Label-free tracking of cytochrome C oxidation state in live cells by resonance Raman imaging.

Free interconversion of cytochrome C (CytC) between native ferrous (Cyt-FeII) and oxidized ferric (CytC-FeIII) states is necessary to maintain the respiratory function of mitochondria. Disturbances in CytC-FeIII to total CytC ratio may indicate mitochondrial dysfunction and apoptosis. Thus, tracking CytC oxidation state delivers important information about cellular physiology. In this work, we propose a novel methodology based on resonance Raman (rR) imaging optimized uniquely to track and qualitatively analyze the transition of Cyt-FeII to CytC-FeIII within live cells without affecting their morphology. None of the commonly used excitation lines allows such clear-cut differentiation, contrary to the 405 nm applied in this work. The presented methodology provides a novel pathway in the label-free detection of ferrous and ferric heme proteins.

Surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) in label-free characterization of erythrocyte membranes and extracellular vesicles at the nano-scale and molecular level.

The manuscript presents the potential of surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) for label-free characterization of extracellular microvesicles (EVs) and their isolated membranes derived from red blood cells (RBCs) at the nanoscale and at the single-molecule level, providing detection of a few individual amino acids, protein and lipid membrane compartments. The study shows future directions for research, such as investigating the use of the mentioned techniques for the detection and diagnosis of diseases. We demonstrate that SERS and TERS are powerful techniques for identifying the biochemical composition of EVs and their membranes, allowing the detection of small molecules, lipids, and proteins. Furthermore, extracellular vesicles released from red blood cells (REVs) can be broadly classified into exosomes, microvesicles, and apoptotic bodies, based on their size and biogenesis pathways. Our study specifically focuses on microvesicles that range from 100 to 1000 nanometres in diameter, as presented in AFM images. Using SERS and TERS spectra obtained for REVs and their membranes, we were able to characterize the chemical and structural properties of microvesicle membranes with high sensitivity and specificity. This information may help better distinguish and categorize different types of EVs, leading to a better understanding of their functions and potential biomedical applications.

Rapid, label-free classification of glioblastoma differentiation status combining confocal Raman spectroscopy and machine learning.

Label-free identification of tumor cells using spectroscopic assays has emerged as a technological innovation with a proven ability for rapid implementation in clinical care. Machine learning facilitates the optimization of processing and interpretation of extensive data, such as various spectroscopy data obtained from surgical samples. The here-described preclinical work investigates the potential of machine learning algorithms combining confocal Raman spectroscopy to distinguish non-differentiated glioblastoma cells and their respective isogenic differentiated phenotype by means of confocal ultra-rapid measurements. For this purpose, we measured and correlated modalities of 1146 intracellular single-point measurements and sustainingly clustered cell components to predict tumor stem cell existence. By further narrowing a few selected peaks, we found indicative evidence that using our computational imaging technology is a powerful approach to detect tumor stem cells in vitro with an accuracy of 91.7% in distinct cell compartments, mainly because of greater lipid content and putative different protein structures. We also demonstrate that the presented technology can overcome intra- and intertumoral cellular heterogeneity of our disease models, verifying the elevated physiological relevance of our applied disease modeling technology despite intracellular noise limitations for future translational evaluation.

Zero- to low-field relaxometry of chemical and biological fluids.

Nuclear magnetic resonance (NMR) relaxometry is an analytical method that provides information about molecular environments, even for NMR "silent" molecules (spin-0), by analyzing the properties of NMR signals versus the magnitude of the longitudinal field. Conventionally, this technique is performed at fields much higher than Earth's magnetic field, but our work focuses on NMR relaxometry at zero and ultra-low magnetic fields (ZULFs). Operating under such conditions allows us to investigate slow (bio)chemical processes occurring on a timescale from milliseconds to seconds, which coincide with spin evolution. ZULFs also minimize T2 line broadening in heterogeneous samples resulting from magnetic susceptibility. Here, we use ZULF NMR relaxometry to analyze (bio)chemical compounds containing 1H-13C, 1H-15N, and 1H-31P spin pairs. We also detected high-quality ULF NMR spectra of human whole-blood at 0.8 µT, despite a shortening of spin relaxation by blood proteomes (e.g., hemoglobin). Information on proton relaxation times of blood, a potential early biomarker of inflammation, can be acquired in under a minute using inexpensive, portable/small-size NMR spectrometers based on atomic magnetometers.

Sulfhemoglobin under the spotlight - Detection and characterization of SHb and HbFeIII-SH.

Sulfhemoglobinemia is an incurable disease caused by an overdose of sulfur-containing drugs with oxidizing properties. Its diagnosis remains hindered due to the similarity of symptoms to other pathological state - methemoglobinemia, as well as contradictory information on the structure and characteristics of sulfhemoglobin. Herein, we present sulfhemoglobinemia model on living functional human erythrocytes, designed to recreate processes which could take place in a patient body in order to complement missing information and highlight distinctiveness of two hemoglobin (Hb) adducts formed after interaction with sulfur donors. Employed techniques, UV-Vis absorption, Raman, Fourier transformed infrared (FT-IR) and electronic circular dichroism (ECD) spectroscopies, allowed to distinguish and characterize Hb adduct with sulfur atom bounded directly to the iron ion (HbFeIII-SH), and irreversibly connected to the porphyrin ring (SHb - sulfhemoglobin). Presented herein results provided also new evidence on formation of both these hemoglobin adducts inside functional erythrocytes under oxidative conditions and during sulfur-containing drug presence, what can be further translated into future physiological studies. Moreover, we found that sulfur attachment to the porphyrin ring altered Hb structure and lead to changes in protein packing inside RBCs, eventually. Interestingly, measurement of blood drop smear by Raman spectroscopy occurred the most accurate method to differentiate HbFeIII-SH and SHb, indicating potential of this technique in sulfhemoglobinemia diagnosis.

Secondary structure alterations of RBC assessed by FTIR-ATR in correlation to 2,3-DPG levels in ApoE/LDLR-/- Mice.

In the present study, we characterized the secondary structure alterations of intact red blood cells (RBCs) cytosol with special attention to the sex-related alterations in 8- and 24-week-old female and male ApoE/LDLR-/- mice, compared to age-matched female and male C57BL/6J control animals. Results were obtained with previously established methodology based on Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Additionally, we evaluated 2,3-DPG levels in the RBCs and showed its potential link to the hemoglobin (Hb) secondary structure alterations. Considering Hb structure alterations probed by FTIR-ATR, the ratio of turns to α-helices in 8-week-old ApoE/LDLR-/- mice suggested more pronounced secondary structure alterations within the RBCs than in the age-matched control. Sex-related differences were observed solely in 24-week-old male ApoE/LDLR-/- mice, which showed statistically significant increase in the secondary structure alterations compared to 24-week-old female ApoE/LDLR-/- mice. Similar to the secondary structure alterations, no sex-related differences were observed in the levels of 2,3-DPG in RBCs, except for 24-week-old male ApoE/LDLR-/- mice, which showed significantly higher levels compared to the age-matched female ApoE/LDLR-/- mice. Considering the age-related alterations, we observed significant increases in the intracellular 2,3-DPG of RBCs with animals' age in all studied groups, except for female ApoE/LDLR-/- mice, where a significant difference was not reported. This suggests the clear correlation between secondary structure of Hb alterations and 2,3-DPG levels for male and female murine RBC and proves a higher resistance of older female RBCs to the secondary structure changes with progression of atherosclerosis. Moreover, it may be concluded that higher 2,3-DPG levels in RBCs occurred in response to the secondary structure alterations of Hb in ApoE/LDLR-/- mice.

Label-free testing strategy to evaluate packed red blood cell quality before transfusion to leukemia patients.

Patients worldwide require therapeutic transfusions of packed red blood cells (pRBCs), which is applied to the high-risk patients who need periodic transfusions due to leukemia, lymphoma, myeloma and other blood diseases or disorders. Contrary to the general hospital population where the transfusions are carried out mainly for healthy trauma patients, in case of high-risk patients the proper quality of pRBCs is crucial. This leads to an increased demand for efficient technology providing information on the pRBCs alterations deteriorating their quality. Here we present the design of an innovative, label-free, noninvasive, rapid Raman spectroscopy-based method for pRBCs quality evaluation, starting with the description of sample measurement and data analysis, through correlation of spectroscopic results with reference techniques' outcomes, and finishing with methodology verification and its application in clinical conditions. We have shown that Raman spectra collected from the pRBCs supernatant mixture with a proper chemometric analysis conducted for a minimum one ratio of integral intensities of the chosen Raman marker bands within the spectrum allow evaluation of the pRBC quality in a rapid, noninvasive, and free-label manner, without unsealing the pRBCs bag. Subsequently, spectroscopic data were compared with predefined reference values, either from pRBCs expiration or those defining the pRBCs quality, allowing to assess their utility for transfusion to patients with acute myeloid leukemia (AML) and lymphoblastic leukemia (ALL).

Sex-Specific Differences of Adenosine Triphosphate Levels in Red Blood Cells Isolated From ApoE/LDLR Double-Deficient Mice.

In this study for the first time, we investigated the correlation between sex-specific differences in adenosine triphosphate (ATP) levels in red blood cells (RBCs) and their mechanical, biochemical, and morphological alterations during the progression of atherosclerosis in ApoE/LDLR double-deficient (ApoE/LDLR-/-) mice. Our results indicate that both sex and age affect alterations in RBCs of both ApoE/LDLR-/- and C57BL/6J mice. When compared with male RBCs, female RBCs were characterized by lower basal ATP and mean corpuscular hemoglobin concentration (MCHC), higher hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), deformability, and phosphatidylserine (PS) exposure levels, regardless of age in both, ApoE/LDLR-/- and C57BL/6J mice. ApoE/LDLR-/- mice compared with age-matched controls showed lower basal ATP levels regardless of age and sex. Intracellular ATP level of RBCs was decreased solely in senescent female C57BL/6J mice, while it was elevated in males. Basal extracellular ATP levels were 400 times lower than corresponding intracellular level. In conclusion, basal ATP levels, RBC morphology, deformability, PS exposure levels alterations are sex-dependent in mice. Changes in basal ATP levels were correlated with PS exposure and trends of changes in MCV. Trends of changes of the most RBC parameters were similar in both sexes of ApoE/LDLR-/- mice compared with age-matched controls; however, their kinetics and levels vary greatly between different stages of disease progression.

The effect of nitric oxide synthase and arginine on the color of cooked meat.

In previous studies, it has been suggested that the NO-synthase enzyme may be responsible for color formation in fermented sausages. Thus, this is the first study in which the aim was to analyze the effects of direct NO-synthase and arginine application to meat on its color after heating. Myoglobin forms as well as the presence of NO-myoglobin were investigated. The color of the meat and myoglobin forms present in the samples were mainly affected by pH differences, caused by a HEPES buffer or arginine. None of the variants demonstrated a bright pink color as in the case of the heated nitrite-cured sample. Based on analysis of the absorption spectra, it can be concluded that there is some evidence of nitroso-complex formation. Therefore, it is probable that optimizing the pH/time/temperature conditions for NO-synthase activity would allow to obtain a desirable color effect. NO-synthase could be used as an alternative curing ingredient.

Identification of inflammatory markers in eosinophilic cells of the immune system: fluorescence, Raman and CARS imaging can recognize markers but differently.

Eosinophils (Eos) play an important role in the immune system's response releasing several inflammatory factors and contributing to allergic rhinitis, asthma, or atopic dermatitis. Since Eos have a relatively short lifetime after isolation from blood, usually eosinophilic cell line (EoL-1) is used to study mechanisms of their activation and to test therapies. In particular, EoL-1 cells are examined in terms of signalling pathways of the inflammatory response manifested by the presence of lipid bodies (LBs). Here we examined the differences in response to inflammation modelled by various factors, between isolated human eosinophils and EoL-1 cells, as manifested in the number and chemical composition of LBs. The analysis was performed using fluorescence, Raman, and coherent anti-Stokes Raman scattering (CARS) microscopy, which recognised the inflammatory process in the cells, but it is manifested slightly differently depending on the method used. We showed that unstimulated EoL-1 cells, compared to isolated eosinophils, contained more LBs, displayed different nucleus morphology and did not have eosinophilic peroxidase (EPO). In EoL-1 cells stimulated with various proinflammatory agents, including butyric acid (BA), liposaccharide (LPS), or cytokines (IL-1ß, TNF-α), an increased production of LBs with a various degree of lipid unsaturation was observed in spontaneous Raman spectra. Furthermore, stimulation of EoL-1 cells resulted in alterations of the LBs morphology. In conclusion, a level of lipid unsaturation and eosinophilic peroxidase as well as LBs distribution among cell population mainly accounted for the biochemistry of eosinophils upon inflammation.

Tauroursodeoxycholic Acid (TUDCA)-Lipid Interactions and Antioxidant Properties of TUDCA Studied in Model of Photoreceptor Membranes.

Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid containing taurine conjugated with the ursodeoxycholic acid (UDCA), has been known and used from ancient times as a therapeutic compound in traditional Chinese medicine. TUDCA has recently been gaining significant interest as a neuroprotective agent, also exploited in the visual disorders. Among several mechanisms of TUDCA's protective action, its antioxidant activity and stabilizing effect on mitochondrial and plasma membranes are considered. In this work we investigated antioxidant activity of TUDCA and its impact on structural properties of model membranes of different composition using electron paramagnetic resonance spectroscopy and the spin labeling technique. Localization of TUDCA molecules in a pure POPC bilayer has been studied using a molecular dynamics simulation (MD). The obtained results indicate that TUDCA is not an efficient singlet oxygen (1O2 (1Δg)) quencher, and the determined rate constant of its interaction with 1O2 (1Δg) is only 1.9 × 105 M-1s-1. However, in lipid oxidation process induced by a Fenton reaction, TUDCA reveals substantial antioxidant activity significantly decreasing the rate of oxygen consumption in the system studied. In addition, TUDCA induces slight, but noticeable changes in the polarity and fluidity of the investigated model membranes. The results of performed MD simulation correspond very well with the experimental results.

Spectroscopic Signature of Red Blood Cells in a D-Galactose-Induced Accelerated Aging Model.

This work presents a semi-quantitative spectroscopic approach, including FTIR-ATR and Raman spectroscopies, for the biochemical analysis of red blood cells (RBCs) supported by the biochemical, morphological and rheological reference techniques. This multi-modal approach provided the description of the RBC alterations at the molecular level in a model of accelerated aging induced by administration of D-galactose (D-gal), in comparison to natural aging. Such an approach allowed to conclude that most age-related biochemical RBC membrane changes (a decrease in lipid unsaturation and the level of phospholipids, or an increase in acyl chain shortening) as well as alterations in the morphological parameters and RBC deformability are well reflected in the D-gal model of accelerated aging. Similarly, as in natural aging, a decrease in LDL level in blood plasma and no changes in the fraction of glucose, creatinine, total cholesterol, HDL, iron, or triglycerides were observed during the course of accelerated aging. Contrary to natural aging, the D-gal model led to an increase in cholesterol esters and the fraction of total esterified lipids in RBC membranes, and evoked significant changes in the secondary structure of the membrane proteins. Moreover, a significant decrease in the phosphorous level of blood plasma was specific for the D-gal model. On the other hand, natural aging induced stronger changes in the secondary structures of the proteins of the RBCs' interior. This work proves that research on the aging mechanism, especially in circulation-related diseases, should employ the D-gal model with caution. Nonetheless, the D-gal model enables to imitate age-related rheological alterations in RBCs, although they are partially derived from different changes observed in the RBC membrane at the molecular level.

Probing Heme Active Sites of Hemoglobin in Functional Red Blood Cells Using Resonance Raman Spectroscopy.

The UV-vis absorption, Raman imaging, and resonance Raman (rR) spectroscopy methods were employed to study cyanohemoglobin (HbCN) adducts inside living functional red blood cells (RBCs). The cyanide ligands are especially optically sensitive probes of the active site environment of heme proteins. The rR studies of HbCN and its isotopic analogues (13CN-, C15N-, and 13C15N-), as well as a careful deconvolution of spectral data, revealed that the ν(Fe-CN) stretching, δ(Fe-CN) bending, and ν(C≡N) stretching modes occur at 454, 382, and 2123 cm-1, respectively. Interestingly, while the ν(Fe-CN) modes exhibit the same frequencies in both the isolated and RBC-enclosed hemoglobin molecules, small frequency differences are observed in the δ(Fe-CN) bending modes and the values of their isotopic shifts. These studies show that even though the overall tilted conformation of the Fe-C≡N fragment in the isolated HbCN is preserved in the HbCN enclosed within living cells, there is a small difference in the degree of distortion of the Fe-C≡N fragment. The slight changes in the ligand geometry can be reasonably attributed to the high ordering and tight packing of Hb molecules inside RBCs.

Eosinophils and Neutrophils-Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy.

Leukocytes are a part of the immune system that plays an important role in the host's defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells' types. To prove this hypothesis, UV-Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.

Age-related and atherosclerosis-related erythropathy in ApoE/LDLR-/- mice.

In this work we applied a multimodal approach to define the age- and atherosclerosis-related biochemical and functional alterations in red blood cells (RBCs) in ApoE/LDLR-/- mice. Our results revealed that age-related changes in RBCs, such as decreases in RBC deformability and mean height, were more pronounced in ApoE/LDLR-/- mice than in age-matched control mice (C57BL/6J). The decreases in phospholipid content and level of lipid unsaturation were accompanied by an increase in cholesterol esters and esterified lipids in RBC membranes in aged C57BL/6J mice. The age-related decrease in the phospholipid content was more pronounced in ApoE/LDLR-/- mice. In contrast, the increase in the total lipid content in RBC membranes occurred only in ApoE/LDLR-/- mice with advanced atherosclerosis. The age-related alterations also included a decrease in the ratio of turns to α-helices in the secondary structure of hemoglobin (Hb) inside intact RBCs. On the other hand, an increase in the ratio of unordered conformations to α-helices of Hb was observed only in ApoE/LDLR-/- mice and occurred already at the age of 5-weeks. This was related to hypercholesterolemia and resulted in an increased oxygen-carrying capacity. In conclusion, progressive mechanical and functional alterations of RBCs in aged ApoE/LDLR-/- mice were more pronounced than in age-matched C57BL/6J mice. Although, several biochemical changes in RBCs in aged ApoE/LDLR-/- mice recapitulated age-dependent changes observed in control mice, some biochemical features of RBC membranes attributed to hypercholesterolemia were distinct and could contribute to the accelerated deterioration of RBC function in ApoE/LDLR-/- mice.

Irreversible alterations in the hemoglobin structure affect oxygen binding in human packed red blood cells.

The ability of hemoglobin (Hb) to transport respiratory gases is directly linked to its quaternary structure properties and reversible changes between T (tense) and R (relax) state. In this study we demonstrated that packed red blood cells (pRBCs) storage resulted in a gradual increase in the irreversible changes in the secondary and quaternary structures of Hb, with subsequent impairment of the T↔R transition. Such alteration was associated with the presence of irreversibly settled in the relaxed form, quaternary structure of Hb, which we termed R'. On the secondary structure level, disordered protein organization involved formation of ß-sheets and a decrease in α-helices related to the aggregation process stabilized by strong intermolecular hydrogen bonding. Compensatory changes in RBCs metabolism launched to preserve reductive microenvironment were disclosed as an activation of nicotinamide adenine dinucleotide phosphate (NADPH) production and increased reduced to oxidized glutathione (GSH/GSSG) ratio. For the first time we showed the relationship between secondary structure changes and the occurrence of newly discovered R', which through an artificial increase in oxyhemoglobin level altered Hb ability to bind and release oxygen.

Probing the structure-function relationship of hemoglobin in living human red blood cells.

Hemoglobin (Hb) is a key component of respiratory system and as such plays important role in human physiology. The studies of Hb's structure and functions are usually performed on cell-free protein; however, it has been shown that there are functionally relevant differences between isolated Hb and Hb present inside red blood cells (RBCs). It is clear that new experimental approaches are needed to understand the origin of these differences and to gain insight into the structure-function relationship of Hb within intact living cells. In this work we present a novel application of Resonance Raman spectroscopy to study heme active site of different forms of human Hb within living RBCs using laser excitation lines in resonance with their Soret absorption bands. These studies revealed that there are no significant changes in the disposition of the Fe-O-O fragment or the Fe-NHis linkage for Hb molecules enclosed in RBCs and these in free isolated states. However, some changes in the orientation of the heme vinyl groups were observed which might account for the differences in the protein activity and ligand affinity. This work highlights importance of protein-based studies and presents a new opportunity to translate these results to physiological cell systems.

Temporal sequence of the human RBCs' vesiculation observed in nano-scale with application of AFM and complementary techniques.

Based on the multimodal characterization of human red blood cells (RBCs), the link between the storage-related sequence of the nanoscale changes in RBC membranes in the relation to their biochemical profile as well as mechanical and functional properties was presented. On the background of the accumulation of RBCs waste products, programmed cell death and impaired rheological properties, progressive alterations in the RBC membranes including changes in their height and diameter as well as the in situ characterization of RBC-derived microparticles (RMPs) on the RBCs surface were presented. The advantage of atomic force microscopy (AFM) in RMPs visualization, even at the very early stage of vesiculation, was shown based on the results revealed by other reference techniques. The nanoscale characterization of RMPs was correlated with a decrease in cholesterol and triglycerides levels in the RBC membranes, proving the link between the lipids leakage from RBCs and the process of vesiculation.

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a "standardization" process of SERS protocols, not proposed by a single laboratory but by a larger community.