ABSTRACT
BSTS epitaxial thin film topological insulators were grown using the MBE technique on two different types of substrates i.e., Si (111) and SiC/graphene with Bi0.7Sb1.6Te1.8Se0.9 and Bi0.9Sb1.5Te1.8Se1.1, respectively. The crystallographic properties of BSTS films were investigated via X-ray diffraction, which showed the strongest reflections from the (0 0 l) facets corresponding to the rhombohedral phase. Superior epitaxial growth, homogeneous thickness, smooth surfaces, and larger unit cell parameters were observed for the films grown on the Si substrate. Polarization dependent Raman spectroscopy showed a weak appearance of the Ag mode in cross--polarized geometry. In contrast, a strong Eg mode was observed in both parallel and cross-polarized geometries which correspond to the rhombohedral crystal symmetry of BSTS films. A redshift of Ag and Eg modes was observed in the Raman spectra of BSTS films grown on the Si substrate, compared to those on SiC/graphene, which was directly associated with the unit cell parameter and composition of the films. Raman spectra showed four fundamental modes with asymmetric line shape, and deconvolution of the peaks resulted in additional modes in both the BSTS thin films. The sum of relative ratios of linewidths of fundamental modes (Ag and Eg) of BSTS films grown on Si substrate was lower, indicating a more ordered structure with lower contribution of defects as compared to BSTS film grown on SiC/graphene substrate.
ABSTRACT
Advances in cryobiology techniques commonly target either the cooling or the warming cycle, while little thought has been given to âªrepairâ« protocols applicable during cold storage. In particular, crystallization is the dominant threat to cryopreserved samples but proceeds from small nuclei that are innocuous if further growth is forestalled. To this end, we propose a laser editing technique that locally heats individual crystals above their melting point by a focused nanosecond pulse, followed by amorphization during rapid resolidification. As a reference, we first apply the approach to ice crystals in cryoprotected solution and use Raman confocal mapping to study the deactivation of crystalline order. Then, we examine dimethyl sulfoxide trihydrate crystals that can germinate at low temperatures in maximally freeze concentrated regions, as commonly produced by equilibrium cooling protocols. We show how to uniquely identify this phase from Raman spectra and evidence retarded growth of laser-edited crystals during warming.
ABSTRACT
Under physiological conditions, the membranes and lipid droplets of germ cells are in a conformationally disordered phase. Typically, during cooling, lipids undergo the transition to ordered phases and, upon heating, melt into a disordered phase. In this communication, we report the lipid phase transition in lipid droplets observed in porcine oocytes. Upon cooling, a sharp lipid phase transition from conformationally disordered to ordered state was detected within the temperature range between 20 and 15 °C. Subsequent heating to 45 °C does not return lipids to their original phase state. To the best of our knowledge, this is the first observation of an irreversible phase transition in lipid droplets of biological cells with native lipid composition.
Subject(s)
Cryopreservation , Oocytes , Animals , Swine , Cryopreservation/methods , Phase Transition , Freezing , LipidsABSTRACT
The ability of salts to change the macroscopic viscosity of their aqueous solutions is described by the Jones-Dole equation with B-coefficient for the linear concentration term. The sign and value of this coefficient are often considered as a measure of the salt's structure-making/breaking ability, while the validity of this assignment is still under discussion. Here, by applying Raman and Brillouin scattering spectroscopy to various salts from the Hofmeister series, we studied a possible relation between macroscopic Jones-Dole's B-coefficient and the microscopic dynamic response. Raman spectroscopy provides information about molecular vibrations and Brillouin spectroscopy about acoustic phonons with wavelengths of hundreds of nanometers. It has been found that Jones-Dole's B-coefficient correlates linearly with the coefficients, describing the concentration dependences of the average OH stretching frequency, real and imaginary parts of gigahertz elastic modulus. These relationships have been interpreted to mean that the OH stretching frequency is a measure of the ion-induced changes in the water network that cause changes in both viscosity and gigahertz relaxation. Depolarized inelastic light scattering revealed that the addition of structure-making ions not only changes the frequency of the relaxation peak but also increases the low-frequency part of the relaxation susceptibility. It was shown that the ion-induced increase in the gigahertz elastic modulus can be described by changes in the relaxational susceptibility without a noticeable change in the instantaneous elastic modulus. The isotropic Raman contribution associated with the tetrahedral-like environment of H2O molecule does not correlate with Jones-Dole's B-coefficient, suggesting a minor influence of these tetrahedral-like configurations on viscosity.
Subject(s)
Salts , Water , Elastic Modulus , Water/chemistry , Ions , Spectrum Analysis, RamanABSTRACT
Cryopreservation of preimplantation embryos is a widely used technique, but this procedure might impact the subsequent embryo development. The effect of slow freezing and vitrification on the lipid metabolism in preimplantation mammalian embryos is not well studied. In this work, we applied Raman spectroscopy of isotopically labeled molecules to address the effects of cryopreservation on fatty acid accumulation in mouse embryos. Embryos after slow freezing or vitrification were cultured for 20 h in a medium supplemented with bovine serum albumin saturated with deuterated stearic acid (dSA). After this period the concentration of dSA estimated from Raman spectra of frozen-thawed and vitrified-warmed embryos at the morula stage was almost twice higher compared to non-cryopreserved morulas. At the same time, frozen-thawed and vitrified-warmed 4-cell embryos did not demonstrate any difference in the level of stearic acid uptake from non-cryopreserved embryos of the same stage. After an additional 24 h culture, cryopreserved and non-cryopreserved embryos demonstrated similar dSA uptake.
Subject(s)
Cryopreservation , Embryo Transfer , Mice , Animals , Cryopreservation/methods , Embryo Transfer/methods , Vitrification , Blastocyst , MammalsABSTRACT
We present a comprehensive theoretical and experimental Raman spectroscopic comparative study of bulk Phosphorus allotropes (white, black, Hittorf's, fibrous) and their monolayer equivalents, demonstrating that the application of the Placzek approximation to density functional theory calculated frequencies allows reliable and accurate reproduction of the bulk spectra at a relatively low computational cost. As well as accurate frequencies, peak intensities are also reproduced with reasonable accuracy. Having established the viability of the method we apply it to other less well characterised phosphorus forms such as isolated P4 cages and the planar blue-phosphorus phase. There are several speculative structural models in the literature for amorphous red phosphorus, and we predict Raman spectra for several of these. Via comparison with experiment this allows us to eliminate many of them such as the P2P2-zigzag chain and connected P4 models. The combination of Density functional theory (DFT) modelling, Placzek approximation for intensities with experimental Raman spectroscopy is demonstrated as a powerful combination for accurate characterisation of phosphorus species.
ABSTRACT
Fatty acid uptake and accumulation in lipid droplets are essential processes of lipid metabolism. Oocyte in vitro culture in media enriched with fatty acid is used to modify the lipid content and composition, aiming to study the consequences of obesity and enhance cell cryotolerance. We applied Raman spectroscopy and deuterium labeling approach to quantify stearic acid uptake and investigate its incorporation within oocytes. Our data suggest that deuterium labeling does not affect oocyte maturation rates. The efficiency of deuterated stearic acid (dSA) uptake was shown to decrease with the increase of its concentration in culture medium and the duration of in vitro culture. The molar ratio between dSA and bovine serum albumin has no significant effect on the dSA uptake for 200 µM but modifies concentration dependence of the lipid uptake. dSA accumulates in all the lipid droplets inside oocytes. Different lipid droplets within the same oocyte exhibit different concentrations of dSA. The scatter in the dSA concentration in lipid droplets decreases with the culture time. Using dSA as an example, we provide a comprehensive description of how fatty acid concentration, its molar ratio versus bovine serum albumin, and culture time affect the uptake of the fatty acids in oocytes. Raman microspectroscopy of deuterium-labeled fatty acids is a nondestructive tool providing information about fatty acid uptake and heterogeneity of their accumulation between lipid droplets within the single oocyte.
Subject(s)
Deuterium , Lipid Droplets/metabolism , Oocytes/metabolism , Stearic Acids , Animals , Cats , Deuterium/chemistry , Deuterium/pharmacokinetics , Deuterium/pharmacology , Female , Isotope Labeling , Oocytes/cytology , Stearic Acids/chemistry , Stearic Acids/pharmacokinetics , Stearic Acids/pharmacologyABSTRACT
Although lipid droplets are believed to play an important role in cryopreservation of mammalian embryos and oocytes, the effect of low temperatures on lipid droplets and related mechanisms of cryodamage are still obscure. Here, we provide Raman spectroscopy evidence of lipid separation inside the lipid droplets in domestic cat oocytes during slow freezing. It was shown that at -25 °C lipids coexist in two separated phase states inside lipid droplets. The scale of detected domains was a few micrometers size. We also found that under certain conditions these areas have a specific spatial distribution. Lipids with high melting temperatures are distributed near the surface of lipid droplets while fusible lipids are located deep inside. Raman spectroscopy was found to be a prospective approach to study inhomogeneity of lipid phase transition in cells and to reveal effects of this inhomogeneity on cryopreservation of biological cells.
Subject(s)
Cryopreservation , Spectrum Analysis, Raman , Animals , Cats , Cryopreservation/methods , Freezing , Lipids , OocytesABSTRACT
Embryos cryopreservation is a widely used technology for genetic resources storage. Cryopreservation suppresses cell respiration, but very little is known about the changes that occur with mitochondria at low temperatures. We used Raman spectroscopy to investigate photoresponse and redox state of cytochromes in the respiratory electron transport chain (ETC) in early mouse embryos during cooling. Redox state of cytochromes was probed by the intensity of cytochrome resonance Raman lines. Photoinduced reactions of cytochromes were used to study the changes in the rates of redox reactions. It is found that the rate of cytochrome photoresponse detected by Raman spectra abruptly changes when embryos are cooled below -50⯰C. Raman mapping revealed that the average intensity of cytochrome Raman peaks at -65⯰C is higher than at -40⯰C. Cytochrome b reduction was found in embryos frozen below -50⯰C when irradiated with 532â¯nm laser radiation. These effects were observed for cells frozen in aqueous solutions of two different cryoprotectants: glycerol and propylene glycol. Raman spectroscopy of cytochromes reveals the abrupt changes in the ETC work of frozen mouse embryos at temperatures below -50⯰C. We suggest that similar phenomena can be found in various cell types.
Subject(s)
Cytochromes b/metabolism , Cytochromes c/metabolism , Embryo, Mammalian/metabolism , Animals , Cold Temperature , Cytochromes b/radiation effects , Cytochromes c/radiation effects , Embryo, Mammalian/radiation effects , Female , Freezing , Light , Male , Mice/embryology , Oxidation-Reduction , Pregnancy , Spectrum Analysis, RamanABSTRACT
Although lipid phase transition is believed to be among the major damaging factors in oocytes and preimplantation embryos cryopreservation, lack of the appropriate experimental methods limits investigation of this phenomenon. Herein, we demonstrate the capabilities of Raman spectroscopy to detect the lipid phase transition within the freezing preimplantation mouse embryos. We exploit the sensibility of antisymmetric CH2 Raman peak to the phase state of lipids. It is shown that during the freezing of the mouse embryos the lipid phase transition occurs at the temperatures between -7 and 0 °C. Similar temperature dependences of CH2 mode intensities are found for lipids in the preimplantation embryos and a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, implying the similarity in the occupation rules of conformational states. Raman spectroscopy is considered as a method of choice to study the lipid phase transition during preimplantation mammalian embryos freezing and cryopreservation.
Subject(s)
Blastocyst/chemistry , Cryopreservation/methods , Lipid Bilayers/chemistry , Lipids/chemistry , Phosphatidylcholines/chemistry , Animals , Female , Freezing , Mice , Molecular Conformation , Phase Transition , Spectrum Analysis, Raman/methodsABSTRACT
The distribution of cryoprotectant (10% glycerol) and ice along the frozen plastic straw (the most useful container for freezing mammalian semen, oocytes and embryos) was studied by Raman scattering technique. Raman spectroscopy being a contactless, non-invasive tool was applied for the straws filled with the cryoprotectant solution and frozen by controlled rate programs commonly used for mammalian embryos freezing. Analysis of Raman spectra measured at different points along the straw reveals a non-uniform distribution of the cryoprotectant. The ratio between non-crystalline solution and ice was found to be increased by several times at the bottom side of the solution column frozen by the standard freezing program. The increase of the cryoprotectant fraction occurs in the area where embryos or oocytes are normally placed during their freezing. Possible effects of the cooling rate and the ice nucleation temperature on the cryoprotectant fraction at the bottom side of the solution column were considered. Our findings highlight that the ice fraction around cryopreserved embryos or oocytes can differ significantly from the averaged one in the frozen plastic straws.
Subject(s)
Cryopreservation/methods , Cryoprotective Agents/pharmacology , Glycerol/pharmacology , Semen Preservation/methods , Spectrum Analysis, Raman/methods , Animals , Embryo, Mammalian/drug effects , Freezing , Ice/analysis , Male , Oocytes/drug effects , Spermatozoa/drug effects , TemperatureABSTRACT
Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm(-1) . When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate.
Subject(s)
Cell Nucleus/ultrastructure , DNA/analysis , Erythroblasts/ultrastructure , Lymphocytes/ultrastructure , Spectrum Analysis, Raman , Animals , Chickens , Humans , Phosphates/chemistry , Rosaniline Dyes , Salamandridae , ZebrafishABSTRACT
Raman scattering spectra were obtained at 25-320 K for bilayers prepared from saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and mono-unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipids, with and without cholesterol. Raman intensities were measured at modes sensitive to lipid inter-chain interactions and/or intra-chain torsional motion (asymmetric CH2 stretching at 2880 cm(-1)) and to the conformational state of lipids (C-C stretching at 1130 cm(-1)). These intensities decreased with temperature, which could be ascribed to increased lipid flexibility. For cholesterol-free and cholesterol-containing DPPC bilayers, the decrease of Raman intensities observed above â¼200 K could be related to the phenomenon of dynamical transition known for biological systems near these temperatures. For a cholesterol-free POPC bilayer, the decrease of intensity for the asymmetric CH2 stretching mode started at a lower temperature, above 100 K, while the addition of cholesterol shifted this starting temperature to a more normal â¼200 K value. The low-temperature lipid flexibility in the case of POPC was related to the abundance of free-volume holes, which disappeared in presence of cholesterol. Near gel-fluid phase transitions, Raman intensities for cholesterol-free bilayers dropped sharply, while for cholesterol-containing bilayers, they changed smoothly.
Subject(s)
Cholesterol/chemistry , Lipid Bilayers/chemistry , Phospholipids/chemistry , Cold Temperature , Gels/chemistry , Molecular Conformation , Phase Transition , Physical Phenomena , Spectrum Analysis, RamanABSTRACT
Low-frequency (down to 30 GHz) inelastic light scattering is studied in a multicomponent glass ZBLAN (ZrF4-BaF2-LaF3-AlF3-NaF) in a wide temperature range. The contributions of the THz vibrational spectrum (boson peak) and of the fast relaxation are extracted and analyzed. It is shown that the fast relaxation spectrum is described by a distribution of relaxation times leading to a power-law ν(α) dependence in the frequency range 30-300 GHz. Temperature dependence of α(T) is well described by the Gilroy-Phillips model, while the integrated intensity of the fast relaxation increases significantly with the temperature. This feature distinguishes the fast relaxation in ZBLAN from the case of most single-component glasses. Thermodynamic and kinetic fragility indexes are significantly different for the ZBLAN glass. The correlations between the boson peak intensity, elastic moduli, and fragility index, found earlier for single-component glasses, are fulfilled for the thermodynamic fragility index of ZBLAN. In contrast, the correlation between the fast relaxation intensity at Tg and the fragility holds better for the kinetic fragility index of ZBLAN. We propose that thermodynamic and kinetic fragilities reflect different aspects of glassy dynamics in the case of glass formers with the complex chemical composition and structure topology: the former correlates with the elastic properties and the boson peak, the latter with the relaxation.
ABSTRACT
There is the rapid growth in application of Brillouin scattering spectroscopy to biomedical objects in order to characterize their mechanoelastic properties in this way. However, the possibilities and limitations of the method when applied to tissues have not yet been clarified. Here, applicability of Brillouin spectroscopy for testing the elastic response of medically relevant tissues of bovine jugular vein and pericardium was considered. Parameters of the Brillouin peak were studied for samples untreated, diepoxide-fixed, and preserved after treatment in alcohol solutions. It was found that diepoxide cross-linking resulted to a slight tendency to increase the Brillouin position for hydrated tissues. The variations in the position and width of the Brillouin peaks, associated with local fluctuations in water concentration, were reduced after diepoxide treatment in the case of the pericardium, but not in the case of the vein wall. To obtain more information about the elastic response of the protein scaffold without the participation of water, dried samples were also studied. Brillouin spectra of the dried pericardium and vein wall revealed a significant increase in the Brillouin peak position (elastic modulus) after conservation in alcohol. In the case of the vein wall, this effect was found for both collagen and elastin-related peaks, which were identified in the Brillouin spectrum. This result corresponds to a denser packing of fibrous proteins after preservation in alcohol solutions. The ability of Brillouin spectroscopy to independently characterize the effect of treatment on the instantaneous elastic modulus of various tissue components is also attractive for its application in the development of new materials for bioimplants. A comparison of the Brillouin longitudinal and Young's elastic moduli determined for the hydrated samples of the vein and pericardium showed that there is no clear correspondence between these material parameters. The usefulness of using both experimental methods to obtain new information about the elastic response of the material is discussed.
Subject(s)
Jugular Veins , Pericardium , Animals , Cattle , Pericardium/chemistry , Spectrum Analysis/methods , Elastin/analysis , Elastin/chemistry , Elastic Modulus , Collagen/analysis , Collagen/chemistryABSTRACT
We studied yeast cells in physiological solution during freezing by Raman microspectroscopy technique. The purpose was to find out the origin of a sharp peak near â¼3430cm(-1) in Raman spectrum of frozen mammalian cells, observed earlier (J. Dong et al., Biophys. J. 99 (2010) 2453), which presumably could be used as an indicator of intracellar ice appearance. We have shown that this line (actually doublet of 3408 and 3425cm(-1)) corresponds to Raman spectrum of hydrohalite (NaClâ 2H(2)O), which is formed as the result of the eutectic crystallization of the liquid solution around the cells. We also show that the spatial distribution of hydrohalite in the sample significantly depends on the cooling rate. At lower cooling rate (1°C/min), products of eutectic crystallization form layer on the cell surface which thickness varies for different cells and can reach â¼1µm in thickness. At higher cooling rate (20°C/min), the hydrohalite distribution appears more homogeneous, in the sample, and the eutectic crystallization layer around the cells was estimated to be less than â¼20nm. These experimental results are consistent with scenarios predicted by the two-factor hypothesis for freezing induced cell injury. This work demonstrates a potential of Raman microspectroscopy to study peculiarities of the eutectic crystallization around single cells in vivo with the high spatial resolution.
Subject(s)
Ice/analysis , Saccharomyces cerevisiae/chemistry , Sodium Chloride/metabolism , Crystallization , Freezing , Spectrum Analysis, RamanABSTRACT
Conformational states of phospholipid chains in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), deuterated 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPCd62), and cholesterol (Chol) were studied by Raman spectroscopy. Parameters of Raman peaks sensitive to conformational order have been used to determine chain order for mixtures over a wide range of compositions. A ternary diagram of fractions of phospholipid chains in conformationally ordered and disordered states has been constructed. It was found that the addition of POPC and cholesterol increases the fraction of DPPC chains in disordered conformations. The so-called liquid-ordered phase includes DPPC molecules in both ordered and disordered states in comparable proportions. It was found that POPC chains are partially ordered in mixtures with DPPC and cholesterol, in contrast to the case of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This maybe the underlying reason why ternary mixtures with POPC have different miscibility behavior compared to DOPC.
Subject(s)
Phosphorylcholine , Spectrum Analysis, Raman , Phospholipids , Phosphatidylcholines/chemistry , Cholesterol/chemistry , Lipid Bilayers/chemistryABSTRACT
Here, we examined the gigahertz sound velocities of hydrated multibilayers of saturated (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC) and unsaturated (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) phospholipids by Brillouin spectroscopy. Out-of-plane and in-plane (lateral) phonons were studied independently of each other. Similar strong temperature dependences of the sound velocities were found for phonons of both types. The sound velocities in the low-temperature limit were two-fold higher than that at physiological temperatures; a significant part of the changes in sound velocity occurs in the solid-like gel phase. The factors that may be involved in the peculiar behavior of sound velocity include changes in the chain conformational state, relaxation susceptibility, changes in the elastic modulus at infinite frequencies, and lateral packing of molecules.
Subject(s)
Lipid Bilayers , Phospholipids , Phospholipids/chemistry , Temperature , Lipid Bilayers/chemistry , Molecular Conformation , Phosphatidylcholines/chemistryABSTRACT
Precursor dynamics of a cubic to tetragonal ferroelectric phase transition in BaTiO3 is studied by the accurate measurement of the second harmonic generation (SHG) integral intensities. A finite signal holds for the SHG integrated intensity above the ferroelectric Curie temperature T(c)=403 K. Above the Burn's temperature T(d)≈580 K, the power law with the exponent γ=1 shows normal SHG nature originating from the hyper-Raman scattering by dynamical polar excitations, while, below T(d), a SHG signal from polar nanoregions becomes dominant with the larger exponent γ=2. Such a crossover of the power law exponent near T(d) is discussed on the basis of the effective Hamiltonian method and Monte Carlo simulation.
ABSTRACT
Glycine phases formed when aqueous solutions were frozen and subsequently heated under different conditions were studied by Raman scattering, x-ray diffraction, and differential scanning calorimetry (DSC) techniques. Crystallization of ice I(h) was observed in all the cases. On cooling at the rates of 0.5 K∕min and 5 K∕min, glassy glycine was formed as an intermediate phase which lived about 1 min or less only, and then transformed into ß-polymorph of glycine. Quench cooling of glycine solutions (15% w∕w) in liquid nitrogen resulted in the formation of a mixture of crystalline water ice I(h) and a glassy glycine, which could be preserved at cryogenic temperatures (80 K) for an indefinitely long time. This mixture remained also quite stable for some time after heating above the cryogenic temperature. Subsequent heating under various conditions resulted in the transformation of the glycine glass into an unknown crystalline phase (glycine "X-phase") at 209-216 K, which at 218-226 K transformed into ß-polymorph of glycine. The "X-phase" was characterized by Raman spectroscopy; it could be obtained in noticeable amounts using a special preparation technique and tentatively characterized by x-ray powder diffraction (P2, a = 6.648 Å, b = 25.867 Å, c = 5.610 Å, ß = 113.12[ordinal indicator, masculine]); the formation of "X-phase" from the glycine glassy phase and its transformation into ß-polymorph were followed by DSC. Raman scattering technique with its power for unambiguous identification of the crystalline and glassy polymorphs without limitation on the crystallite size helped us to follow the phase transformations during quenching, heating, and annealing. The experimental findings are considered in relation to the problem of control of glycine polymorphism on crystallization.