Raman spectroscopy of yeast cells cultured on a deuterated substrate.

Raman spectroscopy of cells cultured in a deuterated substrate is a promising approach to the characterization of mass transfer and enzymatic reactions in living cells. Here, we studied the potential of this approach using the example of yeast cells cultured under aerobic and anaerobic conditions. In our experiments, unadapted to D2O Saccharomyces cerevisiae were cultured in a medium with different concentrations of deuterium oxide and deuterated glucose. It has been shown that the addition of even 10% heavy water leads to a general decrease in the amount of lipids in cells. In the Raman spectra of cells cultured at high concentrations of D2O, additional peaks are found, which are associated with the deuteration of entire chemical groups. We observed a similar effect in the ethanol synthesized by yeast fermentation, the deuteration of which also depends on the concentration of D2O. The results on the characterization of cell deuteration turned out to be in qualitative agreement with the known estimate that aerobic metabolism is 15 times more active than ethanol fermentation. The results of our work determine new limitations and prospects for further application and development of the Raman method of spectroscopy of deuterium tags.

Influence of Single-Wall Carbon Nanotube Suspension on the Mechanical Properties of Polymeric Films and Electrospun Scaffolds.

Carbon nanotubes (CNTs) are used in applications ranging from electrical engineering to medical device manufacturing. It is well known that the addition of nanotubes can influence the mechanical properties of various industrial materials, including plastics. Electrospinning is a popular method for fabricating nanomaterials, widely suggested for polymer scaffold manufacturing. In this study, we aimed to describe the influence of single-walled carbon nanotube (SWCNT) suspensions on polymeric poured films and electrospun scaffolds and to investigate their structural and mechanical properties obtained from various compositions. To obtain films and electrospun scaffolds of 8 mm diameter, we used poly-ε-caprolactone (PCL) and poly(cyclohexene carbonate) (PCHC) solutions containing several mass fractions of SWCNT. The samples were characterized using tensile tests, atomic force and scanning electronic microscopy (AFM and SEM). All the studied SWCNT concentrations were shown to decrease the extensibility and strength of electrospun scaffolds, so SWCNT use was considered unsuitable for this technique. The 0.01% mass fraction of SWCNT in PCL films increased the polymer strength, while fractions of 0.03% and more significantly decreased the polymer strength and extensibility compared to the undoped polymer. The PHCH polymeric films showed a similar behavior with an extremum at 0.02% concentration for strength at break.

Integral Algorithms to Evaluate TiO2 and N-TiO2 Thin Films' Cytocompatibility.

Titanium oxide (TiO2) and oxynitride (N-TiO2) coatings can increase nitinol stents' cytocompatibility with endothelial cells. Methods of TiO2 and N-TiO2 sputtering and cytocompatibility assessments vary significantly among different research groups, making it difficult to compare results. The aim of this work was to develop an integral cytocompatibility index (ICI) and a decision tree algorithm (DTA) using the "EA.hy926 cell/TiO2 or N-TiO2 coating" model and to determine the optimal cytocompatible coating. Magnetron sputtering was performed in a reaction gas medium with various N2:O2 ratios and bias voltages. The samples' morphology was studied by scanning electron microscopy (SEM) and Raman spectroscopy. The cytocompatibility of the coatings was evaluated in terms of their cytotoxicity, adhesion, viability, and NO production. The ICI and DTA were developed to assess the cytocompatibility of the samples. Both algorithms demonstrated the best cytocompatibility for the sample sputtered at Ubias = 0 V and a gas ratio of N2:O2 = 2:1, in which the rutile phase dominated. The DTA provided more detailed information about the cytocompatibility, which depended on the sputtering mode, surface morphology, and crystalline phase. The proposed mathematical models relate the cytocompatibility and the studied physical characteristics.

Coexistence of lipid phases in multilayer phospholipid films probed by Raman mapping.

Biomimetic phospholipid mixtures are actively used as models of biological membranes and materials for drug delivery in biomedical tasks. One of the essential properties of membranes formed from complex phospholipid mixtures is the equilibrium coexistence of domains of different phases. Studying the conformational state and chemical content of different phases is of great interest in membrane biophysics. We propose an approach for studying phase coexistence in stacked phospholipid bilayers using Raman mapping. For this purpose, phospholipid multilayer films were formed in which the domains of the same phase were self-aligned in stacks. Raman spectra with a high spectral resolution and signal-to-noise ratio obtained on these samples made it possible to estimate the chemical composition and conformational state of lipids in domains of different phases. For the ternary mixture 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC-d62)/cholesterol (Chol) used in our demonstration, the phase diagram was studied and the effect of hydration on lipid phase separation was revealed. For the hydrated films, the obtained phase diagram is in qualitative agreement with the previous data obtained using 2H NMR. In dry films, phase separation is observed for all investigated compositions, with a tendency to form a phase with a high fraction of DPPC-d62. The use of multilayer phospholipid films makes it possible to release the potential of Raman microspectroscopy to study the phase diagrams of phospholipid mixtures under various experimental conditions.

Doping of Carbon Nanotubes with Encapsulated Phosphorus Chains.

Single-walled carbon nanotubes (SWCNTs) are a perfect host for the formation of one-dimensional phosphorus structures and to obtain hybrid materials with a large P-C ratio. This work presents a procedure for high-yield phosphorus filling of commercial Tuball SWCNTs and efficient removal of phosphorus deposits from the external nanotube surface. We probed white and red phosphorus as precursors, varied the synthesis temperature and the ampoule shape, and tested three solvents for sample purification. High-resolution transmission electron microscopy and Raman spectroscopy indicated crystallization of interior phosphorus in a form resembling fibrous red phosphorus. An aqueous sodium hydroxide solution allowed removing the majority of external phosphorus particles. Thermogravimetric analysis of the product determined ∼23 wt % (∼10 atom %) of phosphorus, and the X-ray photoelectron spectroscopy (XPS) data showed that ca. 80% of it is in the form of elemental phosphorus. Externally purified SWCNTs filled with phosphorus were used to study the interaction between the components. Raman spectroscopy and core-level XPS revealed p-type SWCNT doping. Valence-band XPS data and density functional theory calculations confirmed the transfer of the SWCNT electron density to the encapsulated phosphorus.

Brillouin Spectroscopy of Binary Phospholipid-Cholesterol Bilayers.

Multicomponent lipid bilayers are used as models for searching the origin of spatial heterogeneities in biomembranes called lipid rafts, implying the coexistence of domains of different phases and compositions within the lipid bilayer. The spatial organization of multicomponent lipid bilayers on a scale of a hundred nanometers remains unknown. Brillouin spectroscopy providing information about the acoustic phonons with the wavelength of several hundred nanometers has an unexplored potential for this problem. Here, we applied Brillouin spectroscopy for three binary bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol. The Brillouin experiment for the oriented planar multibilayers was realized for two scattering geometries involving phonons for the lateral and normal directions of the propagation. The DPPC-DOPC mixtures known for the coexistence of the solid-ordered and liquid-disordered phases had bimodal Brillouin peaks, revealing the phase domains with sizes more than a hundred nanometers. Analysis of the Brillouin data for the binary mixtures concluded that the lateral phonons are preferable for testing the lateral homogeneity of the bilayers, while the phonons spreading across the bilayers are sensitive to the layered packing at the mesoscopic scale.

A monoclinic semiorganic molecular crystal GUHP for terahertz photonics and optoelectronics.

In this paper we describe the properties of the crystal of guanylurea hydrogen phosphate (NH[Formula: see text])[Formula: see text]CNHCO(NH[Formula: see text])H[Formula: see text]PO[Formula: see text] (GUHP) and propose its application in terahertz photonics and optoelectronics. GUHP crystal has a wide window of transparency and a high optical threshold in the visible and NIR spectral regions and narrow absorption bands in the terahertz frequency range. The spectral characteristics of absorption and refraction in the THz range were found to be strongly dependent on crystal temperature and orientation. Computer simulations made it possible to link the nature of the resonant response of the medium at THz frequencies with the molecular structure of the crystal, in particular, with intermolecular hydrogen bonds and the layered structure of the lattice. The possibility of application of the crystal under study for the conversion of femtosecond laser radiation from visible an NIR to terahertz range was demonstrated. It was shown that dispersion properties of the crystal allow the generation of narrow band terahertz radiation, whose spectral properties are determined by conditions close to phase matching. The properties of the generated terahertz radiation under various temperatures suggest the possibility of phonon mechanism of enhancement for nonlinear susceptibility of the second order.

Lipid phase transitions in cat oocytes supplemented with deuterated fatty acids.

Cryopreservation of oocytes has already been used to preserve genetic resources, but this technology faces limitations when applied to the species whose oocytes contain large amounts of cytoplasmic lipid droplets. Although cryoinjuries in such oocytes are usually associated with the lipid phase transition in lipid droplets, this phenomenon is still poorly understood. We applied Raman spectroscopy of deuterium-labeled lipids to investigate the freezing of lipid droplets inside cat oocytes. Lipid phase separation was detected in oocytes cryopreserved by slow-freezing protocol. For oocytes supplemented with stearic acid, we found that saturated lipids form the ordered phase being distributed at the periphery of lipid droplets. When an oocyte is warmed to physiological temperatures after cooling, a fraction of saturated lipids may remain in the ordered conformational state. The fractions of monounsaturated and polyunsaturated lipids redistribute to the core of lipid droplets. Monounsaturated lipids undergo the transition to the ordered conformational state below -10°C. Using deuterated fatty acids with a different number of double bonds, we reveal how different lipid fractions are involved in the lipid phase transition of a cytoplasmic lipid droplet and how they can affect cell survival. Raman spectroscopy of deuterated lipids has proven to be a promising tool for studying the lipid phase transitions and lipid redistributions inside single organelles within living cells.

On the Mutual Relationships between Molecular Probe Mobility and Free Volume and Polymer Dynamics in Organic Glass Formers: cis-1,4-poly(isoprene).

We report on the reorientation dynamics of small spin probe 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) in cis-1,4-poly(isoprene) (cis-1,4-PIP10k) from electron spin resonance (ESR) and the free volume of cis-1,4-PIP10k from positron annihilation lifetime spectroscopy (PALS) in relation to the high-frequency relaxations of cis-1,4-PIP10k using light scattering (LS) as well as to the slow and fast processes from broadband dielectric spectroscopy (BDS) and neutron scattering (NS). The hyperfine coupling constant, 2Azz '(T), and the correlation times, τ c(T), of cis-1,4-PIP10k/TEMPO system as a function of temperature exhibit several regions of the distinct spin probe TEMPO dynamics over a wide temperature range from 100 K up to 350 K. The characteristic ESR temperatures of changes in the spin probe dynamics in cis-1,4-PIP10k/TEMPO system are closely related to the characteristic PALS ones reflecting changes in the free volume expansion from PALS measurement. Finally, the time scales of the slow and fast dynamics of TEMPO in cis-1,4-PIP10k are compared with all of the six known slow and fast relaxation modes from BDS, LS and NS techniques with the aim to discuss the controlling factors of the spin probe reorientation mobility in polymer, oligomer and small molecular organic glass-formers.

Raman Spectroscopic Study of Phase Coexistence in Binary Phospholipid Bilayers.

Binary phospholipid bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) were studied by Raman spectroscopy and differential scanning calorimetry (DSC). We examined features in Raman scattering spectra that are sensitive to the lipid phase and, therefore, could indicate the phase coexistence. It was found that the low-frequency half-width of half-maximum (LHWHM) of the 2850 cm-1 Raman line, corresponding to the symmetric CH2 stretching vibrations, unequivocally reveals the coexisting phospholipids in ordered and disordered conformational states, which correspond to ordered and disordered phases coexistence, in the DPPC mole concentration range from 0.4 to 0.9. The phase coexistence in this concentration range was supported by the particular concentration behavior of the ratio between the intensities of the 2880 cm-1 antisymmetric CH2 vibration line and the 2850 cm-1 symmetric one. It was also shown that the spectral shape of the 1300 cm-1 Raman line, corresponding to the CH2 twisting vibrations, is a good indicator for the phase state and phase coexistence in the phospholipid bilayers. Comparison with the DSC curves confirmed that in the DPPC mole concentration range from 0.4 to 0.9, the two phase transition peaks are observed in DSC curve, those positions are independent of the DPPC concentration. The outcome of the study is the robust label-free contactless approach for the detection of the lipid phase separation, which can be realized with the micrometer resolution.

Nanocage formation and structural anomalies in imidazolium ionic liquid glasses governed by alkyl chains of cations.

Intriguing nanostructuring anomalies have been recently observed in imidazolium ionic liquids (ILs) near their glass transition points, where local density around a nanocaged solute progressively grows up with temperature. Herewith, we for the first time demonstrate experimentally and theoretically, that these anomalies are governed by alkyl chains of cations and crucially depend on their length. Electron Paramagnetic Resonance (EPR) spectroscopy on a series of ILs [Cnmim]BF4 (n = 0-12) shows that only the chains with n = 3-10 favor anomaly. Moreover, remarkable even vs. odd n peculiarities were systematically observed. Finally, similar anomaly was for the first time observed for a non-IL glass of dibutyl phthalate, which structurally mimics cations of imidazolium ILs. Therefore, such anomalous density behavior in a glassy state nanocage goes far beyond ILs and proves to be a more general phenomenon, which can be structurally tuned and rationally adjusted for various potential applications in nanoscale materials.

Membrane-Sugar Interactions Probed by Low-Frequency Raman Spectroscopy: The Monolayer Adsorption Model.

Small sugars are known to stabilize biological membranes under extreme conditions of freezing and desiccation. The proposed mechanisms of stabilization suggest membrane-sugar interactions to be either attractive or repulsive. To obtain new insight into the problem, we use a recently developed low-frequency Raman scattering approach which allows detecting membrane mechanical vibrations. For model membranes of palmitoyl-oleoyl-glycero-phosphocholine (POPC) hydrated in aqueous sucrose and trehalose solutions, we studied the Raman peak between 12 and 15 cm-1 that is attributed to an eigenmode of the normal mechanical vibrations of a lipid monolayer. For both sugars, similar results were obtained. With an increase in sugar concentration in solution, the frequency position of the peak was found to decrease by ∼13% which was interpreted as a consequence of the membrane thickening due sugar monolayer adsorption on the membrane surface. The concentration dependence of the peak frequency position was satisfactorily described by a Langmuir monolayer adsorption model. It is concluded that, at small sugar concentrations (less than 0.2 M), the membrane-sugar interactions are attractive, while at higher concentrations (more than 0.4 M) the attraction disappears. The data obtained show that one sugar molecule on the surface interacts with approximately 3-4 polar lipid heads.

Exploration of the Structural and Vibrational Properties of the Ternary Molybdate Tl5BiHf(MoO4)6 with Isolated MoO4 Units and Tl+ Conductivity.

The phase relations in the subsolidus region of the Tl2MoO4-Bi2(MoO4)3-Hf(MoO4)2 system were studied with the "intersecting cuts" method. The formation of the novel ternary molybdate Tl5BiHf(MoO4)6 is found in this ternary system. The compound has a phase transition at Tpt = 731 K (ΔH = -3.15 J/g) and melts at Tm = 871 K (ΔH = -41.71 J/g), as determined by a thermal analysis. Tl5BiHf(MoO4)6 single crystals were obtained by the spontaneous nucleation method. The crystal structure of Tl5BiHf(MoO4)6 was revealed by structure analysis methods. This molybdate crystallizes in the trigonal space group R3̅c with the unit cell parameters a = 10.6801(4) Å, c = 38.5518(14) Å, V = 3808.3(2) Å3, and Z = 6. The vibrational characteristics of Tl5BiHf(MoO4)6 were determined by Raman spectroscopy. The Tl5BiHf(MoO4)6 conductivity was measured at frequencies of 0.1, 1.0, and 10 kHz in the temperature range of 293-773 K; in this temperature range, the conductivity level was 10-12-10-7 S/cm.

The nano-structural inhomogeneity of dynamic hydrogen bond network of TIP4P/2005 water.

A method for studying the time dependence of the short-range molecular order of water has been proposed. In the present study, water is considered as a dynamic network between molecules at distances not exceeding 3.2 Å. The instantaneous configurations obtained with the molecular dynamics method have been sequentially analyzed. The mutual orientation of each molecule with its neighboring molecules has been studied and the interaction energy of each pair of neighbor molecules has been calculated. The majority of mutual orientation angles between molecules lie in the interval [0°; 20°]. More than 85% of the molecular pairs in each instantaneous configuration form H-bonds and the H-bond network includes all water molecules in the temperature range 233-293 K. The number of H-bonds fluctuates near the mean value and increases with decreasing temperature, and the energy of the vast majority of such bonds is much higher than the thermal energy. The interaction energy of 80% of the H-bonding molecular pairs lies in the interval [-7; -4] kcal/mol. The interaction energy of pairs that do not satisfy the H-bond angle criterion lies in the interval [-5; 4] kcal/mol; the number of such bonds does not exceed 15% and decreases with decreasing temperature. For the first time it has been found that in each instantaneous configuration the H-bond network contains built-in nanometric structural heterogeneities formed by shorter H-bonds. The fraction of molecules involved in the structural heterogeneities increases from 40% to 60% with a temperature decrease from 293 K to 233 K. Each heterogeneity has a finite lifetime and changeable structure, but they are constantly present during the entire simulation time.

Deciphering the orientation of lipid molecules by principal component analysis of Raman mapping data.

The orientation of lipid molecules is an essential characteristic of supported phospholipid layers, synthetic lipid structures, and biological specimens. Here, we perform Raman spectroscopy to analyze the orientation order in lipid structures. For this purpose, we studied dry oriented planar DMPC samples and multilamellar DPPC vesicles in water using Raman mapping. Principal component analysis (PCA) was applied to extract the information about the orientational order of lipid molecules. Using PCA, we revealed the features observed in the phospholipid spectra that are sensitive to hydrocarbon chain orientation relative to the polarization of laser radiation. These spectral features include Raman peaks corresponding to stretching C-C, twisting CH2, rocking and stretching CH3 modes. We suggest to use them as markers of hydrocarbon chain orientation along with light polarization. The proposed Raman analysis can be used to study samples with different levels of hydration.

Normal vibrations of ternary DOPC/DPPC/cholesterol lipid bilayers by low-frequency Raman spectroscopy.

A lipid bilayer containing a ternary mixture of low- and high-melting lipids and cholesterol (Chol) can give rise to domain formation, referred to as lipid rafts. Low-frequency Raman spectroscopy at reduced temperatures allows detection of normal membrane mechanical vibrations. In this work, Raman spectra were obtained in the spectral range between 5 and 90 cm-1 for bilayers prepared from dioleoyl-glycero-phosphocholine (DOPC), dipalmitoyl-glycero-phosphocholine (DPPC) and Chol. A narrow peak detected between 13 and 16 cm-1 was attributed to the vibrational eigenmode of a lipid monolayer (a leaflet). For the equimolar DOPC/DPPC ratio, the Chol concentration dependence for the peak position, width and amplitude may be divided into three distinct ranges: below 9 mol%, the intermediate range between 9 mol% and 38 mol%, and above 38 mol%. In the intermediate range the peak position attains its minimum, and the peak width drops approximately by a factor of two as compared with the Chol-free bilayers. Meanwhile, this range is known for raft formation in a fluid state. The obtained results may be interpreted as evidence that bilayer structures in the raft-containing fluid state may be frozen at low temperatures. The drop of peak width indicates that at the spatial scale of the experiment (∼2.5 nm) the intermolecular bilayer structure with raft formation becomes more homogeneous and more cohesive.

Lipid Droplet Phase Transition in Freezing Cat Embryos and Oocytes Probed by Raman Spectroscopy.

Embryo and oocyte cryopreservation is a widely used technology for cryopreservation of genetic resources. One limitation of cryopreservation is the low tolerance to freezing observed for oocytes and embryos rich in lipid droplets. We apply Raman spectroscopy to investigate freezing of lipid droplets inside cumulus-oocyte complexes, mature oocytes, and early embryos of a domestic cat. Raman spectroscopy allows one to characterize the degree of lipid unsaturation, the lipid phase transition from the liquid-like disordered to solid-like ordered state, and the triglyceride polymorphic state. For all cells examined, the average degree of lipid unsaturation is estimated as ∼1.3 (with ±20% deviation) double bonds per acyl chain. The onset of the lipid phase transition occurs in a temperature range from -10 to +4°C and does not depend on the cell type. Lipid droplets in cumulus-oocyte complexes are found to undergo abrupt lipid crystallization shifted in temperature from the ordering of the lipid conformational state. In the case of mature oocytes and early embryos obtained in vitro, the lipid crystallization is broadened. In the frozen state, lipid droplets inside cumulus-oocyte complexes have a higher content of triglyceride polymorphic ß and ß' phases than estimated for mature oocytes and early embryos. For the first time, to our knowledge, the temperature evolution of the phase state of lipid droplets is examined. Raman spectroscopy is proved to be a promising tool for in situ monitoring of the lipid phase state in a single embryo/oocyte during its freezing.

Structural Anomalies in Ionic Liquids near the Glass Transition Revealed by Pulse EPR.

Unusual physical and chemical properties of ionic liquids (ILs) open up prospects for various applications. We report the first observation of density/rigidity heterogeneities in a series of ILs near the glass transition temperature ( Tg) by means of pulse electron paramagnetic resonance (EPR). Unprecedented suppression of molecular mobility is evidenced near the glass transition, which is assigned to unusual structural rearrangements of ILs on the nanometer scale. Indeed, pulse and continuous wave EPR clearly indicate the occurrence of heterogeneities near Tg, which exist in a rather broad temperature range of ∼50 K. The two types of local environments are evidenced, being drastically different by their stiffness. The more rigid one suppresses molecular mobility, whereas the softer one instead promotes diffusive molecular rotation. Such properties of ILs near Tg are of general importance; moreover, the observed density/rigidity heterogeneities controlled by temperature might be considered as a new type of tunable reaction nanoenvironment.

Effect of glycerol on photobleaching of cytochrome Raman lines in frozen yeast cells.

We applied a Raman spectroscopy approach to investigate the effect of a cryoprotectant on the redox state of cytochromes on freezing yeast cells. The redox activity of cytochromes was studied using time-resolved photobleaching of the resonance Raman lines. It is found that ice formation causes a drastic change in the redox state of cytochromes in cells frozen without cryoprotectant, whereas in the presence of glycerol the effects of ice formation are more gradual. The photobleaching rate of cells frozen in glycerol solution shows a gradual slowing with temperature decrease and an abrupt slowdown below - 48 °C. This abrupt decrease was interpreted as originating from changes in protein conformational dynamics. Our findings provide important new insights into the transition from active to inactive cytochrome states as cells undergo freezing in the presence and absence of cryoprotectant.