating from the effective screening interactions. Such situation is possible at low temperatures in the quantum dots placed between the conducting and superconducting reservoirs, where the proximity induced electron pairing cooperates with the correlations amplifying the spin-exchange potential. The emerging Abrikosov-Suhl resonance, which is observable in the Andreev conductance, can be significantly enhanced by increasing the coupling to superconducting lead. We explain this intriguing tendency within the Anderson impurity model using: the generalized Schrieffer-Wolff canonical transformation, the second order perturbative treatment of the Coulomb repulsion, and the nonperturbative numerical renormalization group calculations. We also provide hints for experimental observability of this phenomenon.

Effects of Low Carbohydrate High Protein (LCHP) diet on atherosclerotic plaque phenotype in ApoE/LDLR-/- mice: FT-IR and Raman imaging.

Low Carbohydrate High Protein (LCHP) diet displays pro-atherogenic effects, however, the exact mechanisms involved are still unclear. Here, with the use of vibrational imaging, such as Fourier transform infrared (FT-IR) and Raman (RS) spectroscopies, we characterize biochemical content of plaques in Brachiocephalic Arteries (BCA) from ApoE/LDLR(-/-) mice fed LCHP diet as compared to control, recomended by American Institute of Nutrition, AIN diet. FT-IR images were taken from 6-10 sections of BCA from each mice and were complemented with RS measurements with higher spatial resolution of chosen areas of plaque sections. In aortic plaques from LCHP fed ApoE/LDLR(-/-) mice, the content of cholesterol and cholesterol esters was increased, while that of proteins was decreased as evidenced by global FT-IR analysis. High resolution imaging by RS identified necrotic core/foam cells, lipids (including cholesterol crystals), calcium mineralization and fibrous cap. The decreased relative thickness of the outer fibrous cap and the presence of buried caps were prominent features of the plaques in ApoE/LDLR(-/-) mice fed LCHP diet. In conclusion, FT-IR and Raman-based imaging provided a complementary insight into the biochemical composition of the plaque suggesting that LCHP diet increased plaque cholesterol and cholesterol esters contents of atherosclerotic plaque, supporting the cholesterol-driven pathogenesis of LCHP-induced atherogenesis.